In silico assisted identification of peppery aroma compound ‘rotundone’ backbone genes from black pepper

Pepper is a major spice of commerce widely used both for its flavoring properties and as a traditional medicine in the orient. Pepper berries are commercially available in three main forms, namely, green pepper (dried unripe fruit), black pepper (cooked and dried unripe fruit), and white pepper (derived from ripe fruit seeds). Black pepper is the major spice of commerce and is one of the most common spices used in cuisines all over the world. Quality of pepper berries is decided both by their microbial status and the chemical parameters such as the content of essential oil, oleoresin, and piperine content. Various postharvest processing and preservation methods can have impact on the aforementioned parameters, thereby affecting the final quality of the product. Chemistry of black pepper has been extensively investigated. The characteristic aroma of the spice is mainly due to the inherent essential oils constituted mainly of terpenes along with other minor volatile constituents. Its spiciness/pungency has been shown to be due to the chemical compound piperine, an alkaloid. Black pepper is also rich in glycosides of phenolic acids and flavonol glycosides. Phenolic compounds, namely, 3,4-dihydroxy-phenyl ethanol glucoside and 3,4-dihydroxy-6-(N-ethyl amino) benzamide, have been identified in unripe green pepper berries and shown to contribute to the color of black pepper berries. Black pepper has been extensively used in traditional indigenous Asian system of medicine such as Ayurveda. The reported health benefits of black pepper include relieving sinus, asthma, and nasal congestion, reducing the risk of cancer, heart, and liver ailment and aiding in weight loss. It also aids in improving digestion, enhances bioavailability of food nutrients, improves cognitive functions, and reduces gastric mucosal damage and peptic ulcers due to its antioxidant and anti-inflammatory properties. While extensive studies have demonstrated the medicinal properties of black pepper, there is a need to have more focused studies on other pepper products, particularly green pepper. This is due to the fact that some of the newer phenolic compounds identified in green pepper and absent in black and white pepper may have novel pharmaceutical activities not reported so far. Thus, from a nutraceutical perspective besides black pepper, green pepper can also play a major role in prevention of diseases. This chapter focuses on the chemistry of black and green pepper with special emphasis on green pepper berries. Various quality parameters that can have an impact on the final acceptability of pepper products of commerce, effect of various postharvest processing methods, and quality control measures adopted will be discussed. The pharmaceutical properties of different pepper products will be discussed with a focus on the biological activities of the newer phenolic compounds identified in green pepper. Finally, the possible development of novel nutraceutical products from different pepper products for health promotion will be deliberated.

Spices are considered as a valuable food material owing not only to their special aroma, but also a myriad of nutritional and health benefits. Black pepper (Piper nigrum L.; Piperaceae) is known as the “king of spices”, being commonly used worldwide in its two forms: black and processed white pepper. The main goal of this study was to perform multi-targeted comparative metabolite profiling and fingerprinting approaches targeting primary and secondary metabolites using gas chromatography mass-spectrometry (GC-MS) post-silylation and ultra-performance liquid chromatography (UPLC-MS/MS) coupled to multivariate analyses and molecular networking. A total of 51 metabolites were annotated using GC-MS belonging to fatty acids/esters (9), alkaloids/nitrogenous (6), sugars (3), sugar alcohols (5), organic acids (15), alcohols (4), and aliphatic hydrocarbons (6) in addition to phenols (3). Fatty acids/esters were enriched in black and white pepper at ca. 23.4 mg g−1. Moreover, piperine was detected at higher levels in white pepper at 5.9 mg g−1 compared to 3.4 mg g−1 in black pepper. A total of 71 metabolites were annotated using UPLC-MS/MS, with piperamides as the most abundant class, of which 6 are first time to be detected in P. nigrum fruit “types A, E and O”. In addition, 7 fatty acids were recoded along 4 flavonoids exhibiting novel glycosidic linkage of kaempferol and apigenin. Furthermore, 5 hydroxycinnamic acids have been detected; some were identified for the first time from P. nigrum fruit. Clusters of fatty acids, flavonoids and phenylamides were detected by negative mode GNPS molecular networking, whereas clusters representing the majority of alkaloids were detected in positive mode. Assay of total phenolics and flavonoids revealed higher levels in black compared to white pepper, with values of 45.6 and 37.5 mg GAE per g for total phenolics and 9.4 & 8.5 mg RE per g for flavonoids, respectively. Assessment of antioxidant capacity using DPPH, ABTS scavenging assays, and FRAP assay revealed moderate effects at 49.79, 20.6, and 104.6 (black pepper), 29.0, 11.5, and 77.5 mg TE per g (white pepper), respectively. Moreover, black and white pepper extracts inhibited α-glucosidase enzyme with an IC50 of 0.77 and 0.62 mg mL−1, compared with acarbose.

