The rancidity of fats. Quick rancidity as a research method. The effect of antioxidants

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1 Volume 21(3), , 2017 JOURNAL of Horticulture, Forestry and Biotechnology The rancidity of fats. Quick rancidity as a research method. The effect of antioxidants Bujancă G. 1*, David I. 1, Danci M. 2 1 University of Agricultural Sciences and Veterinary Medicine of Banat "King Mihai I of Romania" Timisoara, Faculty of Food Processing Technology, Calea Aradului 119, Timisoara, RO , Romania; 2 University of Agricultural Sciences and Veterinary Medicine of Banat "King Mihai I of Romania" Timisoara, Faculty of Horticulture and Forestry, Calea Aradului 119, Timisoara, RO , Romania; *Corresponding author. gabrielbujanca@yahoo.com Abstract In order to track the rancidity process, the method of determining the peroxidic index is also suitable for rapid rancidification. We have established the relationship between the emergence of organoleptic rancidification and the peroxidic index, the pork fat, the sunflower oil, as well as the mixtures. Such relationships can only be established for each fat or for fat blends. For the determination of the storage life I compared several fats, using Swift's fast-pacing method. Based on the results we have made the following findings: To pig fat. From the point of view of fatty tissues, the most stable is the fat of the intestines, and from the point of view of the extraction process the lower and well-melted fat is more stable. Sunflower oil. The molded, heat-treated and alkali-refined seeds shorten the incubation time of the oil. Oxidation products that are born during storage also accelerate the rising of fresh fats. Key words antioxidants, rancidity of fats, pork fat, sunflower oil The production of unpleasant and specific changes in the taste and smell that can be detected organoleptically during fat storage is called rancidity. This is a phenomenon very well known and because of it, a large amount of fat can become inedible. Apart from this, many researchers have shown that besides the property of being unpleasant, the rancid fat also has a particularly harmful effect, especially on the young and hypersensitive organism(11,12,13). After eating rancid fats, nausea, digestive problems, anemia and urticaria have occurred. These findings have not yet been fully elucidated. Instead, it is entirely safe to note that any combination produced during the rancidity has a destructive effect on vitamins and other essential substances.(1,4,10). So, as a general result of the research so far, it can be said that by consuming the rancid fats, at least indirect damage is produced. This determines the need to fight the rancidity. Determination of the evolution of the rancidity process In order to answer this question, we should be fully aware of the rancidity process. Until now, however, we are not clear about the complete mechanism of the reaction that is taking place. Some points may, however, be fixed. a) The rancidity speed is influenced by internal and external factors. Internal factors are the composition of fatty acids from fat and the natural content of antioxidants. The external factors are: the temperature, the amount and the light wavelength, the oxygen concentration, the humidity and, besides them, the substances with porous or antioxidant behavior, which are produced in fat during the processing. b) The above factors strongly influence not only the speed of the rancidity, but also the mechanism. To a certain extent, the mechanism is different, for example, to oleic acid and linoleic acid. Also, after Hilditch (2) it is different at low temperature and high temperature. Generally, it is believed that the decisive part of the rancidity process is due to the structure of fatty acids and, in particular, of unsaturated fatty acids. In fact, other combinations of fat, for example vitamin E, are oxidized, but the flavor and smell come from the decomposition products of fatty acids. It can be taken as a fact that the process involves at least two phases: oxygen is added to the fatty acids, and then the oxygen-rich compound decomposes or transforms. The first phase is known, but the second is more or less unclear. So, the process begins by adding oxygen. The oxygen molecule is added to the unsaturated fat bonds or to two neighboring methyl groups. Thus, 153

