Authentication of traditional meat products (Haleem) sold in India: a first report confirming species mislabelling

Abstract

Traditional meat products like Haleem play a pivotal role in the culinary landscapes of Indian consumers, along with high economic value and business potential. Due to anticipated gains associated with adulterating ‘Haleem’ and constant evasion from regulatory oversight, the susceptibility to adulteration has substantially increased. Furthermore, no reports/surveillance regarding their labelling compliance has been reported. Hence, we conducted a 2-year surveillance using 100 samples collected from Hyderabad, India, using the Chipron™ DNA macroarray analysis technique. The method was validated for sensitivity (1%), specificity, and with proficiency test samples. Following this, the surveillance samples (beef, chicken, and mutton Haleem) were tested. The surveillance revealed an alarming adulteration of 46% of the samples, with different proportions of adulterant species. Adulteration of unconventional meat like camel meat was also found. These concerning results necessitate the requirement of stricter and constant regulatory surveillance to safeguard consumer trust and preserve the authenticity of traditional meat products.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-024-05947-9.

Introduction

Foods of animal origin, like meat, have an ever-lasting demand in terms of consumer preferences. Meat and meat products have been utilized throughout the ages and still contribute to economic and consumer expectations (Tonsor and Lusk 2022). India is a major meat producing country, and total meat production in the country is 9.29 million tonnes (DAHD 2022). Meat and meat product consumption in India has significantly risen over the years, and it is expected to increase further (OECD/FAO 2021). This surge in demand for meat and its related products results in fraudulent activities by sellers for economic profit.

Meat is cherished by most people in the country due to its highly appetizing taste and high nutritional content (Laranjo et al. 2017). Several value-added and traditional meat products have gained importance and consumer preferences over the years. Due to increasing demand and less authenticity emanating from the removal of identifiable morphological characteristics, it becomes easy for fraudulent people to practice illegal activities (Vishnuraj et al. 2023). Economically motivated food fraud is defined as the partial/entire substitution of more expensive meat, with cheaper alternatives (Everstine et al. 2013). Monitoring the meat supply chain for such adulteration is of great concern and poses difficulties for authentication, especially due to high processing (Soman et al. 2020). Since adulteration of cheaper quality meat in misbranded names can lead to pathogenic/toxin responses in humans, a complete vulnerability assessment of the meat supply chain should be conducted regularly (Li et al. 2020).

Traditional meat products have gained importance throughout history, both in religious and savoury aspects. Several Asian countries have the practice of preparing traditional meat products, which provides high demand during the festive seasons (Zhang et al. 2017). One of the highly famous traditional meat products in the Middle East, Central Asia, and the Indian subcontinent is Haleem, a product that relates to Islamic people (TOI 2019). Although of religious importance, Haleem is cherished by all meat-loving consumers. Due to a significant increase in demand for the product, several mislabelling practices and adulteration occur in a substantial manner. Furthermore, due to the limited food safety regulations and inspection applied to traditional meat products, several fraudulent practices go undetected (Soman et al. 2020).

To detect and mitigate such fraudulency, contemplating all the above points, the current research was effectuated as a surveillance study on local Haleem products available in the region of Hyderabad, Telangana, India, over a period of 2 years, utilizing a highly sensitive DNA microarray analysis technique (MEAT 5.0, Chipron GmbH, Germany).

Experimental

Test samples

A hundred samples of Haleem were collected from various outlets in Hyderabad, Telangana State, India, during the Ramzan month of the Muslim Hijri calendar in two years (2016 & 2017). Quality Control Materials (QCMs) of various targeted food animal species like Bos indicus (beef), Bubalus bubalis (buffalo), Ovis aries (sheep), Capra hircus (goat), Sus scrofa domestica (pig), Gallus gallus domesticus (chicken) and Camelus dromedarius (camel) were prepared in-house as per ISO guide 80: 2014. Further, for performing verification of the limit of detection (LoD) of the test (Meat 5.0, Chipron GmbH), beef and buffalo meat obtained from the municipal slaughterhouse in Hyderabad, India, were used. All these samples were made genetically traceable by bi-directional Sanger sequencing (Genetic Analyzer, 3500) using two animal-specific barcodes—12S rRNA and mitochondrial cytochrome B region and was compared with NCBI database. All the experiments were carried out in our ISO/IEC 17025: 2017 accredited laboratory.