As a medicinal and edible herb, Piper nigrum L. is abundant in volatile organic compounds (VOCs), and its essential oil has antibacterial properties. Notably, the aromatic profiles of black pepper (BP) and white pepper (WP) are markedly distinct. Consequently, it is essential to comprehensively characterize the VOCs of BP and WP, and analyze the differences in their VOCs and antibacterial efficacy. This study analyzed the VOCs of BP and WP using headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The fingerprint of HS-GC-IMS was established. Random forest analysis, orthogonal partial least squares discriminant analysis and heatmap were used to analyze differences in BP and WP. Furthermore, the antibacterial efficacy of volatile oils derived from BP and WP was assessed using the antibacterial zone diameter method. A total of 108 and 123 VOCs were identified by HS-GC-IMS and HS-SPME-GC-MS, respectively. The results of multivariate statistical analysis showed that the VOCs of BP and WP are significantly different. In contrast, WP has a milder smell than BP, while BP has a more pungent odor. Eight differential markers were selected. Both BP and WP had inhibitory effects on Staphylococcus aureus and Candida albicans. This study helps to decipher the flavor differences between BP and WP, and provides a material basis for their quality control and pharmacodynamics. It is beneficial to enhance its utilization within the domains of nutrition and traditional Chinese medicine.

Black pepper (Piper nigrum L.) is a tropic plant with large applications due to its commercial, economic, nutritional, and medicinal value. Pepper essential oil is a valuable product from pepper with diverse applications. The extraction of essential oil from pepper is a potential option to enhance its economic value. For this study, the pilot scale hydrodistillation process was employed to extract essential oil from two types of pepper from Vietnam, including whole black pepper (570 g/l) and light berries black pepper (300 g/l). Extraction parameters such as time, temperature, rate, and the material to water ratio were optimized. The quantitative analyses of the essential oils were performed by Gas Chromatography-Mass Spectrometry. The optimum yield was achieved up to 2.38% for light berries black pepper when the extract conditions were set up at a material-water ratio of 1:12.5 (kg/l) for 180 minutes under the extraction temperature of 130 °C. For whole black pepper (570 g/l), the maximum yield was 1.65% at distillation conditions: material-water ratio of 1:15 (kg/l), distillation time of 210 minutes, and temperature of 140 °C. The analysis of essential oils quality showed that all the sensory criteria, physical properties, and chemical composition of the two types of black pepper essential oils satisfied the regulation of ISO 3061: 2008 and TCVN 11881: 2017 black pepper essential oil. B-caryophyllene (28.28%; 21.94%), 3-carene (23.27%; 26%), and D-limonene (14,74%; 20.96%) were the three main components with the highest content in both types of black pepper essential oil compositions.

Black pepper (Piper nigrum L.) is one of the most popular and oldest spices in the world with culinary uses and various pharmacological properties. In order to satisfy the growing worldwide demand for black pepper, improved productivity of pepper is highly desirable. A primary constraint in black pepper production is the non-synchronous nature of flower development and non-uniform fruit ripening within a spike. The uneven ripening of pepper berries results in a high labour requirement for selective harvesting contributes to low productivity and affects the quality of the pepper products. In Malaysia, there are a few recommended varieties for black pepper planting, each having some limitations in addition to the useful characteristics. Therefore, a comparative study of different black pepper varieties will provide a better understanding of the mechanisms regulates fruit development and ripening. Plant hormones are known to influence the fruit development process and their roles in black pepper flower and fruit development were inferred based on the probe-based gene expression analysis and the quantification of the multiple plant hormones using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). In this study, jasmonic acid and salicylic acid were found to play roles in flowering and fruit setting, whereas auxin, gibberellin and cytokinins are important for fruit growth. Abscisic acid has positive role in fruit maturation and ripening in the development process. Distinct pattern of plant hormones related gene expression profiles with the hormones accumulation profiles suggested a complex network of regulation is involved in the signaling process and crosstalk between plant hormones was another layer of regulation in the black pepper fruit development mechanisms. The current study provides clues to help in elucidating the timing of the action of each specific plant hormone during fruit development and ripening which could be applied to enhance our ability to control the ripening process, leading to improving procedures for the production and post-harvest handling of pepper fruits.

Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are the two classes of non-coding RNAs (ncRNAs) present predominantly in plant cells and have various gene regulatory functions at pre- and post-transcriptional levels. Previously deemed as “junk”, these ncRNAs have now been reported to be an important player in gene expression regulation, especially in stress conditions in many plant species. Black pepper, scientifically known as Piper nigrum L., despite being one of the most economically important spice crops, lacks studies related to these ncRNAs. From a panel of 53 RNA-Seq datasets of black pepper from six tissues, namely, flower, fruit, leaf, panicle, root, and stem of six black pepper cultivars, covering eight BioProjects across four countries, we identified and characterized a total of 6406 lncRNAs. Further downstream analysis inferred that these lncRNAs regulated 781 black pepper genes/gene products via miRNA–lncRNA–mRNA network interactions, thus working as competitive endogenous RNAs (ceRNAs). The interactions may be various mechanisms like miRNA-mediated gene silencing or lncRNAs acting as endogenous target mimics (eTMs) of the miRNAs. A total of 35 lncRNAs were also identified to be potential precursors of 94 miRNAs after being acted upon by endonucleases like Drosha and Dicer. Tissue-wise transcriptome analysis revealed 4621 circRNAs. Further, miRNA–circRNA–mRNA network analysis showed 432 circRNAs combining with 619 miRNAs and competing for the binding sites on 744 mRNAs in different black pepper tissues. These findings can help researchers to get a better insight to the yield regulation and responses to stress in black pepper in endeavor of higher production and improved breeding programs in black pepper varieties.

To isolate, fermentatively evaluate and identify black pepper (Piper nigrum L.)-associated bacteria for the microbial decortication of fresh ripened berries and dried black pepper for preparation of off-odour-free white pepper. Among 45 bacterial isolates obtained from black pepper, seven of them were found to decorticate black pepper (>60%) and fresh pepper berries (98-100%) into white pepper within 5days of immersion in bacterial suspension. The 16S rRNA genes (1500-bp amplicon) of these bacteria were sequenced, and species identity was established by closest match in GenBank. Superior-quality white pepper was obtained with Bacillus subtilis (IISR WP 33, 34, 38), Bacillus licheniformis (IISR WP 43), Acinetobacter baumanii (IISR WP 35), Klebsiella pneumoniae (IISR WP 19) and Microbacterium barkeri (IISR WP25). The bacterial isolates were found to secrete multiple hydrolytic enzymes such as cellulase, pectinase, amylase, protease and xylanase. Bacterial cultures were deposited with International Depository Authority at Microbial Type Culture Collection, India, as patent deposits as prescribed in Budapest Treaty for microbial deposits. The white pepper, thus obtained from bacterial decortication process, was free from off-odour compound, especially skatole. Other biochemical constituents such as oleoresin, piperine and essential oils were found in the acceptable range. The bacterial decortication did not affect inherent constituents of pepper such as essential oil constituents, oleoresin and piperine content. One of the most significant findings of the work is identification of specific bacterial species for decortication of fresh berries or black pepper berries into value-added white pepper. This work paved way for developing a technological process for microbial decortication of fresh/black pepper for the production of superior-quality white pepper.

Black pepper (Piper nigrum L.) is one of the most widely used spices in food, beverage, cosmetics, and medicine. Black pepper production has suffered from various fungal diseases. Microbial biological control is an essential part of integrated disease management to reduce the heavy reliance on chemical fungicides. Trichoderma fungi comprise a large group of rhizocompetent filamentous fungi widely used in the biocontrol of plant pathogens. Three field surveys conducted on five black pepper farms in Belaga, Sarawak, identified three fungal diseases: yellowing, black berry, and foot rot. Based on the morphological and molecular characterisation, the identified fungal causal agents were Fusarium solani (yellowing disease), Colletotrichum gloeosporioides (black berry disease), and Phytophthora palmivora (foot rot disease). Twenty isolates of Trichoderma spp. were isolated from secondary forest and Biopark in Bintulu, Sarawak. Trichoderma isolates were characterised based on the morphological characteristics and molecular phylogenetic analysis using the rDNA internal transcribed spacer (ITS) region. Trichoderma isolates were separated into five distinct species, namely T. harzianum, T. virens, T. brevicompactum, T. tawa, and telomorphic Hypocrea lixii. Among the Trichoderma fungi, T. harzianum was the most frequently (65%) isolated species. Trichoderma harzianum (Isolates of TJ9, 10, and 16) showed antagonistic and inhibitory effects by 61 to 70% on in vitro mycelial growth against three common fungal pathogens of black pepper, P. palmivora, C. gloeosporioides, and F. solani. This study highlights the potential of using native Trichoderma fungi as biocontrol agents in the black pepper integrated disease management program.

