Identification of pork in beef meatballs using Fourier transform infrared spectrophotometry and real-time polymerase chain reaction

ABSTRACT

A study on development of Fourier-transform infrared spectrophotometric method combined with principle component analysis as well as real-time polymerase chain reaction for determination of pork–beef mixture in meatballs has been performed. A lipid component extracted from pork and beef in meatballs is analyzed using Fourier-transform infrared spectroscopy, while DNA extracted from meatball was analyzed using real-time polymerase chain reaction. The correlation between actual and predicted concentration of lard using Fourier-transform infrared spectroscopy was performed by aid of partial least squares, while grouping of lard and beef fat components in meatball was carried out by Fourier-transform infrared spectra coupled with principle component analysis. The results showed that Fourier-transform infrared spectra at wavenumbers of 1000–1200 cm⁻¹ coupled with partial least square and principle component analysis are successfully used for quantification and classification of pork in beef meatballs. The relationship between actual value and predicted value of lard (lipid fraction obtained from meatballs containing pork) with Fourier-transform infrared spectrophotometric method revealed good correlation, with coefficient determination (R²) value of 0.997 and standard error of calibration of 0.04%. Principle component analysis is able to classify samples containing pork and beef meatballs. Fourier-transform infrared spectroscopy using normal spectra is fast technique for identification and quantification of lard extracted from pork in meatball. In addition, real-time polymerase chain reaction using Leptin Primer–AJ 865080 can be used for amplification of pork DNA specifically in meatballs containing pork.

KEYWORDS:

• Pork
• Meatball
• FTIR spectroscopy
• Polymerase chain reaction
• Halal authentication

Introduction

Beef meatball is one of favorite food for Indonesian community and is a good source of protein.^[1] However, due to the high price of beef, some producers blended or even substituted beef with pork. For the Muslim community, pork is non-Halal, and Muslima are prohibited to consume any food containing pork.^[2] Currently, the implementation of Halal assurance system has become a global issue. Consuming Halal food is a must for every Muslim.^[3] The adulteration of food products is a major problem in the food industry, because it causes confusion and harm to consumers and food producers. Losses incurred due to the adulteration of food is not only the loss of the material, but also spiritual losses, because Muslims are prohibited from consuming any food products containing pork.^[4,^5] As a consequence, some Muslim scholars develop analytical methods capable of detecting the presence of non-Halal components in any products, including meatballs.

The detection and quantification of adulteration is very important to protect the welfare and health of consumers as well as to assure the Halal authenticity of food products.^[6] Some methods based on physico-chemicals and molecular biology have been reported for analysis of pork in food. Such methods used for analysis of pig derivatives including lard, pork, and porcine gelatin are the electronic nose,^[7] differential scanning calorimetry,^[8] gas chromatography coupled with time of flight mass spectrometer,^[9] high-performance liquid chromatography,^[10] nuclear magnetic resonance spectroscopy,^[11] infrared spectroscopy,^[12,^13] and polymerase chain reaction (PCR).^[14]

Because of its property as a fingerprint technique, Fourier-transform infrared (FTIR) spectroscopy coupled with a several chemometric techniques merge as powerful tools for quantification and classification of pig derivatives in consumer goods (food, cosmetics, and pharmaceuticals). FTIR spectroscopy combined with multivariate calibration has been used for analysis of lard in lotion cosmetics,^[15] lard in meatball broth,^[16] and analysis of porcine gelatin.^[17] FTIR spectroscopy coupled with partial least square (PLS) was also used for quantification of rat’s meat^[18] and wild boar meat^[19] in meatball formulation. However, FTIR spectroscopy has one main drawback, namely if the the composition of the sample to be analyzed is different, FTIR spectra of the analyte in the mixture will also be different. Consequently, the presence of pig derivatives in the different food samples must be quantified using different spectral regions and different chemometrics techniques.

Several methods based on PCR have been proposed as useful means for identifying species of origin in foods, due to their high specificity and sensitivity^[20] from qualitative PCR (conventional PCR) over restriction fragment length polymorphism (RFLP) to quantitative PCR known as real-time PCR.^[21] Our group has used PCR for analysis of pig derivatives, especially pork in food products^[14,²²,^23] and gelatin in pharmaceutical products.^[24] Furthermore, real-time PCR is also used for analysis of rat’s meat DNA in meatball products.^[25] This research was aimed to develop a method of FTIR spectrophotometry combined with chemometrics of PLS and principle component analysis (PCA) for analysis of pork and beef in the meatballs and to use real-time PCR for identification of pig DNA present in meatball products.

Materials and methods

Pork, beef, spices, preservatives, and other additives for making meatballs are obtained from local market in Yogyakarta. The meatball samples were purchased from the several markets around Yogyakarta, Indonesia. All reagents and chemicals used were of pro-analytical grade.

