Development and Application of SCAR Marker for the Detection of Papaya Seed Adulteration in Traded Black Pepper Powder

The present study reports the development of a specific, sensitive, and reproducible Sequence Characterized Amplified Region (SCAR) marker to detect papaya seed powder adulteration in traded black pepper powder. A putative RAPD marker (449 bp) specific to papaya seed was identified, cloned, and sequenced to design the SCAR primers. This specific SCAR marker could detect the presence of papaya seed in all the analyzed simulated standards and in one of five branded market samples of black pepper powder tested. The analytical strategy being very simple could be used for large scale screening of powdered black pepper market samples intended for export and domestic uses.     

DNA Barcoding for Discriminating the Economically Important Cinnamomum verum from Its Adulterants

Traded forms of spice and spice powders are often subjected to admixing with inferior substances by design or default, affecting public health and national prestige. Cinnamomum verum (true cinnamon), a high-value spice, is often adulterated with its inferior species such as C. cassia and C. malabatrum. The presence and detection of the spurious species in traded barks (whole or powder) of true cinnamon is posing problems. DNA markers are now used to detect such adulteration. Here we report the application of a DNA barcoding method to detect these adulterants in traded market samples of true cinnamon using the barcoding loci rbcL, matK and psbA-trnH. The PCR success rate, sequencing efficiency, inter and intra specific divergence, and occurrence of single nucleotide polymorphisms (SNPs) were utilized to assess the potential of each barcode loci to authenticate C. verum from its related adulterants. The amplification and sequencing success was 100% for rbcL and psbA-trnH while matK failed to amplify in the market samples. The locus of rbcL showed higher interspecific divergence while psbA-trnH exhibited lower interspecific divergence. SNPs specific to C. cassia were detected in rbcL locus in seven out of the ten market samples studied thereby confirming the presence of C. cassia adulteration in commercial samples of true cinnamon. Out of the three loci, rbcL locus proved to be efficient in tracing out adulterants in traded cinnamon. The SNP sites in this locus can be exploited in designing C. cassia specific primers, enabling kit development for easy detection of adulterants at the band level itself thereby bypassing the cost of sequencing.     

A new TLC method to detect the presence of ground papaya seed in ground black pepper

A simple TLC method to detect the adulteration of black pepper powder with ground papaya seed was developed. Ethylene dichloride extracts showed a fluorescent spot at 366 nm at R_f 0.943 that proved to be a promising marker for the presence of papaya seed powder in black pepper powder even at a level of 20 g kg^?1. Partial purification by column chromatography followed by GC/MS analysis indicated that this fluorescent marker was a mixture of aldehydes such as n‐nonanal, 2‐decenal and trans‐2‐undecenal which were absent from black pepper. © 2001 Society of Chemical Industry     

Supercritical carbon dioxide extraction for identification of adulteration of black pepper with papaya seeds

Supercritical fluid extraction (SFE) using carbon dioxide was used to detect the adulteration of black pepper powder with ground papaya seed. Thin‐layer chromatography analysis of the SFE extracts showed a fluorescent band at 366 nm at R_f 0.172 that proved to be a promising marker for the presence of papaya seed powder in black pepper powder even at a level of 20 g kg^?1. The straight‐chain aldehydes n‐nonanal, n‐decanal and n‐dodecanal were tentatively identified by gas chromatography–mass spectrometry analysis as components of this fluorescent marker and were not present in black pepper extracts. Copyright © 2003 Society of Chemical Industry     

Sequence characterized amplified region markers: A reliable tool for adulterant detection in turmeric powder

