Monitoring of sausage products sold in Sichuan Province, China: a first comprehensive report on meat species’ authenticity determination

Presently, there is growing concern worldwide regarding the adulteration of meat products. However, no reports on determining meat authenticity have been reported in China. To verify labelling compliance and evaluate the existence of fraudulent practices, 250 sausage samples were purchased from local markets in Sichuan Province and analysed for the presence of chicken, pork, beef, duck and genetically modified soybean DNA using real-time and end-point PCR methods, providing a Chinese case study on the problem of world food safety. In total, 74.4% (186) of the samples were properly labelled, while the other 25.6% (64) were potentially adulterated samples, which involved three illicit practices: product removal, addition and substitution. The most common mislabelling was the illegal addition of, or contamination with, duck. Therefore, meat authenticity monitoring should be routinely conducted. Additionally, the strict implementation of the nation’s food safety laws, along with regular surveillance, should be compulsory to alleviate and deter meat adulteration.


Materials and Methods
Sample materials. A total of 250 sausage products, representing a variety of meat origins (pork, chicken, duck, beef or mixtures) were purchased, between July and August 2016, from 77 local markets and 89 restaurants across 21 counties in Sichuan Province (Fig. 1). These samples represented six different types of meat or meat mixtures, including products labelled as containing chicken (n = 16; Type I) and mixtures of chicken and duck (n = 47; Type II), chicken and pork (n = 156; Type III), chicken, duck and pork (n = 29; Type IV), chicken, pork and beef (n = 1; Type V), and chicken, duck and beef (n = 1; Type VI). Sample details are shown in Table 1. Manufacturer names are not disclosed. All the samples were frozen and maintained at 4 °C in our laboratory. The authentic meat samples (reference samples) of swine, chicken, duck and cattle used as positive controls were purchased from the Chinese Academy of Inspection and Quarantine (Beijing, China). The reference materials for GM soybean GTS40-3-2 were purchased from the Institute for Reference Materials and Measurements in Geel, Belgium, and the reference DNAs for GM soybean A2704-12 and MON89788 were purchased from the American Oil Chemists Society (Champaign-Urbana, IL, USA). DNA extraction. Genomic DNAs were isolated and purified from the reference materials and test samples, and sterilised ultrapure water was used as a negative control (blank) for DNA extraction using a genomic DNA purification kit (Tiangen Biotech Co., Ltd., Beijing, China) following the manufacturer's instructions. The DNA concentrations were measured using a NanoDrop ND-1000 UV Spectrophotometer (Thermo Fisher Scientific, Waltham City, MA, USA).
Primers and PCR conditions. Species-specific DNA segments for duck, pork and beef, and the 18S rRNA of eukaryotes (control for species DNA) were amplified using primer and probe sequences as described in the Inspection and Quarantine Industry Standard of the People's Republic of China 35,36 and the National Standard of the People's Republic of China 37 (Table 2). DNA species-specific fragments of duck, pork and beef, as well as the 18S rRNA of eukaryotes, in samples and reference materials were amplified using real-time PCR on an ABI 7500 fluorometric thermal cycler (Applied Biosystems, Foster City, CA, USA), with simultaneous amplification of the blank from the DNA extraction and non-template PCR control. All the samples were tested for the presence of duck-, pork-and beef-specific DNA fragments. The real-time PCR assays were carried out in a final volume of 25 µL containing 1× THUNDERBIRD ™ Probe qPCR Mix (Toyobo Co., Ltd., Osaka, Japan), 1× ROX reference dye, 500 nM each primer, 250 nM probe and approximately 50-100 ng genomic DNA per sample or reference material (control) as the template [35][36][37] . The thermocycling settings for beef were as follows: initial incubation at 95 °C for 10 min and then 45 cycles of 95 °C for 15 s and 60 °C (annealing and extension, respectively) for 1 min. Fluorescence measurements were taken after annealing and extension. For the thermal amplification programs of pork and duck, the annealing temperature was set to 58 °C for 15 s, and all other program parameters were the same as those for beef.
The sequences of primer pairs for the amplification of chicken, the three GM soybean events (GTS40-3-2, A2704-12 and MON89788) and the endogenous Lectin gene (soybean taxon-specific gene used as a control for Figure 1. Geographical distribution of markets from which the sampled sausage products were purchased for this study. Locality codes correspond to the city names in Table 1.   [38][39][40][41] . The amplification of the blank for DNA extraction and non-template PCR control were performed simultaneously with the same primers to determine whether contamination occurred during DNA extraction and PCR set up. The thermocycling program of the conventional PCR assays for the Lectin gene and two GM soybean lines, GTS-40-3-2 and A2704-12, were as follows: initial denaturation for 5 min at 95 °C; 35 cycles of amplification for 30 s at 94 °C, 30 s at 58 °C and 30 s at 72 °C; and a final extension for 7 min at 72 °C. The annealing temperatures of amplification for the MON89788 line and chicken species-specific DNA were 56 °C and 63 °C, respectively. Except for the annealing temperatures, the other program parameters were the same as those of the GM soybean lines GTS-40-3-2 and A2704-12. As indicated by the standards [35][36][37][38][39][40][41] , the specificity of the methods described was highly specific, and the limit of detection was 0.1%. The specificity (high specificity) and sensitivity (five copies) levels of the conventional and real-time PCR methods used to analyse the sausage samples were not evaluated in this study because they have been previously confirmed as the national standards for GM organism detection in China. PCR products were analysed by 2.5% agarose gel electrophoresis containing ethidium bromide in 1× Tris-acetate EDTA buffer for 20 min at 110 V.

