RSS Feed (xml)
Scientific Methods for Differentiating Porcine and Bovine Skin (Kikil) Using Molecular Analysis, Spectroscopy, and Morphological and Histological Characteristics: A Scientific Review for Food Authentication and Halal Assurance
ABSTRACT
The authentication of animal-derived food ingredients is an essential aspect of ensuring food safety, consumer protection, and halal compliance. One of the challenges frequently encountered is the difficulty of distinguishing bovine skin (Bos taurus) from porcine skin (Sus scrofa) after heating and processing. Their similar physical characteristics make visual identification less reliable, necessitating laboratory-based scientific approaches. This article aims to review various scientific methods for differentiating bovine and porcine skin through DNA analysis using the polymerase chain reaction (PCR), Fourier-transform infrared (FTIR) spectroscopy, immunological assays based on the enzyme-linked immunosorbent assay (ELISA), and morphological and histological examination of skin tissues. This review was conducted through a literature review of international and national scientific publications, primarily those published within the past five years. The findings indicate that PCR offers high analytical sensitivity and specificity because it can detect species-specific DNA, even in samples subjected to heating. FTIR spectroscopy can differentiate gelatin profiles based on the infrared spectral characteristics of protein functional groups, whereas ELISA is effective in detecting porcine-specific proteins in food products. Morphological and histological examinations can serve as preliminary screening methods by identifying hair follicle patterns, collagen structure, dermal thickness, and subcutaneous tissue characteristics, although their accuracy is generally lower than that of molecular methods. Combining these complementary approaches can provide a high level of confidence in food authentication and support halal certification systems.
Keywords: food authentication, bovine skin, porcine skin, PCR, FTIR spectroscopy, ELISA, halal assurance.
1. INTRODUCTION
Food species authentication has become an important issue in food safety, consumer protection, and halal assurance. The globalization of food trade has increased the complexity of food supply chains, creating opportunities for the substitution or adulteration of raw materials derived from different animal species (Codex Alimentarius Commission, 2023).
From a halal perspective, the presence of ingredients derived from pigs (Sus scrofa) is a major concern because pork and its derivatives are prohibited for consumption by Muslims. Therefore, scientific species identification is essential for halal certification and food surveillance (BPJPH, 2024).
Kikil, or animal skin, is a widely traded food commodity. After boiling, bovine and porcine skin may exhibit relatively similar appearances, making them difficult to distinguish through visual inspection alone. Furthermore, boiling may alter or eliminate some of their original morphological characteristics, thereby increasing the risk of misidentification (Rohman et al., 2021).
Advances in molecular analytical technologies have led to the development of various highly accurate authentication methods, including polymerase chain reaction (PCR), real-time PCR, DNA barcoding, ELISA, liquid chromatography–mass spectrometry (LC–MS), and Fourier-transform infrared (FTIR) spectroscopy (Ballin, 2022; Rohman & Windarsih, 2024).
In addition to laboratory-based techniques, preliminary identification can be performed by examining the anatomical and histological characteristics of skin tissue, including hair follicle patterns, dermal thickness, hair follicle distribution, collagen structure, and subcutaneous tissue morphology (Jiang et al., 2022).
This article aims to review the scientific methods available for differentiating bovine and porcine skin using molecular analytical techniques and tissue-based morphological and histological approaches. It also highlights the potential complementary roles of these methods in food authentication and halal assurance.
2. METHODOLOGY
This article employed a narrative literature review approach to identify, collect, and synthesize relevant scientific publications from the following databases and scholarly search platforms:
Scopus
PubMed
ScienceDirect
SpringerLink
Wiley Online Library
Google Scholar
The literature search primarily covered publications published between 2020 and 2025, supplemented by selected seminal references that underpin the development and application of polymerase chain reaction (PCR), Fourier-transform infrared (FTIR) spectroscopy, and enzyme-linked immunosorbent assay (ELISA).
The search terms included the following keywords and combinations thereof:
Pork authentication
Beef authentication
Pork detection
PCR pork identification
FTIR gelatin authentication
ELISA pork detection
Halal authentication
Skin histology pig cattle
Publications were selected based on their relevance to species authentication using molecular, spectroscopic, immunological, and histological methods. Particular attention was given to studies addressing the differentiation of bovine and porcine materials, the analytical performance of authentication techniques, and their applicability to processed food products. The selected literature was then synthesized to compare the principles, advantages, limitations, and potential applications of these methods in food authentication and halal assurance.
3. RESULTS AND DISCUSSION
3.1. DNA Analysis Using Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) is widely recognized as a reference method for species authentication because it enables the detection of DNA fragments specific to individual organisms (Ballin, 2022).
Mitochondrial DNA is frequently selected as a molecular target for several reasons:
Its high copy number per cell.
Its relative stability under certain food-processing conditions.
Its genetic variation among animal species.
Commonly used mitochondrial gene targets include:
Cytochrome b (cyt b).
12S ribosomal RNA (12S rRNA).
16S ribosomal RNA (16S rRNA).
The mitochondrial DNA displacement loop (D-loop).