2 compounds with two kinds of peroxide bonds can be produced: CH CH CH CH CH O O O OH cyclic peroxide or hydroperoxide Some thought that epoxides and moloxides would be produced, but more recent research considers probable only the existance of the first two compounds. The cyclic peroxides were analyzed, and the hydroperoxides were even prepared from Farmer's rancid fats, by crystallisation at low temperature, chromatographically. Therefore, their presence has been definitively confirmed. (3) According to the above, the rancidity process can be pursued in two ways. By determining the added oxygen or the formed peroxides. For both ways there have been developed methods, but the latter is faster and more convenient. The basic principle is that peroxides oxidize the iodide to iodine and thus their quantity can be determined with thiosulfate. The determinations only make sense if the amount of peroxides increases proportionally to the progress of the rancidity and only if the method is quantitative and not disturbed by other compounds. Indeed with the advancement of incubation time, the breakdown of peroxides (this would be the second phase of the rancidity) will begin and various compounds of the aldehyde and acid type will be obtained.the research on this issue has provided satisfactory answers. They have shown that the peroxide content increases continuously with the rancidity process; therefore, in order to control this process, its determination provides a support point which, although not complete, is satisfactory(8,9). The appearance of the by-products alters the other fat value indices (eg the acidity index, iodine index, viscosity, etc.), but the finding of these changes or the quantitative determination of the compounds that have occurred (eg aldehydes) it is not appropiate for the description of the level of rancidity, at least so demands the level of knowledge reached to date(5,6,7). We also investigated the variation of the peroxide index and the other value indices, especially from the point of view of the increase in the peroxide index. For this purpose, we caused natural rancidity of the sunflower oil and pork fat, by blowing air at 100 C. We will discuss later about the changes that occurred in natural rancidity. The results obtained at the artificial rancidity of the sunflower oil are shown in Fig.1. Fig 1. Results obtained at the artificial rancidity of the sunflower oil It is apparent from the figure that in fact the amount of peroxides increases continuously until the rancidity can be found organoleptically (the broken line) and even afterwards for a longer time. So, the 154

3 determination of the peroxidic index may be indicated for the follow-up of the rancidity process and for artificial rancidity. The other value indexes undergo changes especially after the organoleptic rancidity occurs, so in fact they are not conclusive for tracking the process of rancidity. Decreasing the iodine index, increasing the refractive index and viscosity indicates polymerization. In the case of artificial rancidity of the pork fat, the result was similar, although it has other value indices. During the rancidity process we also investigated the changes in the ultraviolet spectrum (fig. 2). We have noticed that, with the start of the rancidity process, there is a steady increase in the maxima, indicating the occurrence of conjugate bonds, but the value changes did not lend themselves to accurate quantitative conclusions. Fig 2. During the rancidity process we also investigated the changes in the ultraviolet spectrum For the determination of the peroxidic index we used the modified Lea method (4). This differs from the usual determination of the peroxide index, since determination takes some time in artificial light and is carried out in an inert gas background (carbon dioxide or nitrogen). We expressed the result in milliliters of normally consumed thiosulphate, related at 1000 g of fat. In the following, the peroxide index is understood as this value. The value thus obtained is double the usual Lea index. Determination of incubation time of different fats. Fast rancidity The determination of incubation time for some fats stored at room temperature requires several months. For shortening the time, numerous artificial methods have been developed everywhere, so-called fast-rancidity methods. Each of these methods seeks to use the action of any of the external factors that influence the rancidity, such as light, temperature, oxygen, or different catalysts. Joiner keeps the fat at 63⁰C (5). Others catalyze with light or preoxidants. Swift causes rancidity without light, by increasing the temperature and the amount of air (6). In our experiences we have applied this latter process. Its advantage is that it fixes the external circumstances that influence the rancidity (temperature, light, air quantity, humidity) and thus allows the examination of the action of the structure of fatty acids as well as of anti- and pre-oxidants. The principle of the method is shown in Fig