DNA extraction

Extraction of DNA from meat and Haleem samples was performed using Vertebrate Lysis Buffer (VLB) digestion followed by spin column extraction (Nischala et al. 2022) The quality and concentration of all the extracted DNAs were measured using a Spectrophotometer (DeNovix, USA) and an A_260/280 value of 1.7 to 1.9 was considered optimum for downstream testing.

Chipron assay verifications

Chipron macro array analysis kit (Meat 5.0) was used following the manufacturer’s instructions after proper standardization and verification (CAC/GL 74: 2010). The PCR protocol was standardized for 25 µL with the following components: 0.12 μL of 10X NEB Taq buffer, 0.5 μL of dNTP mix, 1.5 μL of biotinylated primer mix (16S rRNA target and labelled as ‘MEAT’), 3 μL of template DNA and remaining being adjusted with nuclease-free water in a C1000 touch thermal cycler (Bio-Rad, USA). The PCR conditions followed were 35 cycles of initial denaturation at 94 °C for 30 s, followed by annealing at 57 °C for 45 s and elongation at 72 °C for 45 s, followed by strand completion at 72 °C for 2 min. The protocol was optimized with a ramp rate of 3 °C/s. To verify the specificity of the Chipron assay, QCMs from six animal species (prepared in-house) were utilized. These six species were selected to verify the ability of the method to detect most possible adulterant species in Haleem. To verify the sensitivity of this method, a binary mixture of beef and buffalo, at 1 and 2% mixture of beef in buffalo meat and vice versa, were prepared and tested using Chipron assay. Also, this assay has been externally validated through participation in relevant proficiency testing (Food Chemistry –2976) from Fapas (Fera Science Ltd, UK) with beef matrix and adulterants of chicken, equine, lamb, and pork.

Investigating market Haleem samples and results interpretation

100 Haleem samples collected over a period of two years were used in this investigation. The results were recorded qualitatively, analyzed, and reported as percentages.

Results and discussion

Quality and quantity evaluation of DNA

Haleem, typically being a highly processed product, provided a very low quantity (or) degraded DNA from many test samples. Hence, the DNA amplifiability was verified using myostatin gene amplification in a real-time PCR set-up (Laube et al. 2007). There have been prior reports on the issue of DNA degradation in highly processed meat products, how it varies depending on the kind and degree of processing, and the need to assess DNA amplifiability (Sreenivasan and Viljoen 2021). The analysis resulted in the amplification of DNA from all Haleem samples with high Cq values, as expected for low quantities of DNA. Hence, the samples were allowed for further investigation in Chipron microarray.

Specificity of DNA macro array

For specificity testing, DNA extracted from QCMs of six species (mentioned previously) was tested at lower concentrations. On the chip, it provided dark visible spots at locations of species-specific capture probes, with excellent fluorescence intensity, indicating the assay’s specificity (Suppl. Fig F1). This is in accordance with several previous studies (Cottenet et al. 2016), where the specificity of the Chipron was evaluated with pure meat. It is also important to know that the occurrence of contamination in Chipron is a high probability since the macro array is a sensitive detection method (Drdolová et al. 2019). This evaluation, which was without any unintended amplification, offers both accurate specificity and a demonstration of the accredited laboratory’s effectiveness in preventing contamination.

Limit of detection

Sensitivity (LoD) was evaluated in relative, using beef and buffalo meat mixtures in the following percentages—99: 1 & 98: 2 buffalo in beef and vice versa. The limit of detection of 1% was reliably established in this experiment, which was the lowest limit intended to be tested in the current study (Fig. 1; Suppl. Table T1). However, the absolute limit of detection (LoD_abs) of this technique and other macro-array-based analyses was reported to be 0.1% (Cottenet et al. 2016).

