Exploring Untargeted metabolomics for halal authentication of Triceps brachii, Longissimus Dorsi, and Biceps femoris of meat muscles

ABSTRACT The demand for authentic meat has increased due to the growing consumption of halal meat. In this study, a combined metabolomics and chemometrics approach was employed to authenticate beef and pork samples from specific muscle groups. By using an untargeted metabolomics analysis, distinct metabolite profiles were observed in TB, LD, and BF muscles, differentiating pork from beef. Subsequently, Principal Component Analysis (PCA) results confirmed the distinguishable metabolite profiles between beef and pork. Cluster analysis further revealed that the metabolites in each muscle of beef and pork have different characteristics. Additionally, Partial Least Squares-Discriminant Analysis identified 15 potential metabolites could be used to authenticate the halal status of meat. Creatine, L-carnitine, carnosine, nicotinamide, L-phenylalanine, DL-lactic acid, acetyl L-carnitine, hypoxanthine, inosine, DL-malic acid, N-methyl-2-pyrrolidone, L-glutathione, Inosine-5-monophosphate (IMP), L-tyrosine, and palmitoylcarnitine is a potential metabolite to differentiate beef and pork. This study offers valuable insights into determining the halal status of meat based on metabolite profiles.


Introduction
Halal meat has become an increasing concern, particularly in Indonesia, where the majority of the population is Muslim [1] .To address this issue, the Indonesian government has made it mandatory for all food products to obtain a halal product certificate by October 2024 (Law No. 33/2014).Halal meat, which includes beef, is a dietary requirement for Muslims.However, a significant concern arises when halal meat is mixed with non-halal meat, such as combining beef with pork.Economic factors often drive this mixing, but it poses a serious issue for the integrity of halal meat products. [2]When beef and pork are combined, the halal status of the meat is compromised.It is important to use a method of halal authentication for meat.[5] This method is selective for analyzing beef and porcine DNA, but it requires complex sample preparation procedures.However, due to the addition of a small quantity of pork to beef mixture, more target DNA extraction is required.As a result, PCR is unable to detect the target DNA, leading to the possibility of false-negative results. [6]There is a need for an alternative method, which is needed for halal authentication in meat through a metabolomics approach using UHPLC-HRMS (Ultra High-Performance Liquid Chromatography-High Resolution Mass Spectrometry). [7]etabolomics is a qualitative and quantitative analysis method that focuses on metabolites with a modest molecular weight of 100 to 1,000 in cells, tissues, and biological fluids.These metabolites are the end products resulting from gene expressions influenced by interactions between the genome system and the environment. [8]Metabolites are made up of an intermediate component as well as products, and by using metabolomics, distinct features that can serve as a fingerprint for metabolites are found in samples, including meat. [9]This approach can assess the components of substances globally with minimum sample preparation.By analyzing metabolites present in both beef and pork, metabolomics offers insights into their respective profiles.Subsequently, the metabolite profiles of pork are likely to differ from those of beef, making the method a valuable guideline for determining the halal status of the meat. [10][13] To determine the halal status of meat, it is crucial to authenticate all three muscles.In this situation, the metabolomics technique utilizes UHPLC-HRMS equipment, known for its good selectivity and sensitivity for halal authentication. [14]The spectral data generated from the analysis using UHPLC-HRMS alone is insufficient for validating halal authentication on TB, LD, and BF of beef and pork.Additional analysis in the form of chemometric analysis is required. [15] chemometric analysis is needed to determine whether beef and pork metabolite profiles are different using PCA (Principal Component Analysis).[16] PCA describes the metabolite distribution in each beef and pork muscle.Partial Least Squares Discriminant Analysis (PLS-DA) is then employed to identify the metabolite profiles that can differentiate between beef and pork.The results of the PLS-DA analysis provide the most influential metabolite profiles to determine the halal status of beef and pork.Metabolite profiles for halal meat verification can be generated by chemometric analyses using PCA and PLS-DA.[17] By examining the TB, LD, and BF muscles of beef and pork, these analyses contribute to the authentication of the halal status of meat, ensuring consumer protection against non-halal products.