Results
There were no targeted DNA bands or amplification signals in the PCR of the blank controls, and the expected DNA bands or amplification signals were obtained in the PCR of the positive controls. In total, 250 sausage samples collected in 166 local markets or restaurants in 21 cities in Sichuan Province, China were first identified using PCR methods. DNA fragments of the 18S rRNA and/or Lectin gene were amplified successfully from templates isolated from all the samples, indicating the efficient extraction of genomic DNA from eukaryotes (meat species) and soybean (plant addition to sausage), which can be used in the subsequent analyses. Among the samples, the mislabelling of 64 products (25.6%) was found. Mislabelled samples collected from 17 cities, except for sausage samples purchased from LZ, BZ, ZY and ZG, were determined ( Table 1). The greatest frequency (75.0%) of mislabelling was observed in PZH, and the lowest frequencies (8.3%) were detected in SN, MS and NC. Although most of the sausage products sampled were from the capital city of Sichuan Province (CD), the frequency of mislabelled samples was less than 19%. Surprisingly, the three highly imported GM soybean lines, GTS40-3-2, MON89788 and A2740-12, were not detected in any sample labelled as containing soybean.
Among the 64 mislabelled samples, one sample labelled as Type III (containing chicken and pork) tested positive for chicken and duck (Type II; Table 3), representing a typical meat species substitution. Three samples labelled as Type I (containing chicken, duck and pork), tested negative for duck (Type III; Table 3). Additionally, a high incidence of illegal additions or contamination of undeclared meat species was observed in 60 samples. Duck meat was the most common undeclared animal species found in 58 samples, while unclaimed pork was detected in the 2 samples labelled as Types II and VI. The greatest incidences (75%) of illegal additions of, or   (Table 4).

Discussion
Here, for the first time, the meat authenticity of ham sausages produced in China was investigated to assess the extent of mislabelling in sausage products using two types of detection methods based on PCR. In total, 74.4% of the samples were properly labelled, with the contents corresponding to the product lists. However, 25.6% of the potentially adulterated samples were subject to one of three illicit practices, removal (declared species not detected in the product), addition and substitution with another species. The percentage of mislabelling (25.6%) in this work was relatively small compared with those discovered in American (38.5%) 42 and Malaysian (78.3%) 43 markets. However, the percentage of mislabelling (25.6%) was greater than that reported in Canadian markets (20%) 26 . The most common mislabelling in this study was contamination with, or the illegal addition of, duck.
Altogether, 58 samples were found to contain undeclared duck as determined by real-time PCR, with Ct values ranging from 20.75 to 35.47. However, it was difficult to determine whether duck meat was intentionally added to the 58 samples or these samples were illegally contaminated with duck meat because Ct values are affected by many factors (i.e., inhibitory ingredients in the samples and the level of DNA degradation). Similarly, two samples containing unstated pork had low Ct values of 25.74 and 26.79, which indicated illegal additions or cross-contamination. In the sausage production chain, cross-contamination can arise when improperly cleaned (or uncleaned) equipment is used to process meat from more than one species 44 . Perhaps, a quantitative PCR analysis will aid in determining whether undeclared ingredients are additives or contaminants. Compared with the illegal additives in sausage products reported in previous studies 17,[22][23][24] , in China, the prevalent unlabelled additive to sausage was duck, while in other countries chicken was the major unlabelled additive. Generally, the average price of duck is low in China, while beef is the highest, followed by pork and chicken. Therefore, duck is an economical additive for manufacturers to decrease the production cost of sausages. The different prices of meats worldwide explained why the illegal meat species used as additives differ between the sausages produced in China and those in other countries. They also explained the substitution of duck for pork in the test samples. However, the more expensive pork was added to two samples labelled as Types II and VI, and they had pork-associated Ct values of less than 30. Accordingly, the presence of pork did not seem to be the result of cross-contamination events. Similarly, it is hard to understand why the declared duck was not detected in three samples marketed as Type IV. It may be that the two aforementioned cases were mislabelled as human errors.
Another main reason for performing a survey on the meat authentication of sausage is because of religious concerns. Of the 250 sausage samples, two were clearly listed as meeting Muslim dietary laws (Halal) on their labels. One tested positive for undeclared duck, while the other was correctly labelled. Fortunately, the pork prohibited by Halal was not detected. China is a multiracial country, with a Muslim minority population of more than 10 million. It is estimated that more than 100,000 Hui people live in Sichuan Province, China. The mislabelling of sausage products may lead to the violation of personal or religious beliefs. A previous study reported that pork is the most common meat substitute, and its consumption breaches Muslim dietary restrictions 17 . Unfortunately, pork was found in sausages and ground-meat products in previous investigations 10,17 . Although pork was not found in the Halal sausages tested in this study, the detection of meat origins for Halal products should not be neglected in the future.