Sample preparation

Meatballs were prepared according to Purnomo and Rahardiyan^[1] by emulsifying 90% finely ground meats (beef, pork, and pork–beef mixture) with 10% oats. The mixture was combined with saline 0.01% (wt/wt) and spices, shaped about a ping pong ball size, and finally put into boiling water. Meatballs containing pork at concentration levels of 0, 5, 10, 15, 25, 50, 60, 75, 80, and 100% of pork in a mixture with beef were made for calibration samples. Validation samples taken from a number of meatballs prepared in a laboratory containing certain levels of pork were also prepared. Fats in meatball were extracted for FTIR spectroscopic analysis, while DNA in the samples is extracted to be subjected to real-time PCR analysis.

Extraction of fat from meatballs

The extraction of fat in the meatballs is done by solvent extraction method as in Rohman et al.^[12] using concentrated hydrochloric acid as a hydrolytic agent and petroleum benzene as solvent extraction. A 50 g meatballs was cut into small pieces and incubated into water bath at 70°C for 30 min. After cooling, the samples were filtered with filter paper and then put into a separating funnel. Liquid–liquid extraction is done using a mixture of petroleum ether and petroleum benzene (1:2 v/v). An extraction is done three times. The solvent of the extract was evaporated in a vacuum rotary evaporator at 40°C under reduced pressure. If necessary, the evaporation is done with the aid of nitrogen gas. The fats were then analyzed by FTIR spectrophotometer.

Analysis using FTIR spectrophotometer

THE lipid fraction obtained is scanned using FTIR spectrophotometer (ABB MB3000, Canada), equipped with a ZnSe crystal plate, with sample handling techniques of attenuated total reflectance (ATR) using deuterated triglycine sulfate detector (DTGS) and germanium as beam splitter. The lipid fraction was placed on ATR crystal at a controlled temperature (20°C). Measurements were taken at 32 scans and at resolution of 4 cm⁻¹ with a strong apodiziasiation. The spectrum of air is used as reference (background). Each sample is read in absorbance in triplicates.

Real-time PCR analysis

The primers for real time-PCR were obtained by designing a primer using the NCBI (National Center for Biotechnology Information) software. Primer used is Leptin Primer - AJ 865080, i.e., forward: 5’-CTT AGC ACC TCG ATC AAG CA-3’; reverse: 5’-GCT TCC TTG AAC TGC TGT GT-3’. DNA isolation was performed with Proteinase K and lysis buffer. A 250 mg of sample was transferred into 2 mL Eppendorf tube and was extracted using 1 mL of TNE lysis buffer (50 mM TrisHCl pH 8, EDTA 0.1 M, NaCl 0.1 M, and SDS 1%) and 15 μL of proteinase K (20 mg/mL). This mixture was incubated at 55°C for 1 h and subjected to centrifugation. The supernatant was extracted using phenol-chloroform followed by DNA precipitation in absolute ethanol. The DNA obtained was subsequently washed using 70% ethanol and dissolved in TE buffer (Tris HCl 10 mM; pH 8 and EDTA 1 mM). The isolated DNA was analyzed using real-time PCR with SYBR green^® universal PCR master mix fluorescent dye. For each reaction, a total of 20 μL of mixture containing of 10 μL SYBR Green master mix, 1 μL primer forward and 1 μL primer reverse, 1 μL 50 ng isolated DNA was prepared. The amplification was performed with a real-time PCR using PCR CFX96 (Biorad, USA). The thermal cycler protocol was as follows: initial denaturation at 95°C for 15 s, annealing, and extension at 56 and 72°C for 10 s, respectively.

Statistical analysis

The multivariate analysis of PLS and PCA for FTIR spectra data was analyzed using Minitab software version 16 (Minitab Inc., USA). Accuracy of PLS model was evaluated by coefficient of determination (R²), while the precision of analytical method was assessed using root mean square error in calibration (RMSEC) and root mean square error of prediction (RMSEP).

Results and discussion

Analysis of pork in meatball using FTIR spectra

FTIR spectra of the lipid component extracted from meatballs containing 100% pork and 100% beef is depicted in Fig. 1. Both spectra did not show any peak/special pattern, and revealed the typical characteristic bands for general IR absorption of triglycerides, because fats are basically dominated by triglycerides (95–98%) and other minor components such as sterols and vitamins. ^[27] However, because of the nature and composition of the different fatty acids in pork and beef, both spectra can be differentiated in terms of peak intensity, especially in the area of wavenumbers of 3006, 1117, and 1098 cm⁻¹. FTIR spectrophotometry can be used as a potential way to distinguish lard from beef fat, because FTIR spectrum is considered as a fingerprint, which means no two fats with the same FTIR spectrum, in the number of peaks or the peak intensity.^[28] Peaks at 1111 and 1197 cm⁻¹ appear the two adjacent peaks with different intensities, as assigned with circle, which indicate the presence of vibration of ester linkages of triglycerides. Indeed, this region in which a variation observed between pork and beef was optimized for quantification.

Figure 1. FTIR spectra of beef and lard in meatballs. Peak marked with a circle indicated the significant absorption bands for the differentiation.