Materials
Methanol, water, formic acid, and acetonitrile for LC-MS grade were all pro-analysis quality chemical reagents (Merck, Germany).In this study, TB, LD, and BF muscles of beef and pork used were obtained from the local market in Bandung (Pasar Bandung), Indonesia.Beef TB, LD, and BF are segregated from pork to avoid cross-contamination.Separation of packaging containers begins at the point of meat purchase and continues to the laboratory prior to metabolite extraction.

Extraction of metabolites from meat
The meat sample (50 mg) was placed in a microcentrifuge tube to which 1 ml of methanol was added.The mixture was vortexed for 30 seconds and then sonicated for 30 minutes at room temperature.The sample was centrifuged at 1,400 × g for 5 minutes to separate the supernatant.The supernatant was taken and filtered using a 0.22 µm PTFE filter.The resulting metabolites were stored in vials at −20°C before further analysis.This process was conducted for the TB, LD, and BF muscles of beef and pork.

Metabolomics analysis using UHPLC-HRMS
The instruments used in the metabolomics approach were UHPLC and Q Exactive (Thermo Scientific, USA) from the Advance Research Laboratory of IPB University.The UHPLC conditions involved using a C18 Accucore (Thermo Scientific, USA) 100 × 2.1 mm x 1.5 μm stationary phase.The mobile phase consisted of two components: mobile phase A, which was 0.1% formic acid in water, and mobile phase B, which was 0.1% formic acid in methanol.The mobile phase in the metabolomics analysis was a gradient where minutes 0-16: 5% B up to 90%, 10% A; minutes 17-20: 90% B, 10% A; and minutes 20-25: 5% B, 95% A, with a flow rate of 0.3 μL/min.The temperature was 40°C, and the injection volume was 3 µL.The data were read at m/z mass intervals of 66.7-1,000 m/z, and the spectral fragments were obtained at a resolution of 17,500-75,000 Hz.The metabolite spectra obtained were further matched with Discover Compound data, and the process was replicated three times.The detailed parameters of the LC gradient and MS instrument are shown in Supplementary F1.

Statistical and Chemometric Analysis
The statistical analysis of the metabolite compounds was conducted using one-way variance (ANOVA) in SPSS 16.0 software (SPSS, Chicago, IL).If the results were significant (P < .05),then, chemometrics analysis was performed, including PCA, PLS-DA, and heatmap analysis for classification using Metaboanalyst 5.0 (https://www.metaboanalyst.ca/).Each sample was replicated three times.

Selectivity mobile phase of metabolomics
The metabolomics analysis of beef and pork on TB, LD, and BF muscles for halal authentication proved that UHPLC-HRMS is a selective method.The number of metabolites discovered shows the method's selectivity.Among the different mobile phase combinations tested, the gradient mixture of water and methanol exhibited the highest level of selectivity, surpassing water and acetonitrile.Specifically, the analysis using the water and acetonitrile mobile phase provided 31 metabolites, while the analysis using the water and methanol mobile phase generated 177 metabolites.As a result, selecting the water and methanol mobile phase is the best UHPLC-HRMS mobile phase condition for halal authentication using metabolomics techniques, as it enables the detection of a greater number of metabolites (Supplemental F1).Water and methanol mobile phases can extract metabolites from beef and pork more than water and acetonitrile mobile phases. [18]