4 Fig.3 Examination of the action regarding the structure of fatty acids as well as of anti- and pre-oxidants The machine consists, of an oil bath set in a sea bath with constant temperature. By keeping the boiling water, the temperature of the oil bath varies according to the atmospheric pressure between 97.5 and 98.5 C. The tubes containing the fat samples sink into the oil bath. The amount of passing air is ensured by water columns of determined height and capillaries of a certain cross section. During research we passed through the fat an air stream of 140 ml per minute. In the machine, the rancidity occurs within hours, so incubation time is generally expressed in hours. The advance of the rancidity process is controlled by the peroxidic and organoleptic index. Research done with different fats Fig. 4 shows the results of fast rancidity on pork fat, goose fat, sunflower oil and flaxseed oil. Fig. 4 Results of fast rancidity on pork fat, goose fat, sunflower oil and flaxseed oil Fig. 4 has to be considered from four points of view: from the point of view of the speed of growth of the peroxidic index, the appearance of the curve representing the increase, the relationship between the organoleptic rancidification and the peroxidic index and the duration of the incubation time. 156

5 1. It has been shown that the rate of growth of the peroxide index varies according to the nature of the fat. It is maximum in linseed oil, followed by goose fat, pork fat and sunflower oil. For the fats examined by us, 2. the peroxidic index of 100 was reached in the times shown in Chart 1. There is also the composition of fatty acids from fat. The oleic, linoleic and linolenic acid content of different fats and the duration of air blowing until the peroxide index is 100 Name The duration of air blowing until the peroxide index reaches 100 in hours Linolenic acid content Linoleic acid content Oleic acid content Percentage Pork fat 7 Under Goose fat 4 ½ Under Sunflower oil 8 ½ Linseed oil 1 1/ From the chart it can be seen that fats that have more unsaturated bonds grow faster. Indeed, the double bonds of the chains of the acids, respectively the methyl groups near them, activate slightly (7). However, many fats contain natural antioxidants or prooxidants, and for that reason, they deny that regularity. For example, vitamin E is an antioxidant; instead, the squalene content of the unsaponifiable part has a pro-oxidant effect. Among our oils, sunflower oil owes its remarkable value (its content of linoleic acid is high, though its incubation period is long) to its natural antioxidant content. The antioxidant action is proved that in the mixtures of pork fat and sunflower oil ( % oil), the incubation time is longer than pure pork fat. 4. Dura-tion of air blowing 2 hours 30 minute Pork fat Table 1 3. According to our research, the aspect of the graph represents the values of the peroxide index that also depends on the fatty acid content of the fats (Figure 4). In the case of fat containing few unsaturated fatty acids (goose, pig fat), the curves are probably exponential because the addition of oxygen by oleic acid is catalysed by the resulting peroxides of linoleic acid and the acids with many unsaturated bonds. In their absence, oleic acid peroxides are born but very slowly. To demonstrate this assumption, we examined on the Swift device the increase in the peroxide index of pork fat and various crude vegetable oils. The results are summarized in Chart 2. The peroxidic index of some pig fat and oil blends after an identical blowing time Pork fat + Pork fat + Pork fat + Pork fat + 5% Sunflower oil r oil Casto 5% castor 5% linseed 5% tung sunflower oil oil oil oil Lins eed oil Table 2 Tung oil 5,1 5,0 4,2 16,6 81,3 22,0 10, hours 7,3 7,0 7,8 95, ,2 15, In particular, blowing the air for 4 hours in blends prepared with linseed oil and tung oil (Chinese wood), shows the catalyst role of the blended oils. (linseed oil contains 43% linolenic acid with 3 double bonds, and tung oil contains 70-80% elaeostearic acid with 3 conjugated double bonds). 5. From the industrial point of view, the most important is the relationship between the constant organoleptic and the peroxidic index. In FIG. 4 I have marked the values of the peroxide index with the arrows when the taste is rancid. In the pork fat, the rancid taste feels neat. Through a lot of research (made with raw fats, we have established that a secondary taste occurs when the peroxide index becomes 10, and when the peroxide index reaches , a pronounced rancid taste appears. If this is true, it means that the peroxide index can be established for any raw fat, not mixed with other fats, at any stage of incubation. During air blowing, the rancid taste also occurs in goose fat, but at a higher peroxidic index than in pork fat (at ). 157