Fig. 1.

Analysis of relative meat mixtures of beef and buffalo meat for sensitivity evaluation in Chipron macro array MEAT 5.0

Validation through proficiency testing (PT)

The proficiency testing material was detected positive for horse and pork, whereas chicken and lamb were not detected. These results were in accordance with the PT report (Suppl. Fig F2) and this validation unambiguously verified the suitability of the test for the intended purpose.

Haleem surveillance

Haleem samples (n = 100) were tested for the presence of undeclared animal species, in addition to the meat species mentioned for sale/labelling. Different Haleems like mutton, chicken and beef were evaluated. Out of the 100 samples tested, 46 samples (46%) contained undeclared animal species (Table 1).

Table 1.

Complete details of 100 samples of Haleem tested. Various undeclared animal species were detected in the samples, and their signal intensity values from microarray analysis have been reported. The compliance to labelling was also verified, and mislabelled products were identified

Sample	Signal value	Meat species detected						Labelled as	Mislabelling (Yes/No)
		Sheep	goat	beef	buffalo	Chicken	Camel
H-1	60,338	√						Mutton
H-2	57,065		√					Mutton
H-3	60,831	√						Mutton
H-4	60,130							Mutton
H-5	59,560	√						Mutton
H-6	59,699	√						Mutton
H-7	58,018	√						Mutton
	23,103		√
H-8	60,102	√						Mutton	√
	57,003		√
	60,884				√
H-9	60,854	√						Mutton
H-10	60,984	√						Mutton
H-11	61,054	√						Mutton
H-12	59,552	√						Mutton
	46,842		√
H-13	58,116		√					Mutton	√
	35,060					√
H-14	60,850	√						Mutton
H-15	25,488			√				Mutton	√
	46,856	√
	51,877		√
	60,130				√
H-16	60,496	√						Mutton	√
	39,088					√
H-17	60,487	√						Mutton
	2535		√
H-18	60,476	√						Mutton
H-19	58,666	√						Mutton
	2589		√
H-20	57,674	√						Mutton
H-21	56,541			√				Beef	√
	2525	√
	58,774				√
H-22	59,434			√				Beef
	61,125				√
H-23	59,918			√				Beef	√
	5682	√
	12,519		√
	52,468						√
	3176					√
H-24	57,224			√				Beef	√
	10,630	√
	2962		√
	58,737				√
	45,308					√
H-25	57,242	√						Mutton	√
	51,462		√
	59,278					√
H-26	58,820	√						Mutton
	47,610		√
H-27	42,518	√						Mutton
	48,712		√
H-28	50,380	√						Mutton
	57,921		√
H-29	49,454	√						Mutton
	11,768		√
H-30	54,262	√						Mutton
	58,944		√
H-31	55,496	√						Mutton
H-32	54,342			√				Beef	√
	45,869	√
H-33	47,673	√						Mutton
H-34	54,894			√				Beef	√
	14,988	√
H-35	51,686	√						Mutton
H-36	53,766	√						Mutton
H-37	52,104			√				Mutton	√
	49,505	√
	43,922		√
	3980				√
	15,586					√
H-38	54,794	√						Mutton	√