Turmeric powder (Curcuma longa L.), an important medicinal spice product traded internationally, is subjected to adulteration by design or default with powders of related curcumin containing wild species like Curcuma zedoaria and Curcuma malabarica leading to toxicity and poor quality of the produce. The present study aims at development of specific, sensitive and reproducible Sequence Characterized Amplified Region (SCAR) markers to detect these adulterants in traded turmeric powder. Two putative RAPD markers, ‘Cur 01’ and ‘Cur 02’, generated by random primers OPA 01 and OPE 18 were identified as C. zedoaria/C. malabarica specific by comparative RAPD analysis of genuine turmeric and market samples of turmeric powder, C. zedoaria and C. malabarica. These specific RAPD markers were cloned and sequenced. Two pairs of SCAR primers were designed from the RAPD markers ‘Cur 01’ and ‘Cur 02’, respectively. Six market samples of turmeric powder and four simulated standards besides the genuine samples were analyzed using the specific SCAR markers. Both the SCAR markers detected the presence of C. zedoaria/C. malabarica adulteration in four market samples and all the simulated standards prepared in different concentrations. The two SCAR markers developed in the study would be potentially useful for the regulatory agencies to detect C. zedoaria/C. malabarica adulteration in traded turmeric powder. The analytical strategy being very simple could be used for large scale screening of turmeric powder samples intended for export and domestic uses.     

The determination of aflatoxins in spices by immunoaffinity column extraction using HPLC

Seventy‐five samples of different spices marketed in Turkey were purchased from bazaars, herbal shops and supermarkets. Equal amounts of paprika, chilli, black peppers and cumin were purchased and used to test and compare the amount of aflatoxin contamination. Two different analytical methods were examined for their efficacy by adding a known amount of aflatoxin to the blank samples of paprika. Twenty‐seven paprika, all the chilli powder and four ground black pepper samples were contaminated with aflatoxin B₁ in the range of 0.5–116.4, 1.6–80.4 and 0.3–1.2 μg kg^?1 respectively. Twenty‐three (30%) paprika and chilli powder samples were above the regulatory limits used in the European Union. No aflatoxin contamination was detected in the cumin samples at a detection limit of 0.2 μg kg^?1.     

In vitro protein digestibility of legumes cooked with spices

The present study was planned to investigate the effect of spices on in vitro protein digestibility in decorticated forms of bengal gram (Cicer arietinum), black gram (Phaseolus mungo), green gram (Phaseolus radiatus) and red gram (Cajanus indicus). The spices used were chilli (Capsicum annum), pepper (Pepper nigrum L.), coriander (Coriander sativum) and a mixture of these. Legumes were pressure cooked with 5.0% of freshly powdered spices and in vitro protein digestibility determined using pepsin and pancreatin enzymes by standard techniques. Samples without spices served as controls. The results reveal that protein content of legumes ranged from 20.5 to 23.0 g/100 g. The percent protein hydrolysed for legumes without spices were 63.4 for bengal gram, 65.8 for black gram, 60.0 for green gram and 55.4 for red gram. Casein sample could be hydrolysed to the extent of 78.8%. Chilli powder decreased digestibility significantly by 50, 78, 73, 60 and 69% in casein, bengal gram, black gram, green gram and red gram, respectively. Pepper exhibited a variable effect of altering the digestibility to 93% in casein and red gram, 106% in black gram and green gram and 98% in bengal gram, which were not significant. Coriander also decreased digestibility by 48, 76, 87, 77 and 73% and mixture of spices by 74, 91, 96, 96 and 82% in casein, bengal gram, black gram, green gram and red gram, respectively. It can be concluded that spices do influence in vitro protein digestibility in legumes to varying extent.     

Rhodamine B in spices determined by a sensitive UPLC-MS/MS method

Rhodamine B (RhB) is a banned food additive and has been classified as illegal colourant. Therefore, the risk of RhB contamination should be strictly monitored. In this study, a sensitive UPLC-MS/MS method was applied to monitor RhB in 292 various spices such as chilli, pepper and tomato products. The results showed 22.7% of chilli powder samples, 18.5% of pepper powder samples, 11.1% of chilli oil samples and 9.1% of pepper oil samples were contaminated with RhB. Chilli powder contained RhB up to 44,935 μg/kg with an average of 743 μg/kg, pepper powder up to 65.9 μg/kg with an average of 4.1 μg/kg, chilli oil up to 14.6 μg/kg with an average of 1.0 μg/kg and pepper oil up to 1.1 μg/kg with an average of 0.2 μg/kg, respectively. Considering the common consumption of chilli products and pepper products by so many consumers, RhB exposure is significant and should be decreased.     