Group Type
Products labelled as different meat  I  products labelled as chicken  16  4  0  0  0  4  0  0  22.87-35.18   II  products labelled as mixture of  chicken and duck  47  1  0  1  0  0  0  0  26.79   III  products labelled as mixture of  chicken and porcine  156  55  1  0  0  54  0  0 Table 4. Sample groups and mislabelling, the fraudulent substitution, removal and addition of meat ingredients in sausage samples from Sichuan, China. Table 4 is grouped by product category to emphasise the number of mislabelling and the fraudulent substitution, removal and addition events of meat ingredients in sausage samples. (2019) 9:19074 | https://doi.org/10.1038/s41598-019-55612-x www.nature.com/scientificreports www.nature.com/scientificreports/ China has imported large quantities of GM soybeans since 2000. However, the three most imported GM soybean lines (GTS40-3-2, MON89788 and A2704-12) were not detected in any of the sausage samples, as in highly processed foods in previous reports 45,46 . It is challenging to extract and amplify total DNA from heavily processed products (especially fermentation products) because of the degradation of genomic DNA. Thus, the exogenous DNA of GM soybean in the samples tested may have degraded during fermentation; therefore, the targeted DNA segments (flanking DNA fragments) were not amplified 46 .
The observations of illegal additions, removals and substitutions in sausages in this study were similar to those of previous research 17, [22][23][24] . Because of the high market value of meat compared with most plant-derived products, meat products are often targets for adulteration 17 . The sausage production technique often leads to changes in the appearance, colour, texture and flavour of the meat; therefore, additions, removals and substitutions are easily masked 47 . Thus, it is very difficult to visually detect these illicit events during meat processing. Consequently, adulterations are commonly found in sausage and ground meat products 17,[22][23][24] .
The accurate identification of ground-meat products, including sausages, in markets is a growing concern because of the high incidence of adulteration worldwide. This study revealed that the mislabelling of sausages owing to illegal additions, substitutions and removals is a reality in China, and that local consumers are undoubtedly encountering undeclared animal species in sausage products. Our results provide a baseline for the preliminary assessment of meat species' mislabelling in sausage products in China. Owing to recent food fraud scandals, including the intentional contaminating of infant milk formula with melamine [48][49][50] , food safety in China currently focuses on identifying the presence of poisonous chemical substances in foods. The food control authorities in China are not routinely monitoring sausage and ground-meat products for mislabelling. To date, the authentication of meat species in meat products has not been conducted across China. Although the mislabelling resulting from additions, substitutions of undeclared meat species and the removal of declared meat species may not impact food safety and public health, consumers can be deceived regarding the products they are purchasing and consuming 51 . Presently, according to our research, the fraudulent mislabelling of the additional of duck to sausages is becoming an important problem in the meat industry. The meat industry in China will face a crisis in consumer confidence if the adulteration of sausages becomes generalised. Identifying the species' origins in sausages and meat products is important for preventing adulteration and for protecting consumers' health and religious convictions 23 .
In China, the adulteration and misbranding of meat products are prohibited by the related food safety laws. Despite the implementation of more stringent food labelling regulations locally and globally, the adulteration or misrepresentation of food products for illicit financial gain continues to be a common societal problem 52,53 . Accordingly, public safety measures and testing procedures should be implemented by regulatory agencies based on the declared products' contents. This is an ongoing issue that urgently requires monitoring. This survey highlights the importance of increasing national concerns and government efforts in food traceability. The strict implementation of the food safety laws of the People's Republic of China, along with regular surveillance and monitoring programs, are required to alleviate and deter mislabelling issues. Additionally, regular audits of processed meat plants by the regulatory agencies should be implemented to ensure that manufacturing operations comply with Chinese food regulations. While this investigation suggests the occurrence of the addition of undeclared meat species in sausage products, further studies are needed to detect the extent of mislabelling and to identify points in the production chain where mislabelling occurs. Future areas of work also include the expansion of the tested meat species, the ground meat products and the sampling sites across China.

conclusions
Although several regulations and laws related to food and agricultural products are enforced in China, there is still a lack of information on the authentication of meat species. This study found a relatively low incidence (25.6%) of mislabelling owing to the adulteration of sausage products on the commercial market compared with previous reports 17, [22][23][24] . Nevertheless, further studies are needed to determine the extent of adulteration across the whole meat industry in China, which would provide the government with more comprehensive data to be used in decision-making related to controlling the quality and safety of meat products.

Data availability
The datasets generated or analysed during the current study have been provided in this manuscript.