Determining fatty acids and halal authentication of sausage

Sausages are instant food that requires the halal attention for a Muslim. In general, nonhalal issues are usually associated with pig derivatives. One of the pig derivates is lard that can be analyzed using GC-MS via a transesterification catalyzed with a base catalyst like sodium methoxide 2%. This study indicated that pork sausage has different fatty acids compared with beef sausage. The pork sausage contained the dominant fatty acids such as palmitic acid (37.75%), myristic acid (22.24%), oleic acid (25.29%), and lauric acid (8.46%). Whereas, beef sausage has the dominant fatty acids of palmitic acid (42.31%), oleic acid (20.19%), stearic acid (10.92%) and myristic acid (7.66%). The commercial sausages 1, 2, and 3 have similar dominant fatty acids such as palmitic acid, oleic acid, stearic acid, and myristic acid compared with fatty acid types in beef sausage. The discriminant analysis also showed that the beef sausage is separated location from pork sausage and all samples are not containing the pork or lard because they are far away from pork sausage.


Introduction
Food is one of the primary needed by humans because it contains some ingredients that the human body needed. The one ingredient is fat or oil in triglyceride form that can be hydrolyzed into glycerol and fatty acids. The hydrolysis process of triglycerides depends on the chain length and molecular position of the fatty acids (Bauer et al., 2005). Based on triglycerides composition, the different animals will result in the differences in triglycerides composition (Rohman et al., 2012). This evidence can be used as one way for discriminant analysis between halal food and non-halal food. For a Muslim, food consumption is supposed for good or healthy food and also halal foods (Fadzlillah et al., 2011).
In general, non-halal products are the product associated with pig derivatives so a Muslim must be aware and take care to choose the halal product. Either producers or consumers must learn to understand about halal and they ensure the halal certification because it has many advantages in the halal product (Hassan and Awang, 2009). Furthermore, certification halal must be the responsibility of both governments and companies to ensure the products is halal (Khattak et al., 2011). Nakyinsige et al, (2012) stated that the head of the meat industry company must know about the rules of sharia in the production of halal meat. This is because the international market for halal food reaches the US $ 580 billion per year (Qureshi et al., 2012). On the other hand, there is a big contribution from the halal food industry in the development of society and national economic growth (Bohari et al., 2013).
For detection pork or lard in the product, we can analyze the fats content composed of monoglycerides, diglycerides, triglycerides, fatty acids. The biggest component in lard or pork is triglycerides, which is about 95% (Gunstone, 2002;Lobb and Chow, 2007). Triglycerides (TAG) are a reaction formed from one molecule of glycerol and three fatty acids and it can be hydrolyzed to produce glycerol and fatty acids again ( Figure 1). The differences of TGA contained in pork compared with other meat animals cause they can be grouped if the foods contain pork. The analytical methods developed to analyze lard or other components of pork. Ahda et al. (2016) and also Gutierrez and Perona (2000) analyzed pork based on triglycerides component using HPLC. Furthermore, the FTIR spectroscopy is also used as a pork analysis based on fingerprints of functional group (Guntarti et al., 2015;Guntarti and Seshilia, 2017). Besides that, the discriminant analysis also can be grouped based on fatty acid types using KLT and GC-MS (Ismiyarto et al., 2006;Almeida et al., 2006;El-Ghorab et al., 2009;Setiawati and Edward, 2012). This research is aimed to analyze commercial sausage products based on fatty acid contents using GC-MS. This is a way for giving a solution where each sausage producing can cause the hydrolysis of triglycerides in sausage. So, fatty acid analysis can be a solution in halal authentication because it can ensure the discriminant result is only based on fatty acids components, not both mixture between triglycerides and fatty acid components.

The extraction of triglycerides in pork, beef and sausage.
Samples of meat (pork, beef) sausages were sliced into small pieces, put in a glass beaker, put in an oven at a temperature of 50°C until it was melted. Furthermore, the above fat liquid was extracted with n-hexane and then added Na 2 SO 4 anhydride to bind the water. The fat liquid was evaporated before the transesterification process.

Transesterification process of triglycerides of sausages samples
A total of 20 mg of samples fat was added using 50 mL of sodium methoxide solution (NaOH: methanol 2% w/v) and then heated at 60°C for 60 mins. After that, the solution was kept at room temperature and then added the saturated NaCl. The supernatant can be separated using centrifugation method and up layer injected on the GC-MS instrument.