	60,745		√
	58,356					√
H-39	55,574	√						Mutton
	8678		√
H-40	57,171	√						Mutton	√
	25,880				√
H-41	52,443	√						Mutton
H-42	45,840	√						Mutton	√
	60,878		√
	18,567					√
H-43	45,604	√						Mutton	√
	47,819					√
H-44	53,316	√						Mutton
	56,838		√
H-45	4464			√				Mutton	√
	47,740	√
	58,417		√
H-46	49,878	√						Mutton	√
	4262		√
	42,523					√
H-47	48,664			√				Beef	√
	60,626	√
	14,144		√
	13,490				√
	17,494					√
H-48	50,173			√				Beef	√
	58,332	√
	21,119		√
	4626				√
	13,156					√
H-49	53,050			√				Beef	√
	59,156	√
	2396		√
	58,934				√
	12,444					√
H-50	52,496			√				Beef	√
	14,176	√
	48,140				√
H-51	54,410	√						Mutton	√
	7772		√
	3206			√
H-52	57,330	√						Mutton	√
	17,550					√
H-53	53,312					√		Chicken
H-54	54,974		√					Mutton	√
	54,787	√
	5464				√
	8086					√
H-55	51,362					√		Chicken	√
	2747	√
	4496				√
H-56	48,878	√						Mutton
H-57	50,582	√						Mutton	√
	7866					√
	23,103		√
H-58	48,967		√					Mutton	√
	15,608	√
	4866					√
	2465				√
H-59	58,156	√						Mutton	√
	15,366					√
H-60	60,439	√						Mutton
H-61	58,948		√					Mutton	√
	58,907	√
	31,174					√
H-62	61,422	√						Mutton
H-63	43,722	√						Mutton	√
	43,722			√
	8998				√
H-64	60,394				√			Beef	√
	57,976			√
	51,046	√
	40,452					√
	10,522		√
H-65	60,944	√						Mutton
	7822		√
H-66	48,726			√				Beef	√
	45,653	√
	8964				√
H-67	56,954					√		Beef	√
	31,290	√
	8574				√
H-68	32,910				√			Beef	√
	26,560	√
	21,008					√
	9734		√
H-69	51,177			√				Beef	√
	19,702					√
	3236	√
H-70	54,936	√						Mutton	√
	4159					√
	2058				√
H-71	51,944	√						Mutton
H-72	42,148	√						Mutton	√
	2122				√
H-73	41,482			√				Beef	√
	24,812	√
	9734				√
	9402					√
	3510		√
H-74	51,872	√						Mutton	√
	4391					√
	3114				√
H-75	39,675	√						Mutton
H-76	49,882	√						Mutton
H-77	38,998		√					Mutton
H-78	46,849	√						Mutton
H-79	16,576				√			Beef
	3252			√
H-80	38,728			√				Beef	√
	3910	√
H-81	40,074	√						Mutton
H-82	60,118					√		Chicken
H-83	60,008				√			Beef
	59,280			√
H-84	61,570	√						Mutton	√
	56,894					√
H-85	61,218	√						Mutton	√
	11,512				√
	9027					√
H-86	61,190	√						Mutton
H-87	60,686	√						Mutton	√
	5090				√
H-88	58,422	√						Mutton	√
	53,228		√
	17,068				√
	9708						√
H-89	60,644		√					Mutton
	32,138	√
H-90	39,844	√						Mutton
H-91	32,334	√						Mutton
H-92	16,660	√						Mutton
	25,218		√
H-93	30,118	√						Mutton
H-94	14,807					√		Mutton	√
H-95	35,478	√						Mutton
	34,088		√
H-96	22,757	√						Mutton	√
	5374					√
H-97	37,606	√						Mutton
H-98	33,660	√						Mutton
H-99	18,134	√						Mutton
H-100	12,310	√						Mutton
	14,694		√