Species-specific primers designed for Bos taurus are intended to amplify target DNA from bovine samples, whereas primers specific to Sus scrofa are designed to amplify DNA from porcine samples (Rohman & Windarsih, 2024). The specificity of these assays must be established through appropriate analytical validation, including the assessment of cross-reactivity with DNA from other animal species.
The principal advantages of PCR include:
High analytical sensitivity.
High analytical specificity when appropriately designed and validated.
The ability to detect low levels of species-derived contamination.
Applicability to processed products, including boiled and dried materials, as well as gelatin, provided that sufficient amplifiable DNA remains.
Real-time PCR (qPCR) also enables the detection and, under appropriate calibration conditions, quantification of porcine DNA in mixed food matrices. Detection at contamination levels below 0.1% may be achievable with validated assays, although actual performance depends on the target sequence, DNA quality, sample matrix, processing conditions, and quantification strategy (Ali et al., 2023).
Despite these advantages, DNA degradation during intensive heat treatment and gelatin processing may reduce amplification efficiency. Therefore, the use of short DNA targets, appropriate extraction procedures, and suitable positive and negative controls is important when analyzing highly processed products. PCR-based methods can provide strong molecular evidence for species authentication, but their performance should be evaluated for each relevant food matrix.
3.2. Fourier-Transform Infrared (FTIR) Spectroscopy
Fourier-transform infrared (FTIR) spectroscopy is an analytical technique based on the absorption of infrared radiation by molecular bonds and functional groups.
In animal-derived gelatin, FTIR analysis commonly examines characteristic protein-associated absorption bands, including:
Amide A.
Amide I.
Amide II.
Amide III.
Differences in collagen composition, molecular structure, and processing history may influence the infrared absorption profiles of porcine and bovine gelatin, potentially resulting in differences in peak positions, expressed as wavenumbers, and spectral intensities (Rohman et al., 2021).
FTIR data are commonly combined with multivariate statistical and chemometric techniques, including:
Principal component analysis (PCA).
Partial least squares (PLS) methods.
Other chemometric classification and discrimination approaches.
The combination of FTIR spectroscopy and chemometric analysis can facilitate the differentiation of bovine and porcine gelatin based on their spectral fingerprints. However, classification performance depends on the quality and representativeness of the reference samples, the extent of processing-related variation, spectral preprocessing, and model validation procedures (Rohman et al., 2021).
FTIR spectroscopy offers several practical advantages, including rapid measurement, minimal sample preparation in many applications, and the potential for nondestructive analysis. Nevertheless, because the spectra of animal-derived proteins may overlap, FTIR-based classification should be validated using independent samples. Where halal authentication requires definitive species identification, FTIR results may be complemented by molecular or immunological confirmation.
3.3. Protein Detection Using Enzyme-Linked Immunosorbent Assay (ELISA)
The enzyme-linked immunosorbent assay (ELISA) is an immunological method that detects target proteins through specific antigen–antibody interactions.
Its principal advantages include:
Relatively rapid analysis.
Operational simplicity.
Relatively low analytical costs.
Suitability for preliminary screening applications.
Monoclonal antibodies directed against porcine-specific protein targets can bind to the corresponding antigens in a sample. In enzyme-based detection systems, this interaction produces a measurable color change that can indicate the presence of porcine-derived material, provided that the target protein and assay conditions are appropriate (Asensio et al., 2022).
ELISA is therefore potentially useful as a rapid screening tool in halal food authentication. However, its performance may be affected by protein denaturation, degradation during heating, matrix interference, and the availability of suitable species-specific antibodies. These factors are particularly relevant when testing extensively processed products.
ELISA-based rapid test kits can be used for preliminary screening, followed by PCR-based confirmation when necessary. The selection of a confirmatory method should take into account the sample matrix, the intended analytical target, and the validation requirements. A negative ELISA result does not necessarily exclude the presence of porcine-derived material if the target protein has been substantially degraded or altered during processing.
3.4. Morphological and Histological Examination
In addition to laboratory-based molecular, spectroscopic, and immunological analyses, preliminary identification can be performed by examining the biological structures of animal skin. Morphological and histological observations may provide useful information about differences between bovine and porcine skin, particularly when the tissue retains recognizable anatomical features.
Macroscopic examination may reveal differences in skin thickness, surface texture, and the distribution of visible hair follicles. Microscopic examination of tissue sections can provide further information on hair follicle architecture, collagen fiber organization, dermal structure, and the characteristics of subcutaneous tissue.
These characteristics should be interpreted cautiously because they may vary with animal age, anatomical sampling location, breed, tissue preparation, and processing conditions. Boiling and other thermal treatments may alter tissue architecture and reduce the reliability of morphological identification.
