In the determination of the stability of oil-in-water emulsions, the quantification of auto-oxidation usually relies on as an indicative measure of relative stability. There are a number of tests that can be applied, which mainly rely on the oxidation levels during the ordinary storage of the oil-in-water emulsion (Conde, 2001). In this study, there are two techniques that have been used in the determination of this apparent stability, mainly in the form of peroxide values (PV) as well as in thiobarbituric acid-reactive substances (TBARS) assays (Caprioli, Monahan and O’Sullivan, 2001). These assays act as a quantification mechanism after the detection of auto-oxidation, which leads to instability of unsaturated oils. Instability usually causes going bad of these oils through oxidative rancidity, which results in the physical manifestation of a bad taste and bad odors. Food science solutions to this problem include the enhancement of oils through modification of the physical quality of these oil substances to reduce auto-oxidation (Gordon and Wishart, 2010).
In this arrangement, it must be demonstrated that the introduction of the sample proteins samples of choice (Gelatin, Beta-lactoglobulin, Bovine serum albumin) changes the stability considerably (Benjakul et al, 2010). Apparently, the introduction of the phenolic compound acts in the reduction of the auto-oxidative activity in the oil-in-water emulsion. It, therefore, follows that the modification achieved on the reduction of the auto-oxidative activity is further enhanced through the combination of these two antioxidative agents (phenolic compound and protein) (Frankel et al 2002). In ordinary lab tests, the concentration of the antioxidative agent determines the level of reduction of auto-oxidative activity. It, therefore, implies that an increase in the antioxidative agent reduces auto-oxidative activity through the increased antioxidative quality in the oil-in-water emulsion. Proteins effectively provide a preventive action against oxidation by way of formation of a protein film covering the oil droplets (Almajano et al, 2008). In achieving the formation of films around the oil droplet, the protein molecules ensure that oxygen molecules do not easily permeate into the oil molecule. Blocking out oxygen in the oil-in-water emulsion implies that it is not possible for auto-oxidation to take place. Alternatively, this indicates that the development of oxidative rancidity is significantly reduced and effectively increases the stability of the oil (Alemán et al, 2005).
In the mentioned essays, the value of peroxides as well as thiobarbituric acid-reactive substances of the various samples used in the assay is descriptive of the stability of each of the samples. In the results, the higher the value of the peroxide or thiobarbituric acid content in the assay illustrates its effective oxidative potential thereby its instability. Oxidative activity increases with time since the freshly produced oil-in-water sample has little oxidation activity which reaches its peak within some days. In Figure 1., peroxide values are highest in the control sample which contains the oil-in-water emulsion, which indicates that its oxidative activity is also high. The separate introduction of the three protein samples in the assay gives a reduction in the peroxide value in an increasing affinity as follows; Sample mixed with gelatin> Sample mixed with beta-lacoglobulin> Sample mixed with bovine serum albumin. By day 20, there was peroxide value of above 200µ mols for three samples, with only the Sample mixed with bovine serum albumin attaining a value slightly less than these values. This demonstrates that the introduction of proteins slightly reduces antioxidation activity in the sample thereby making a contribution to the stability levels (Chow, Chu and Yang, 2008). Alternatively, the fluctuations in the curves between the first and the last day are considerably reduced which illustrates the stability achieved in terms of oxidation, apart from the Sample mixed with gelatin.
The second phase of peroxide value assay is represented in the results, determining auto-oxidation in oil-in-water samples containing both the protein and caffeic acid as an antioxidant. Separate assays for the three composite samples gave considerable reductions in the peroxide values. The control sample containing just the oil-in-water emulsion mixed with the antioxidant has a value close to the results of the first assay for quantifying peroxide values for protein-alone modification Heinonen, Kivikari and Viljanen 2004). On the contrary, it is clear that peroxide values experience a drastic drop in the addition of the antioxidant in the same sample. Perhaps the defining point of this study is demonstrated by the peroxide value obtained in the Sample containing the antioxidant alone when compared with the first samples containing the protein alone. Their curves almost resemble each other which drastically changes upon the introduction of the protein and antioxidant aspects in the sample. The drastic drop in curve fluctuations, as well as peroxide values, define the impact of protein addition in the stability of oil-in-water emulsions modified with an antioxidant agent (Aewsiri et al, 2009).
In terms of decreasing oxidation activity after the introduction of the protein and antioxidant agent, the peroxide values in the second assay rate as follows; Sample mixed with gelatine and antioxidant> Sample mixed with beta-lactoglobulin and antioxidant> Sample mixed with bovine serum albumin. The drastic drop in peroxide value between the control sample and those containing protein shows a difference of about 150µ mols which illustrates the distinction. In terms of the difference made in reducing the auto-oxidation, it is clear that the introduction of proteins in the oil-in-water emulsions works in making the improvements. The antioxidation mechanism achieved in the addition of the antioxidant is enhanced with the addition of protein. Both molecular reactions and physical reactions are synergistically involved in the reduction of oxidation that occasions instability in the oil-in-water emulsions. Molecular antioxidative activities are achieved in a number of ways thereby ensuring that the oxidation processes are kept at a low level hence reduce oxidative rancidity in oils. Among these molecular mechanisms is the radical scavenging which reduces the free radical effect and considerably reduces oxidative activity (Aewsiri et al, 2009).
According to Aewsiri et al 2009, the addition of these additives results n the formation of adducts which effectively reduce the size of oil droplets.