Out of 100 Haleem samples, 78 were mutton Haleem, 19 were beef Haleem, and 3 were chicken Haleem. Mutton samples where goat DNA was found were not considered as mislabeling, as sheep and goat meat are interchangeably used in India due to lack of knowledge (Nischala et al., 2022). The same was followed for beef Haleem when buffalo DNA was found. Comparative analysis of different meat species in a Haleem was calculated and reported, along with the signal intensity for each species (Table 1). Of the total 78 mutton Haleem tested, 33 (42.3%) were found to be adulterated with chevon, which are closely related species in terms of their local availability and meat characteristics. In a previous study from the region, almost 50% of the sheep and goat meat was reported to be mislabelled (Nischala et al. 2022). Interestingly, five mutton Haleem (6.4%) did not even contain mutton. In the mutton Haleem samples tested, the presence of undeclared chicken was reported in 18 samples (23.07%), buffalo meat in 13 (16.66%), beef in 5 (6.41%), and camel in one sample (Fig. 2a). The increased percentage of both beef and carabeef in mutton Haleem may be due to potential substitution with remnants of beef Haleem, which is largely popular in the area. Hence, health or religiously selective consumers may worry about undisclosed beef/buffalo meat in products labelled as purely mutton/chevon. Owing to its widespread religious acceptance, chicken is the most popular meat both locally and globally (Whitnall and Pitts 2019), and as a cheap alternative, chicken has been mostly incorporated into mutton Haleem. The most unethical and probably one of the unexpected is the adulteration of camel meat, which was also found in one of our previous studies (Vaithiyanathan et al. 2021). The addition of unconventional meats in traditional foods is one of the alarming findings, particularly in the current context where zoonoses and public health risks are of significant concern. Previous studies like Vaithiyanathan et al. (2021) have also stressed the alarming chances of camel meat being adulterated with other meats.

Fig. 2.

a Bar chart showing the adulteration in mutton Haleem samples (n = 78) and the percentage of samples that contain undeclared meat species. b Bar chart showing the adulteration in beef Haleem samples (n = 19) and the percentage of samples that contain undeclared meat species

Regarding beef Haleem, alarming adulteration was observed in the 19 samples analyzed, with 14 samples (73.68%) showing buffalo meat adulteration (Fig. 2b). Such adulteration with closely related species is the most common type of meat fraud, where organoleptic differentiation is not feasible (Vishnuraj et al. 2023). Yet, mutton had the highest part, being adulterated in almost 84.21% of the beef samples. The reason might be due to the easy availability of mutton in the market than beef, which is imperative with the data provided. Additionally, the unintentional use of remnants may further support this observation. Hence, the signal intensities exclude possibilities of any carry-over contaminations. Species like chicken and goat were found to be adulterated in almost 52.63% and 42.1% of beef Haleem, respectively (Fig. 2b). Furthermore, just like in the case of mutton Haleem, beef Haleem was also found to be adulterated with camel meat in one sample tested. Out of the three chicken Haleem samples examined, only one sample exhibited traces of buffalo and sheep, although the signal intensity was notably low. This occurrence might be attributed to inadvertent contamination stemming from inadequate cleaning of the cooking vessels employed during production (Kane and Hellberg 2016). Since traditional products are more likely to be neglected for regular food inspections and safety evaluations, a high amount of adulteration, even with unconventional meat such as camel can occur. To mitigate such food fraud and to safeguard the public from the risks associated with adulterations, continuous and strict monitoring of meat food products is to be taken up by national food regulators like the Food Safety and Standards Authority of India.

Conclusion

This research employed intrinsic surveillance to investigate animal species mislabelling in the traditional dish Haleem. It utilized the validated DNA macroarray method within an ISO/IEC 17025:2017 accredited facility. The macroarray protocol was adapted for convenience while maintaining specificity and sensitivity. Over a 2-year period, the assay uncovered significant adulteration in Haleem samples from local retail outlets, particularly the substitution of closely related species. Additionally, unconventional meat adulteration, including camel meat, raised concerns. This can help the regulatory authorities in maintaining strict food safety enforcement along with vulnerability assessment evaluation in traditional markets with the help of robust and sensitive techniques like DNA macro array.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

VMR Methodology, Investigation, Formal analysis, Visualization, Writing—original draft, Writing—review & editing, Project administration. VS Conceptualization, Formal analysis, Visualization, Resources. BRP Visualization & Investigation. AKN Formal analysis, writing—original draft, Writing—reviewing & editing. BSB Conceptualization, Visualization, Resources.

Funding

No external funding to carry out the research.

Data Availability

The datasets generated during the research study are available from the corresponding author upon reasonable request.

Code availability

Not applicable.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this research article.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.