Table 1. Morphological Differences Between Bovine and Porcine Skin (Kikil)
Characteristic | Bovine Skin | Porcine Skin |
Hair follicle pore patterns | Pores are relatively evenly distributed and densely arranged, without a consistently distinctive pattern. | Three hair follicle pores may appear in clusters forming a triangular pattern (trio follicle arrangement). |
Dermal thickness | Moderate to thick, with densely packed collagen fibers. | May exhibit a relatively thick dermis and more developed subcutaneous tissue, depending on the anatomical site. |
Collagen structure | Dense and compact collagen fiber arrangement. | May exhibit a looser connective tissue arrangement, accompanied by a greater amount of adipose tissue in some regions. |
Tissue color | Milky white to cream. | Pale white, glossy, and sometimes more translucent. |
Texture | Relatively firm, dense, and resilient. | Relatively soft and elastic. |
Odor after heating | Characteristic cooked beef odor. | Characteristic cooked pork odor, influenced in part by its lipid composition. |
Histologically, porcine skin exhibits hair follicles arranged in clusters, whereas bovine skin generally shows a more dispersed distribution of hair follicles. In addition, the subcutaneous adipose tissue in porcine skin is generally thicker than that in bovine skin (Jiang et al., 2022).
Nevertheless, morphological characteristics may be altered by boiling, pressure cooking, or subsequent processing. Consequently, these characteristics should not be used as the sole basis for definitive species identification.
3.5. Combining Methods for Reliable Species Identification
No single analytical method is universally optimal for all sample types and processing conditions. Therefore, a tiered analytical approach is recommended to improve the reliability of species identification:
Preliminary screening: Conduct morphological examination or ELISA-based testing to identify samples that may require further investigation.
Confirmatory analysis: Apply PCR or real-time PCR to detect species-specific DNA sequences.
Additional verification: Use FTIR spectroscopy when the sample consists of gelatin or extracted collagen, provided that the method has been validated for the relevant sample matrix.
This multimodal approach can improve the reliability of food authentication, particularly for processed products that have undergone protein denaturation or tissue degradation. Combining complementary analytical techniques can provide more robust evidence of species identity than relying on a single method (Rohman & Windarsih, 2024).
4. CONCLUSION
The scientific differentiation of bovine and porcine skin cannot rely solely on visual characteristics because food-processing procedures may alter the physical appearance and structural properties of the tissues. DNA-based methods, particularly polymerase chain reaction (PCR) and real-time PCR, provide highly reliable approaches to species identification by detecting species-specific genetic material with high analytical sensitivity and specificity, provided that the methods are appropriately designed and validated.
Fourier-transform infrared (FTIR) spectroscopy offers an effective complementary approach for differentiating bovine and porcine gelatin based on their infrared spectral characteristics, whereas enzyme-linked immunosorbent assay (ELISA) serves as a rapid screening method for detecting porcine-specific proteins. Morphological and histological examinations, including the assessment of hair follicle patterns, dermal thickness, follicle distribution, collagen structure, tissue color, texture, and subcutaneous tissue characteristics, can support preliminary identification but are insufficient to establish species identity conclusively.
Therefore, an integrated approach combining morphological examination with molecular and immunological analyses provides a robust framework for food authentication, food safety surveillance, and halal assurance. The selection of analytical methods should be tailored to the type of sample, its processing history, and the intended purpose of testing. Such an approach can strengthen the scientific basis for identifying animal-derived ingredients and support reliable implementation of halal certification and food control systems.
5. REFERENCES
Ali, M. E., Hashim, U., Mustafa, S., Che Man, Y. B., & Islam, K. N. (2023). Recent advances in DNA-based methods for halal food authentication. Food Control, 150, 109739.
Asensio, L., González, I., García, T., & Martín, R. (2022). Immunological methods for meat species identification: Current advances and future perspectives. Foods, 11(8), 1127.
Ballin, N. Z. (2022). Authentication of meat and meat products using molecular biology methods: A review. Meat Science, 188, 108780.
BPJPH. (2024). Pedoman Penyelenggaraan Jaminan Produk Halal. Badan Penyelenggara Jaminan Produk Halal, Kementerian Agama Republik Indonesia.
Codex Alimentarius Commission. (2023). General Principles of Food Hygiene (CXC 1-1969, Revised 2023). FAO/WHO.
Jiang, H., Zhang, Y., Liu, X., Wang, Z., & Chen, L. (2022). Comparative histological characteristics of mammalian skin and their application in species identification. Journal of Morphological Sciences, 39(4), 245–257.
Rohman, A., Windarsih, A., Erwanto, Y., & Zakaria, Z. (2021). Fourier Transform Infrared Spectroscopy combined with chemometrics for halal authentication of food products: A review. International Journal of Food Properties, 24(1), 1149–1166.
Rohman, A., & Windarsih, A. (2024). Analytical techniques for halal authentication of food products: Recent advances and future trends. Trends in Food Science & Technology, 145, 104374.
Universitas Islam Negeri Ar-Raniry Banda Aceh. (2021). Identifikasi Gelatin Babi dan Gelatin Sapi Menggunakan Spektroskopi FTIR. Repository UIN Ar-Raniry.
World Health Organization. (2022). Food Safety Strategy 2022–2030. World Health Organization.
Zhang, L., Wang, Q., Liu, H., & Chen, Y. (2024). Advances in molecular authentication technologies for animal-derived food products. Comprehensive Reviews in Food Science and Food Safety, 23(2), 1456–1482.
#FoodAuthentication
#BeefSkin
#PorkDetection
#FoodSafety
#HalalAssurance
No comments:
Post a Comment