Establishment of a standard reference material (SRM) herbal DNA barcode library of Vitex negundo L. (lagundi) for quality control measures

The majority of the population in the Philippines relies on herbal products as their primary source for their healthcare needs. After the recognition of Vitex negundo L. (lagundi) as an important and effective alternative medicine for cough, sore throat, asthma and fever by the Philippine Department of Health (DOH), there was an increase in the production of lagundi-based herbal products in the form of teas, capsules and syrups. The efficiency of these products is greatly reliant on the use of authentic plant material, and to this day no standard protocol has been established to authenticate plant materials. DNA barcoding offers a quick and reliable species authentication tool, but its application to plant material has been less successful due to (1) lack of a standard DNA barcoding loci in plants and (2) poor DNA yield from powderised plant products. This study reports the successful application of DNA barcoding in the authentication of five V. negundo herbal products sold in the Philippines. Also, the first standard reference material (SRM) herbal library for the recognition of authentic V. negundo samples was established using 42 gene accessions of ITS, psbA-trnH and matK barcoding loci. Authentication of the herbal products utilised the SRM following the BLASTn and maximum-likelihood (ML) tree construction criterion. Barcode sequences were retrieved for ITS and psbA-trnH of all products tested and the results of the study revealed that only one out of five herbal products satisfied both BLASTn and ML criterion and was considered to contain authentic V. negundo. The results prompt the urgent need to utilise DNA barcoding in authenticating herbal products available in the Philippine market. Authentication of these products will secure consumer health by preventing the negative effects of adulteration, substitution and contamination.     

A survey of the incidence and level of aflatoxin contamination in a range of imported spice preparations on the Irish retail market

The aflatoxin contents of 130 commercial spice preparations, including pepper, chilli, curry powder, cayenne, paprika, cinnamon, coriander, turmeric and cumin, were determined using high performance liquid chromatography (HPLC). Samples were obtained from various retail outlets in Ireland, including supermarkets, shops and market stalls. Aflatoxin B1 gave the highest incidence of contamination in spice preparations and was found in 20 of the 130 samples. The highest concentration of aflatoxin, 27.5 μg/kg, was detected in a sample of chilli powder; next highest was in a sample of cayenne pepper which contained 18.5 μg/kg. Five samples (3.8%), consisting of chilli, cayenne pepper and turmeric pepper, were above the regulatory limits of the European Union. Aflatoxin contamination was not detected in cumin or cinnamon samples at a level of quantitation (LOQ) <0.2, <0.1, <0.5, <0.3 μg/kg for B1, B2, G1 and G2, respectively.     

Degradation of the natural mutagenic compound safrole in spices by cooking and irradiation

Safrole was determined using gas-liquid chromatography in some common spices as star anise, cumin, black pepper and ginger. Safrole concentration in these spices was 9325, 3432, 955 and 500 mg · kg^?1, respectively. Black pepper was chosen to use in the following experiments. Using Ames-test with Salmonella TA 98 and TA 100 proved high cytotoxic effects due to pure safrole and black pepper volatile oil in both of them. The degradation of safrole was obvious after drying of the washed seeds of black pepper especially at 70 °C for 30 min or with sun-drying. Also, high irradiation doses (20 and 30 kGy) caused high degradation of more than 90% of the initial toxic concentration in black pepper. Whereas, microwave caused same effects at 75 s, but unfortunately, the powder was burned due to moisture absence. Boiling whole seeds or powder of black pepper during cooking for few minutes (1-5 min) were more efficient in decreasing safrole content. Finally, these results proved that the mutagenicity of some spices due to presence of safrole can be destructed during drying of the washed seeds or during cooking either with or without any additional treatment as irradiation. But irradiation of these spices became more necessary for using in some food industries as milk products to get more safe for human consumption.     