Quantitative analysis of lard and beef fat in the meatballs was performed with the aid of PLS. Lard’s spectrum revealed the similar peak height at 1117 and 1097 cm⁻¹. While beef fat’s spectrum, the peak height at 1097 cm⁻¹ is lower than that at 1117 cm⁻¹. As a result, with increasing concentrations of lard in meatballs resulted in high-altitude spectral peaks approaching 100% of lard. This difference is exploited for the quantification of lard mixed with beef meatballs. The wavenumbers used for the determination of lard and beef fat that has been optimized is 1200–1000 cm⁻¹ using FTIR normal spectra. The wavenumbers selection is based on its ability to form a linear relationship between actual pork and beef (x-axis) and the predicted value of lard (y-axis) with FTIR spectrophotometry associated with PLS multivariate calibration, as indicated by the value of coefficient of determination (R²) = 0.997 (Fig. 2) with an equation of y = 1.003 x + 0.254. The analysis of variance data revealed that slope value (1.003) is different from zero (p <0.05) and intercept value (0.250) is not different significantly from zero (p > 0.05). Some spectral treatments using normal and Savitzy-Golay derivatization (first and second derivatives) are also performed. The selection of either normal or derivative spectra is based on the capability of spectral treatment at 1200–1000 cm⁻¹ to provide the highest R² and lowest values of RMSEC. Finally, FTIR normal spectra are preferred for quantitative analysis of lard. The validation of the method was done by prediction of independent sample and then calculated using calibration models. The validation model resulted R²-value of 0.995 for the correlation between actual and predicted values of validation samples. The validation error is calculated as the RMSEP using the formula:

RMSEP value obtained is 0.04%. The high value of R² and low value of RMSEP indicated good PLS model for analysis of pork in beef meatballs.

Figure 2. The relationship between the actual value of lard (x-axis) extracted from pork meatball with lard predicted by FTIR spectra-PLS model (y-axis) in the mixture of pork and beef.

Classification of beef and pork meatballs using PCA

PCA is a technique that can be used for classification of samples based on similarities that occur between samples.^[29] Analysis of lard and beef fat was performed using a total of 93 variables derived from the wavenumbers in the range of 1000–1200 and 2900–3050 cm⁻¹. Projections are used to separate properly between the meatballs that containing pork and beef. Further meatball samples purchased from the market are also tested with this grouping. Figure 3 revealed that the tested samples were classified into beef fat, indicating that samples can be identified as beef meatballs.

Figure 3. PCA analysis for classification of pork meatballs, beef meatballs and meatball samples. A: Pork meatballs 100%, B: beef meatballs 100%, and C: meatballs from commercial samples.

The scree plot in Fig. 4 relates the eigenvalue to principle components. PC1 is able to describe 59% of the original data variables, while PC2 is able to describe 23% of the data variables, and PC3 is able to describe 5% of the total original data variables. Other principle components are not used because they do not really represent the original variables. Thus the use of PC1, PC2, and PC3 are able to produce a model that describes the entire original variable data, i.e., nearly 100%.

Figure 4. The scree plot relating the eigenvalue with principle components.

Analysis of pork meatball using real-time PCR

The optimization of Leptin primer to get the right temperature and conditions for PCR amplification is done. In the first time, the primers of leptin were tested at a temperature of 50.9°C, but the primer is not specific. After Leptin primer is being tested at temperature of 56°C, the primer can amplify DNA target. Annealing temperature is an important factor in the process of RT-PCR amplification. This is associated with a primary attachment of a primers on a target DNA, so that the amplification process can run specifically. The results showed that DNA amplification was only observed in pork DNA, therefore it can be stated that the used leptin primer is specific for pork DNA (Fig. 5). With different primers which are specific for pork DNA, the annealing temperature is different. Besides, the detection limit of DNA using different primers is different, as a consequence, some researchers developed primers capable of detecting pork DNA as low as possible. Maryam et al.^[14] reported the annealing temperature of 62°C using primers of loop686 with detection limit of 0.05% of pork in food product. Rahmawati et al.^[30] reported that the optimum annealing temperature for pork DNA amplification is 59oC using D-loop22 primer.

Figure 5. Amplification curves of Leptin Primer on DNA from meatballs. Red: Pork meatball; green: beef meatball.

The samples identified as beef meatball were analysed using real-time PCR using specific primer for pork DNA. There is no amplification found for samples tested, therefore, the samples are suspected to contain beef meatballs.

Conclusion

FTIR spectrophotometry method combined with PLS multivariate calibration can be used and developed for the detection of pork fat in beef meatball samples at wavenumbers of 1200–1000 cm⁻¹, with R² = 0.997 and RMSEC = 0.04%. PCA is able to classify samples containing pork and beef meatballs. Furthermore, real-time PCR using Leptin Primer–AJ 865080 was successfully used for amplification of pork DNA specifically in meatballs containing pork.

Funding

The authors thank to the Ministry of National Education for financial support via project the National Strategic Competitive Research with contract number LPPM/2011/004. Integrated Research and Testing Laboratory (LPPT-UGM) was acknowledged for providing the FTIR spectrophotometer.

Additional information

Funding

The authors thank to the Ministry of National Education for financial support via project the National Strategic Competitive Research with contract number LPPM/2011/004. Integrated Research and Testing Laboratory (LPPT-UGM) was acknowledged for providing the FTIR spectrophotometer.