The protein and the antioxidant additionally increase the droplet state of the oil-in-water emulsion. Oil droplet adsorption on the protein molecules nearly covers the entire droplet which increases the chances of creation of a protein film around the droplet. In creating a film around the droplet, it is possible for the mechanism to block entry of the vital oxygen molecules that facilitate oxidative activity. Besides the creation of the film that physically creates an oxidation barrier, protein adsorption creates stability in that the droplet aggregation is significantly reduced. The effectiveness of aggregation inhibition in reducing oxidative activity is also ensured in that neighboring droplets have little chance to share their oxidative activity since they are further apart. The phenolic compound derivative of carboxyl groups also facilitates the contribution of negatively charged molecules in the emulsion (Miskelly et al, 2011). In terms of contribution from the protein, the charges are likely to be neutrally charged. The negatively charged molecules occasion an electrostatic repulsion among themselves, which creates stability of the emulsion.
The other assay involving the thiobarbituric acid-reactive substances (TBARS) analysis is also presented in the results as Figure 2. In the preparation, the sample is treated as observed in the peroxide value analysis. The sample containing the oil-in-water emulsion is used as the experimental control. In the determination of the oxidation levels with progression in days, the control was assayed alongside the proteins first and then compared with the addition of the antioxidant to the control sample alone as well as with the sample containing the proteins. The first phase of the essay dealt with the sample mixed with proteins and gave a decreasing oxidation valuation as follows; Sample mixed with gelatine> Sample mixed with beta-lactoglobulin> Sample mixed with bovine serum albumin. Interestingly, the second assay phase that using the sample mixed with the antioxidant is nearly masked by the values obtained in the first phase of the assay. This indicates the importance of protein addition in the sample containing antioxidant in explaining the generation of stability and antioxidative activity (Caprioli, Monahan and O’Sullivan, 2001).
The second phase of the assay incorporates both proteins and the antioxidant with the use of the sample mixed with the antioxidant alone being the control. The assay valuation gives reducing oxidation in the sample as follows; Sample mixed with gelatine and antioxidant> Sample mixed with beta-lactoglobulin> Sample mixed with bovine serum albumin. The distinction between the addition of the protein in the sample with antioxidant stands out clearly in the two assays where protein contribution can be said to significantly contribute to a drastic drop in oxidative activity in the sample. The curve on the assay using the sample containing the antioxidant alone is overlapped by the results of the protein phase of the assay, which clearly indicates the defining moment (Heinonen et al, 2005). As illustrated in the results represented by Figure 1 and discussed above, it is clear in both the assays that the introduction of protein in the assay containing the sample and the antioxidant changes the values in a massive way. Curve fluctuations are also reduced in the second phase of the assays which illustrates the achievement of the stable state for the oil-in-water emulsion as well as the reduced oxidation values (Aewsiri et al, 2009).
Oils in fatty foods exist in the form of emulsions and heavily depend on the rancidity quality of the food in terms of their shelf life. Therefore, their shelf life is dependent on the oxidation characteristic that the type of oils in the food have, which implies that their oxidation rate is directly related to the shelf life. The addition of antioxidants in a relatively lower concentration when compared to the actual food substrate is a positive food chemistry indulgence in adding more life to foods of this class. The mechanism in which the antioxidants lengthen the shelf life of fatty foods is mainly through prevention or delaying of oxidation that occasion rancidity (Almajano, Delgado and Gordon, 2007a). Foul odors, as well as flavors, are indicative of such foods that have been tampered with by oxidation. Phenolic substances have been used in different ways as antioxidants thereby making them among the most suitable antioxidants for use in these processes. However, they are not as effective as scientists would like them to be.
The performance of the different types of antioxidants is usually dependent on the type of oils, for instance, fatty acids, triacylglycerols among others. Although proteins alone cannot be used as antioxidants, they possess some potential to reduce oxidation in other foods. They can, therefore, be used to modify the shelf life of most foods of oil-in-water nature. In a dramatic chemical reaction between phenolic substances and proteins, the solution for enhanced antioxidative activity has been found. Food chemistry applies the enhanced combination or synergy between these two substances to achieve high performance in the preservation of fatty foods (Almajano et al, 208).
Proteins application as major emulsifiers is perhaps one of the most important breakthroughs in food science. This phenomenon is based on the protein’s capacity to improve the stable state existence of oil-in-water emulsions without the formation of unwanted by-products such as foams as well as formation products with a reduced interfacial tension (Aewsiri et al, 2009). With regard to the actual impact of proteins on the stability of oil-in-water emulsions with antioxidant agents follows a complex interaction between nucleophilic groups with electrophilic groups available in the molecular configuration of the involved agents. A number of nucleophilic groups on proteins include cysteine, tryptophan, methionine, N-terminal proline, tyrosine as well as histidine which create affinity for amino groups on other substrates (Kataola, Kitora, and Yamamoto, 1998). A reaction of such protein substrates with phenolic compounds that have electrophilic groups facilitates the reduction of the free amino groups. However, intermediate configurations of these substrates might be necessary to ensure that the reactive groups are in their active state in order for the reaction to proceed. Caffeic acid, as well as gallic acid, is an example of the simple molecular compounds referred to as phenolic acids or polyphenols (Almajano, Delgado and Gordon, 2007b). Oil-in-water emulsions having this class of compounds are stabilized by the addition of proteins due to the synergistic impact gained in reducing oxidation.
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