Prevalence of aflatoxin contamination in red chilli pepper (Capsicum annum L.) from India

The aflatoxin contamination of chilli pepper grown and marketed in Tamil Nadu, a southern Indian state, was assessed. Chilli samples were collected at different stages of the value chain and were quantified using the enzyme-linked immunosorbent assay (ELISA) test. Forty-two representative samples were collected from four districts identified as the hub for production, distribution, agro-processing industries and retail stores. In addition, interviews were conducted among the chilli farmers, vendors and agro-industrialists across the hubs to assess their knowledge on aflatoxin contamination and safe handling practices. The maximum aflatoxin content determined in the chilli pepper was 37.8 µg kg⁻¹. Almost 66.7% of samples collected from the retail outlets had aflatoxin values above 10 µg kg⁻¹. The total aflatoxin content in the samples collected across the value chain was in the range of 3.83 to 37.80 µg kg⁻¹. Statistical analysis on aflatoxin contents showed that there were significant differences (P < 0.05) between the districts representing different operations of value chain. The detected aflatoxin content was highest in samples collected from Dindigul district and least in Erode district. The results of the perception study showed that respondents into farming and trade activities had very little or no knowledge of aflatoxin contamination of chilli. The prevalence of unacceptable levels of aflatoxin in the chilli supply chain in the districts studied is probably due to tropical climatic conditions and poor handling practices of chilli.     

Molecular Identification of Economically Motivated Adulteration of Red Pepper Powder by Species-Specific PCR of Nuclear rDNA-ITS Regions in Garlic and Onion

Economically motivated adulteration (EMA) of food is a matter of great concern in Korea due to its unfavorable effects on public distribution orders. EMA of red pepper powder is typically done with a mixture of garlic (Allium sativum) and onion (Allium cepa), leading to a reduction in red pepper powder content. To establish a rapid and accurate detection method for EMA red pepper powder, we designed species-specific PCR primers based on the internal transcribed spacer regions of garlic and onion DNA. Among the examined DNA extraction methods, the DNA extraction kit with a silica gel column was found to be a time-saving, high-purity, and high-yield method. When the designed species-specific PCR primers were used for designed EMA red pepper powder with different mixture ratios of garlic and onion, the target ingredients were detected in a concentration-dependent manner. Furthermore, duplex PCR allowed specific simultaneous detection of garlic and onion. Real-time quantitative PCR assay was successfully used to detect garlic and onion quantitatively in binary mixed samples containing as little as 0.05 % of these components. When we examined EMA red pepper powder collected from the Ministry of Food and Drug Safety using a conventional PCR assay, the newly designed PCR primers successfully detected the target ingredients.     

Exposure assessment to Sudan dyes through consumption of artificially coloured chilli powders in India

In view of detection of Sudan dyes in chillies, the Spices Board of India formulated a mandatory testing programme in all chilli consignments exported from India. However, no surveillance data on use and levels of Sudan dyes in domestic chilli products are available in India. Hence chilli powders were monitored to check the magnitude of artificial coloration and the likely exposure assessment of Sudan dyes. Among 800 non‐branded, loose chilli powder samples, over 66% were found to employ artificial colouration while only 33% were free from Sudan dyes. None of the branded chilli powder samples was found to contain Sudan dyes. The maximum content of Sudan I noted was as high as 11.8 mg g^?1, which at the rough per capita consumption estimates of 0.5–1.0 g chilli powder per day amounts to an intake of 5.8–11.8 mg of Sudan I. This may lead to unwarranted health consequences, hence regular monitoring of the chilli powder samples is advisable to safeguard the health of unsuspecting consumers.     

Effect of Capsicum annuum (Red Sweet and Cayenne) and Piper nigrum (Black and White) Pepper Powders on the Shelf Life of Fresh Pork Sausages Packaged in Modified Atmosphere

ABSTRACT Capsicum annuum (red sweet and hot cayenne) and Piper nigrum (black and white) pepper powders were evaluated for inhibition of oxidative reactions and extension of the shelf life of fresh pork sausages packaged in a modified atmosphere. Sausages containing either red sweet pepper or cayenne pepper (0.1%, 0.5%, or 2%) or black or white pepper (0.1%, 0.5%, or 1%) were packaged in a modified atmosphere of 80% O₂+ 20% CO₂, stored for 16 d in the dark at 2 °C, and analyzed each 4 d for pH, CIE L*, a*, and b*, 2‐thiobarbituric acid‐reactive substances (TBARS), psychrotrophic aerobes, sensory discoloration, and off‐odor. Results demonstrated that Capsicum peppers (sweet red and hot cayenne) enhanced red color but failed to prevent discoloration, whereas they were very effective in inhibiting lipid oxidation, chiefly at the highest concentration used (2%), which resulted in a delay of off‐odor formation. Piper peppers (black and white) significantly delayed discoloration with small modification of sausage color; furthermore, they also inhibited lipid oxidation, which led to a delay of off‐odor formation, particularly in the form of black pepper. Besides this, all those spices inhibited microbial growth when added at the highest concentration (1%Piper and 2%Capsicum).     