6 Compared to pork fat, sunflower oil becomes inedible at a higher peroxidic index. At the same time, the foreign tastes that appear are different. Up to the peroxidic index 40, no foreign taste can be found and before the appearance of the net rancid taste, between the peroxidic indices , there is a metallic, bitter, taste other than the pork fat. Because of this, the oil becomes inedible, although no specific flavor can be observed except for peroxidic indices greater than 200. In the case of flaxseed oil, during a fast rancidification, a whole range of taste (bitter, metallic, pungent, fish lard) appears and the net rancid taste does not appear in the peroxidic index 400. In conclusion, the rancid taste or other tastes that make the fat inedible can be felt at peroxide indices that differ because of the nature of the fat. So the relationship between this and the peroxide index is appreciated for each fat in part. The varied tastes show that, during the rancidification process, at fats with different composition of fatty acids appear different products and different quantities. The main cause of the specific rancid taste is probably as determined by Powick (8), the non-linear and monillic aldehyde resulting from oleic acid. If a fat contains greater amounts of unsaturated acids (linoleic and linolenic acid) compared to oleic acid, then the rancid taste is masked b) Name by other foreign substances that result from these latter compounds. In the case of these oils, the relatively small taste impairment observed at higher peroxidic indices demonstrates that linoleic and linolenic acid peroxides initially cause fewer reactions than oleic acid peroxides, so their taste or the products they produce are not so unpleasant. The duration of the incubation time can be determined by the signs (circles) of Fig.4. By comparing the examined fats, we come to the same conclusion as in point 1: the time in which the rancidification occurs depends, in particular, on the structure of fatty acids, but is largely influenced by pro- and antioxidants in fat. Research done with pork fat In FIG. 4 we have been shown the results of rapid rancidification of typical pork fat. Then, we dealt with pork fat from different fatty tissues, produced by the slaughterhouse and fats, obtained at different melting temperatures. a) Chart 3 summarizes the results of the research on the fats from the various fatty tissues of a single animal. Characteristics of fats derived from different fatty tissues of a single animal Iodine Index Solidification point at ⁰C Chin fat 70,4 26,8 Fat on the stomach Drop point in ⁰C 23,0 Peroxide index Penetration at 25 C Table 3 Hatching time (rancidification) in hours Under , ½ 65,6 28,7 26,5 Under , ½ Fat on the back 65,7 29,0 27,0 Under Fat 57,9 32,5 29,0 Under Fat from the intestines 52,8 35,0 38,7 Under In the chart, fats are indicated in the order of the magnitude of their content in unsaturated fatty acids. The duration of the incubation time is directly proportional to this order: the fastest fat on the chin; Fats inside the body and especially fat in the intestines are stable for almost a double time. Fatty acid analysis: chicken fat: 32.1% saturated fatty acids, 57.2% oleic acid, 10.0% linoleic acid, 3% unsaturated fatty acids 0.7%. In the intestinal fat: 48.4% saturated fatty acids, 44.0% oleic acid, 7.0% linoleic acid, 0.6% unsaturated fatty acids (determined by ultraviolet ray spectrometer). c) Chart 4 shows the results of the rapid rancidification of slaughterhouse products. The chart shows the effect of improving the decantation and the relatively slow rancidification of the fat in the intestines. The fat obtained through the scratching of the skin becomes rancid much faster. This is probably due to the high initial peroxide index and its relative unsaturation (iodine index). It is worth noting the resilient nature of the fat J (having a low melting temperature). 158

7 Name Characteristics of slaughterhouse products Peroxide Iodine Index Acidity index index The duration of incubation in hours Fat J 0,8 59,8 0,45 8 Dehydrated export fat 1,6 68,0 0,51 6 Unprocessed fat 1,1 Fat from the intestines 1,3 66,4 61,1 0,49 5, ½ 0,72 11 Fat obtained from the skin 4,9 72,6 0,20 4 Table 4 d) In order to investigate the effect of the different melting temperatures, we melted the fat from the same bacon from the back in several ways: the fat melted at 130 C in an oil bath, it became rancid in 6 hours. Instead, melted fat under the same conditions, but at 180 C, has already altered after 3 hours. The fat melted at 130 C in the open flame was found to be rancid after 2 hours and ½. So, during melting at high temperatures, either antioxidants are destroyed or - most likely - they are born of very strong, fat-soluble