Untargeted metabolomics
The untargeted metabolomics analysis conducted on TB, LD, and BF muscles of beef and pork yielded a total of 177 compounds.The further examination focused on the 177 metabolites with a mass-tocharge ratio (m/z) percentage value exceeding 70%, resulting in the identification of 61 metabolites.These metabolites exhibited an MZ value of over 70% in both beef and pork samples, indicating their consistency and reliability.[21] This indicated that the results of the untargeted metabolomics study are reliable.The metabolites discovered in pork were the same as those discovered in beef, although at different levels.This is consistent with Kim et al., [22] in which NMR examination of beef and pork metabolites revealed the same metabolites but at different amounts.Subsequently, this study found that leucine, isoleucine, glycine, and phenylalanine were present in both beef and pork but in different amounts, indicating that pork metabolite profiles differ from beef.Distinct characteristics were observed in the metabolite compositions across different muscles of beef and pork.In addition, the results of the one-way ANNOVA statistical analysis test showed that TB, LD and BF of beef and pork had a p value of less than 0.05.This indicates that each part of beef and pork has significant metabolite differences.Table 1 shows the metabolite content profiles in each beef and pork muscle.Distinct metabolite profiles were observed in the TB, LD, and BF muscles of pork.The three most prevalent metabolites in pork TB are creatine, L-phenylalanine, and DL-malic acid.In pork LD, the most prominent metabolites are creatine, carnosine, and nicotinamide.Creatine, L-phenylalanine, and carnosine were pork BF's three most common metabolites.The composition of the metabolites differed between pork TB, LD, and BF.According to Jang et al., [23] the metabolites in each area of pork differed, such as xanthine in the pancreas and acylcarnitine in the leg (muscle).
The profiles of the metabolites in the TB, LD, and BF muscles of beef varied.The three most prevalent metabolites in beef TB are carnosine, L(-)-carnitine, and creatinine.The most abundant metabolites in beef LD are carnosine, L(-)-carnitine, and creatinine.Although the quantities differed, the primary metabolites in beef TB and LD were consistent.The three primary metabolites in beef BF are L(-)carnitine, myristyl sulfate, and dodecyl sulfate.The primary metabolites in the various beef muscles varied.The metabolite content in beef Longissimus and Psoas Muscles exhibited the same metabolites, but their quantities are different, including L-carnitine, D-malic acid, and hypoxanthine. [24]he main metabolites identified in pork TB are creatine, L-phenylalanine, and DL-malic acid, while creatinine, L(-)-carnitine and carnosine are the key metabolites in beef TB.The primary metabolite profiles of beef and pork TB are different.These distinct metabolite profiles can contribute to the differentiation between beef and pork TB.In pork LD, the three primary metabolites are creatine, carnosine, and nicotinamide, while creatinine, L(-)-carnitine and carnosine are the three main metabolites in beef LD.The metabolite profiles of beef and pork LD meat were found to be different, indicating that pork LD differs from beef LD.In pork BF, the main metabolites are creatine, L-Phenylalanine, and carnosine, while L(-)-carnitine, myristyl sulfate, and dodecyl sulfate are the key metabolites in beef BF.This further elucidates the differentiation between beef and pork BF.Trivedi et al. [39] discovered that the distinct metabolites in beef and pork included creatinine, citric acid, and glycine.Chemometrics analysis can be used to clarify the variations in the profiles of metabolites in beef and pork, as well as the metabolites that indicate halal status. [26]