Authentication of Small Berry Fruit in Fruit Products by DNA Barcoding Method

Small berry fruit products are gaining an expanded market due to their high nutrition value. However, the authenticity of products is challenged by adulteration and mislabeling. To establish an accurate and robust method for identifying both known and unknown fruit species in small berry fruit products, DNA barcoding technology based on Sanger sequencing was adopted. To overcome the influence of processing conditions on DNA recovery, mini‐barcodes of rbcL and ITS and a medium‐barcode of psbA‐trnH were applied. To identify ingredients in products containing mixed species, plasmid cloning was applied to separate mixed barcodes. The method established in this paper could detect 1% to 10% target species in mixed fruit juice.     

The use of reverse-phase C18-bonded silica for the trapping,concentration and analysis of headspace vapour from model organic compounds,banana pseudostem and black pepper

A study of recovery efficiency of a series of volatile compounds trapped on reverse-phase C₁₈-bonded silica has shown that reverse-phase silica is a useful addition to the existing range of adsorbents available for headspace volatile concentration and analysis. A gas chromatograph comparison of the airborne volatiles from black pepper (Piper nigrum) and banana pseudostem (Musa sp) recovered from reverse-phase silica, activated charcoal and Porapak Q traps shows no qualitative differences in the components present.     

Assessment of Microbiological Quality of Some Gamma Irradiated Indian Spices

Irradiated (10 kGy) and unirradiated pre-packed whole and ground spices including black pepper, red chilli, and turmeric were examined by six different laboratories for microbiological quality. No colony forming units (CFU) were reported in the largest quantity of irradiated spices used in the study by three out of six laboratories. The other three laboratories reported counts ranging between 0–90 CFU/g in irradiated samples. None of the six laboratories reported the presence of E. coli or B. cereus in the spices exposed to gamma irradiation. These data suggest that a standard plate count of 0–100 CFU/g and a count of zero CFU/g for E. coli and B. cereus be fixed for spices exposed to a 10 kGy dose of gamma rays.     

射频杀菌条件下黑胡椒颗粒中沙门氏菌的替代菌研究

为探究热杀菌过程中屎肠球菌NRRL B-2354作为沙门氏菌ATCC 14028替代菌的可行性,通过等温实验测定黑胡椒颗粒中屎肠球菌NRRL B-2354和沙门氏菌ATCC 14028的耐热性参数,并对黑胡椒颗粒（A_w=0.65）开展射频杀菌实验验证。等温实验结果表明,黑胡椒颗粒中屎肠球菌的耐热性高于沙门氏菌。射频杀菌实验中,将2 g接种微生物的黑胡椒颗粒真空包装后置于45 g黑胡椒中心位置,在40.68 MHz射频场中加热15 min,黑胡椒颗粒中沙门氏菌的减少量相较于屎肠球菌更多,证明在射频加热中屎肠球菌的耐热性高于沙门氏菌,可作为黑胡椒颗粒杀菌过程中沙门氏菌的合适替代菌。     

Aflatoxin B1 and aflatoxins in ground red chilli pepper after drying

In this study, 180 red chilli pepper (RCP) berry samples were obtained from two different croplands of Gaziantep and Kahramanmara? (Turkey) in August, September and October. RCP berry samples were dried under sunlight and grinded. Ground red chilli pepper (GRCP) samples were analysed for aflatoxins (AFs, sum of B₁, B₂, G₁ and G₂) and AFB₁ contamination. According to the results, in 49 of 180 samples, AFB₁ and in 37 samples, AFs were higher than legal limits. The lowest amounts of AFs and AFB₁ were obtained in August and the highest amounts in October. χ² analysis showed that there were no significant differences (p > 0.05) between cities among 3 months according to number of samples with AFs and AFB₁ above legal limits. According to the Duncan multiple-range test, there was no significant difference between all months. Strict measures are necessary to produce high-quality GRCP. RCP berry must be treated to reduce moulds before production of GRCP.