prooxidant tissues. From an industrial point of view, this fact draws attention to the need for melting to be conducted with care and mixing. According to these data, it seems that the low melting temperature is more appropriate, not only to keep the taste of fresh fat, but also in terms of rancidification. Researches with sunflower oil In Fig. 5 we reproduced the results of the research on the rapid sunflower oil rancidification. However, we also investigated the effect of different extraction methods on rancidification and, in particular, on alkaline refining. In FIG. 5 are the results of the artificial rancidification of the pressed raw oil as well as the edible, fresh and stored refined oil obtained from the processing of the same crude oil and finally of the crude oil obtained by pressing the molded seeds. Refined edible oil becomes rancid in front of the raw, and the tastes that appear when becoming rancid are unpleasant. By laboratory refining we have determined that this difference is due to the attack of alkali, probably due to the destruction of a part of natural antioxidants. So alkali refining not only reduces the amount of biological substances important to the body but it is also harmful from the point of view of the rancidity. Of course, the deposited oil became rancid before the fresh one. The same was found in the oil extracted from moldy seeds. Research on mixtures of pork fat and sunflower oil We also researched the effect of sunflower oil mixed in pork fat. Besides investigating the action of the quality of the blended oil, we also followed the effect of oil quality. Fig 5. The results of the research on the rapid sunflower oil rancidification Fig 6. The results of the artificial rancidification of the pressed raw oil 159

8 a) The results obtained with the raw sunflower oil were shown in Fig. 6. By introducing % oil into the mixture, b) The results obtained with the freshly stored oil the incubation time of the pig fat is extended from 6 are shown in Chart 5. hours 30 minutes to 8 hours 40 minutes, so a precise While the incubation time for fat blended with fresh oil result has been found, in the sense of an improvement. did not exceed 70% of the time required to incubate the The difference is probably due to the effect of fat, the oil-mixed fat with the periodic index 47 was antioxidants introduced with the oil. The blending of strongly rancid; as well as that mixed with oil, having a 20, 30, and 50% of the oil has not produced the same peroxidic index of 30, showed a decrease in stability. improvement (the effect of linoleic acid is on the Thus, the quality of the introduced oil greatly foreground), but all the blends have grown in a longer influences the rancidification time of the mixture. The time than oil. oxidation products, which take place during the Using fresh, refined oil we have obtained similar rancidity process, catalyze the rancidification of the results: introducing % oil has brought some fresh fats. improvement. Blends of % have become rancid at the same time as pork fat. The effect of introducing 10% sunflower oil of different qualities on the increase of the peroxide index of the mixture The peroxide index of the Peroxidic index of the mixture after 4 blended oil hours of air blowing 2,0 8,0 30,7 10,8 47,0 30,9 125,0 216,0 The relationship between fast rancidity and storage capacity Table 5 It would be advantageous if we found a numerical relation between the incubation time obtained at rapid rancidification and the shelf life of the refrigerator. For this purpose, we investigated the pork fat stored in the refrigerator (Figure 7) with the Swift device at certain intervals. Fig.7. Pork fat stored in the refrigerator with the swift device at certain intervals 160

9 It can be seen from the figure that the duration of the incubation at the rapid rancidification decreases, the more the fat has been stored for longer. For example, the initial incubation time of 6 hours 20 minutes is reduced after 9 months of storage in 2 hours, but after 3 months of storage it barely shows a change in relation to fresh fat. In connection with this issue, the results of new statistical research are still needed. c) The effect of antioxidants It is known that certain substances delay the rancing of fats. Based on hundreds of researches, the use of various natural and synthetic substances has been recommended, and their action has also been theoretically supported. Their role is mainly in the absorption of activation energy. As mentioned above, the rancidity process begins by reacting the fat with oxygen. But oxygen can not enter the fatty acid chain, only under the action of a so-called activating energy (light or caloric energy). If the antioxidant in this