Data processing
Chemometrics analysis of principal component analysis (PCA) using Minitab 16 software. The variables used for discriminant analysis are fatty acid types and relative fatty acid content, in each sausage will be determined.

Results and discussion
Analysis of fatty acid components in sausage is very important from the difference of carbon atoms length, fatty acids saturation degree, and double bonds position in the chain of fatty acids (Dupuy, 1996). Fatty acids can be classified to become saturated fatty acids, monounsaturated fatty acids, and also polyunsaturated fatty acids (Coltro, 2005). Figure 2 showed a difference in chromatogram profiles of all sausage samples. 100% pork sausage (red color) appeared to have a different chromatogram profile with 100% beef sausage (blue color). Commercial sausage samples (yellow color) have similarities with 100% beef sausage (blue color). The fatty acids in 100% pork sausage are more than the fatty acids contained in 100% beef sausage and commercial sausages.
In Table 1, 100% pork sausage is detected lauric acid on while it's undetected in 100% beef sausage and commercial sausages. This fact has influenced high temperatures that cause fat damage through hydrolysis reaction and oxidation reaction during the process of derivatization. The process of fat oxidation will occur in the unstable hydroperoxide of long-chain fatty acids become short-chain fatty acids. Hence, the lauric composition in pork sausage is very high. The speed of the oxidation process also depended on the type of fat and storage conditions. There are some undetected fatty acids in the samples at the time of the analysis, so the value of the total fatty acids is lower than the supposed FULL PAPER Figure 1 Differences are also seen in palmitic acid, which is 37.75% contained in 100% pork sausage, while 100% beef sausage has a higher value which is 42.31%, Same thing went for the three commercial sausages with a value of 45.99%; 47.29%; and 46.79% respectively, the stearic acid content in 100% beef sausage is 10.92% higher than 100% pork sausage with a content of 6.56%, a different fatty acid composition among samples of 100% beef sausage and 100% pork sausages based on the results of GC-MS analysis in which the saturated fatty acid content of 100% beef sausage is greater than 100% pork sausage. The identification of the three commercial sausages above has a similar fatty acid content with 100% beef sausage. This is consistent with FULL PAPER Figure    the results seen in the grouping of PCA as seen in the commercial sausages plot included in the same quadrant with beef sausage.
In addition, the value of Similarity Index (SI) may indicate the accuracy of the data based on the similarity of fragmentation pattern and basic peak (base chromatogram) which referred to the literature of standardized instruments. The greater its SI value, the more accurate the identification of compounds on the MS instrument. Each of the identified compounds can then obtained information about the relative molecular mass using MS spectra. Then, the data of fatty acids content were analyzed using principal component analysis for discriminant and grouped the commercial sausage products and this method more effective and to be easier to conclude that the commercial sausage product is not containing lard or pork. Based on Figure 3, pork sausage and beef sausage are separate in different quadrants. This is a piece of evidence that fatty acid content in both beef sausage and pork sausage is different. The commercial product 2 made is not containing pork or lard or beef. In contrast, both commercial products 1 and 3 showed that both are located in the same quadrant with beef sausage. It can be concluded that both made from beef because they have similar fatty acid contents as beef sausage.
The results of PCA analysis using Minitab resulted in 8 PCs are presented in Figure 4. Each PC displays eigenvalue, proportion, and cumulative values. Eigenvalue variations can explain the data on each PC and show how much influence a variable on the formation of the characteristics of a matrix (Miller and Miller, 2005). In Figure 4, PC1 with eigenvalue 4.3992 is able to describe 55.5% of the total original data variables while PC2 with eigenvalue 3.0274 is able to describe 37.80% of the total original variables. Thus, 2 PCs described the illustration data for discriminant analysis of sausage samples of 92.80%.

Conclusion
The beef and pork sausages have different fatty acids contents, where pork sausage has palmitic acid (37.75%), myristic acid (22.24%), oleic acid (25.29%), and lauric acid (8.46%). Whereas, beef sausage contains palmitic acid (42.31%), oleic acid (20.19%), stearic acid (10.92%) and myristic acid (7.66%). The sausage commercials contain fatty acid dominant such as palmitic, oleic, stearic, and myristic where they have similarity fatty acids content like fatty acids of beef. The PCA analysis showed all of the commercial sausages did not contain the pork or lard because they located far away from pork sausage based on its fatty acid content.