Metabolomics and Chemometrics Analysis
In this study on metabolic, a total of 18 samples were selected and divided into six groups, each compromising of three biological replicates (Figure 1).A thorough examination of the data revealed that the R2 values ranged from 0.85 to 1.00, indicating a high level of repeatability for each sample.The Pearson correlation coefficient is employed to assess the degree of correlation between variables.The objective is to ensure that the variables exhibit no correlation by the process of grouping and reducing them into new principal components that are mutually uncorrelated.This is done to facilitate further principal component analysis (PCA).The samples within each group showed clear differentiation, as evident from the PCA analysis of the metabolite content, and exhibited satisfactory reproducibility (Figure 1).[29] Based on their metabolites, chemometric analysis can be used to describe the distinctions between beef and pork.[35] Meanwhile, objects are grouped based on shared characters using cluster analysis.According to the cluster analysis, each muscle of beef and pork has the same properties. [25,36]CA provided a clearer picture of the metabolite profiles in each beef and pork muscle in Figure 2. The PCA results showed distinct metabolite profiles for the TB, LD, and BF muscles of both beef and pork.Figure 2 showed that the metabolite profiles of pork TB, LD, and BF form three distinct groups, signifying noticeable differences in the metabolites present in these muscles.The metabolites in beef TB, LD, and BF formed separate groupings, are indicating that these three muscles had distinct metabolite profiles.According to Figure 3, notable differences were observed in the metabolite profiles of beef and pork TB.Similarly, the LD and BF metabolite profiles of beef and pork also exhibited significant distinctions.These various metabolite profiles serve as valuable indicators to elucidate the differences between beef and other types of meat.These various metabolites can distinguish between beef and pork, thereby contributing to halal authentication.According to studies by Saputra et al. [37] and Rahayu et al., [38] PCA may detect dog meat in meatballs.These findings are supported by PCA's ability to identify pork in meat samples in both cases.Therefore, PCA can be used to determine the halal status of beef and pork.
The results obtained from PCA analysis, as shown in Figure 3, present the distinct metabolite profiles when the muscles of beef and pork are combined.The PCA results highlight that the combined TB, LD, and BF components of beef and pork exhibit unique metabolite profiles.This implies that halal authentication can be performed on all three components of beef and pork using metabolomics and PCA.To identify the specific metabolites that contribute to halal authenticity, a PLS-DA analysis is necessary.A chemometric technique called PLS-DA is used to estimate possible metabolites that could be utilized to authenticate halal products.Subsequently, PLS-DA can predict probable substances that meat may include as halal indicators.In the context of this study, PLS-DA identified 15 candidate metabolites that could serve as markers for halal meat, as shown in Figure 4.
According to Figure 4, a total of 15 metabolite chemicals serve as halal authentication indicators in the TB, LD, and BF muscles of beef and pork.Windarsih et al. 2022 reported that there were 15 possible metabolites based on the VIP score for detecting pork in Pangasius hypophthalmus meat, one of which was xanthine. [6]According to Ali et al. [34] there was an increase in amino acids in chicken meat that was slaughtered in a non-halal manner.Furthermore, Trivedi et al. [39] discovered an increase in sphingolipids and fatty acids in a study they conducted.According to a previous study by Jia et al., [40] the radiation process on meat increased L-phenylalanine, L-isoleucine, L-histidine, guanosine, guanine, creatinine, glutathione, and nicotinic acid while decreasing inosine 5′monophosphate (IMP) and guanine 5′-monophosphate.
To further explore the characteristics of beef and pork, a cluster analysis was performed.Figure 4 displays the results of a heatmap-based cluster analysis.The analysis clearly shows that beef TB, LD,  BF, pork TB, LD, and BF exhibit distinct clustering patterns, indicating inherent differences in traits and attributes between beef and pork.Li et al. [41] used cluster analysis to describe pork metabolism during storage.Windarsih et al. [42] reported that cluster analysis was utilized to detect pork in meatballs using a metabolomics technique.Therefore, cluster analysis, which elucidates the variations in TB, LD, and BF between beef and pork, can serve as a means to verify the halal status of meat.

Conclusion
In conclusion, selective UHPLC-HRMS proved to be a successful metabolomics approach for the authentication of beef and pork in TB, LD, and BF muscles.By analyzing the metabolomics data sets of these three muscles, comprehensive information could be obtained to authenticate the halal status of meat.It was observed that the metabolite profiles of pork muscles differed from those of beef.The chemometric investigation revealed that the metabolites profiles in each beef and pork muscle differed.The PCA results visually explained the differences between beef and pork in TB, LD, and BF.Cluster analysis indicated that each muscle of beef and pork had distinct properties enabling their classification based on their metabolites.For halal authentication, PLS-DA analysis provided information on possible metabolites for beef and pork.Furthermore, 15 metabolites were identified as potential markers for halal authentication of beef and pork, including creatine, L-carnitine, carnosine, nicotinamide, L-phenylalanine, DL-lactic acid, acetyl L-carnitine, hypoxanthine, inosine, DL-malic acid, N-methyl-2-pyrrolidone, L-glutathione, Inosine-5-monophosphate (IMP), L-tyrosine, and palmitoylcarnitine.

Figure 1 .
Figure 1.Quality control and analysis of metabolome data of meat muscles.A: Pearson correlation analysis between metabolome samples.B: PCA analysis between metabolome samples.TB: Triceps brachii; LD: Longissimus dorsi; and BF: Biceps femoris.

Figure 3 .
Figure 3. Score Plot of Principal Component Analysis in Each Muscle and TB, LD, BF of the Mixed Meats (Beef and Pork) of Beef and Pork Meat.TB: Triceps brachii; LD: Longissimus dorsi; and BF: Biceps femoris.

Table 1 .
Putative identification metabolites in each muscle of beef and pork meats.