energy does not occur, the rancidification does not occur until the majority of the antioxidant is consumed. To prevent the consumption of antioxidants, so-called synergists (substances that act together), which regenerate antioxidants that have lost their efficacy by absorbing the activating energy, are added. Typically, these are combinations of acidic nature (citric acid, phosphoric acid, phosphatide, etc.); therefore, their role is now believed to be in the removal of traces of metals that promote rancidification. In fact, almost without exception, the most effective antioxidants are liposoluble compounds, of the phenolic type or containing at least several hydrocyclic groups (NDGA), propyl gallate (PG), butylhydroxyanisole (BHA), conidendrine, quercetin, tocopherol, palmitilascorbic acid. The amount needed is generally Among the substances considered to be the best, we investigated the action of palmiticascorbic acid and gallic acid esters. Both were prepared by us: palmitilascorbic acid was prepared by the reaction between sodium ascorbate and palmitic acid chloride and the gallic acid esters by esterification of the acid with the corresponding alcohols in the presence of sulfuric acid. I investigated their action on pork fat. The results are shown in Fig.8. Fig. 8 Action of palmiticascorbic acid and gallic acid esters on pork fat By adding 0,01 ascorbic ester, the duration of the incubation time of the pork fat was increased 3-fold and the propyl gallate used in the same proportion increased 10-fold. So, gallic acid esters are particularly effective. Ethyl gallate has a weaker action than propyl gallate. Citric acid and licitin act as synergists. Conclusions 1. In order to track the rancidity process, the method of determining the peroxidic index is also suitable for rapid rancidification. We have established the relationship between the emergence of organoleptic rancidification and the peroxidic index, the pork fat, the sunflower oil, as well as the mixtures. Such relationships can only be established for each fat or for fat blends. 161

10 2. For the determination of the storage life I compared several fats, using Swift's fast-pacing method. Based on the results we have made the following findings: To pig fat. From the point of view of fatty tissues, the most stable is the fat of the intestines, and from the point of view of the extraction process the lower and well-melted fat is more stable. Sunflower oil. The molded, heat-treated and alkali-refined seeds shorten the incubation time of the oil. For fat blends. Mixing an oil with a higher content of unsaturated fatty acids (especially linolenic acid) promotes (catalyses) the rancidification of pork fat, unless it contains antioxidants in such an amount or so effective that it counterbalances the influence of the fatty acids. Probably, due to its natural antioxidant content, the addition of fresh and crust sunflower oil in a proportion of 5 to 10% increases the stability of pork fat to some extent. Neither fresh and refined sunflower oil, introduced into such a mixture, will cause a reduction in stability. Instead, blending with linseed oil promotes, to a great extent, pork fat (demonstrating the action of unsaturated acids). Oxidation products that are born during storage also accelerate the rising of fresh fats. By adding 0.01% of gallic acid (ethyl gallate and propyl gallate) esters, the duration of incubation is about 10 times longer compared to the initial duration. References 1.Quackenbush F.W., Oil & Soap 22, 1995, Atherton D. și T.P. Hilditch, J. CHEM. Soc. 1994, Farmer E. H. și D. A. Sutters, J. Chem. Soc. 1993, Lea C. U., J. Soc. Chem. Ind. 65,, 1946, Joyner N. T. și J. E. Mo. INTYNE, Oil & Soap 15, 1998, Swift C. E. și Rose W. G., Oil & Soap 19, 1992, Farmer E. H., Koch H. P., J. Chem. Soc. 1993, Powick W. C., J. Aga. Research, 26, 1993, Christianson J. A., Phys. Chem. 28, 1999, Rinovetz,A.,Jianu,C.,Rinovetz,Adina.,Iancu,T.,Caza cu,mihaela.,bujancă,g.,jianu,i.,medium-melting point fraction and the impact of temperature on fractional crystalisation of pork lard,journal of Food,Agriculture- Environment Vol.9(1),ISSN: ,pp , Rinovetz,A.,Jianu,C.,Rinovetz,Adina.,Cazacu,Miha ela.,bujancă,g.,jianu,i.,impact of centrifugation time and speed on dry fractionation of pork lard Journal of Food,Agriculture-Environment Vol.9(2),ISSN: ,pp , Bujancă,G.,TraȘcă,T.,RiviȘ,A.,Jianu,C.,Jianu,I.,Rin ovetz,a.,rinovetz,adina.,changingmechanical and structural features of dough mechanically,research People and actual task son multidisciplinary sciences,lozenec Bulgaria,ISSN: ,pp , Bujancă,G.,PîrȘan,P.,Imbrea,F.,BotoȘ,L.,Hygrosco pic proprieties of oleaginous cattle cakes,symposium Trends in European Agriculture Development,Vol.41(1),ISSN: ,pp ,