Piper nigrum L. is a widely used spice because of its flavour and health effects. It is prepared as black and white pepper, according to the harvest time and inclusion of the outer skin. Pepper pericarp is usually considered waste when making white pepper. In this study, bioactive and flavour compounds and minerals in the pericarp of black pepper were determined to identify its applications. The pericarp contained total phenol, total flavonoid and piperine contents of 1421.95 ± 22.35 mg GAE/100 g, 983.82 ± 8.19 mg CE/100 g and 2352.19 ± 68.88 mg/100 g, respectively. There were higher levels of total phenols and total flavonoids in the pericarp compared with black pepper and white pepper. Piperine content was lower in the pericarp than in black pepper. The principal monoterpene compounds in the pericarp were α-pinene (9.2%), 2-β-pinene (14.3%), δ-3-carene (21.5%) and dl-limonene (18.8%), and the primary sesquiterpenes were α-copaene (5.1%) and caryophyllene (17.2%). The higher percentages of flavour compounds found in the pericarp would impart a more potent odour, and the pericarp exhibited higher minor and tiny differences based on electronic nose analysis. It had more minerals than black pepper and peeled black pepper.

<p>Indonesia is one of the major pepper (Piper nigrum L.) producer countries in the world. The main pepper products are distinguished into black and white pepper. Each region has a tradition to produce each of the products and influencing cultivation practices and cost structure. This research was aimed to analyze the cost of productions of black and white pepper and their relative competitiveness to the pepper price at the farm level with conventional and improved cultivation practices. The survey methods were used to obtain the primary data from respondents selected with the snowball sampling method. Lampung and Bangka Belitung Islands were chosen to represent the black and white peppers of smallholders, respectively. The result showed that the farms with conventional cultivation practices did not have sustainable relative competitiveness, indicating higher production costs than the lowest prices received in the long term. On the other hand, relative competitiveness was relatively better in farms that implemented improved cultivation practices. Therefore, to achieve sustainable relative competitiveness, pepper farms should apply improved cultivation practices. The relative competitiveness of white pepper was better than black pepper because the productivity of white pepper was higher even though the production cost was also a little bit higher than black pepper.</p>

Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.). Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.

A validated qualitative method using oleuropein as a robust biomarker to confirm ground black pepper (Piper nigrum L.) adulteration with olive (Olea europaea L.) by-products

Black pepper is one of the most valued and widely used spices in the world and dominates multi-billion dollar global spices trade. India is amongst the major producers, consumers and exporters of black pepper. In spite of its commercial and cultural importance, black pepper has received meagre attention in terms of generation of genomic resources. Availability of markers distributed throughout the genome would facilitate and accelerate genetic studies, QTL identification, genetic enhancement and crop improvement in black pepper. In this perspective, the sequence information from the recently sequenced black pepper (Piper nigrum) genome has been used for identification and characterisation of Simple Sequence Repeats (SSRs). Total 69,126 SSRs were identified from assembled genomic sequence of P. nigrum. The SSR frequency was 158 per MB making it, one SSR for every 6.3 kb in the assembled genome. Among the different types of microsatellite repeat motifs, dinucleotides were the most abundant (48.6%), followed by trinucleotide (23.7%) and compound repeats (20.62%). A set of 85 SSRs were used for validation, of which 74 produced amplification products of expected size. Genetic diversity of 30 black pepper accessions using 50 SSRs revealed four distinct clusters. Further, the cross species transferability of the SSRs was checked in nine other Piper species. Out of 50 SSRs used, 19 and 31 SSRs were amplified in nine and seven species, respectively. Thus the identified SSRs may have application in other species of the genus Piper where genome sequence is not available yet. Present study reports the first NGS based genomic SSRs in black pepper and thus constitute a valuable resource for a whole fleet of applications in genetics and plant breeding studies such as genetic map construction, QTL identification, map-based gene cloning, marker-assisted selection and evolutionary studies in Piper nigrum and related species.

Black pepper (Piper nigrum L.) is a tropical plant, best known for its fruit, used as a spice all around the world. The fruits of black pepper can be processed in various ways, so there are end products such as white, black, red, and green pepper. Black pepper contains many substances such as terpenes, alkaloids, lignans, phenylpropanoids, etc., which are responsible for some of the most important biological activities: antioxidant, antimicrobial, anticancer, anti-inflammatory, analgesic, antipyretic, hepatoprotective, bio-enhancing and enzyme inhibitory activities. These activities have also been proven in clinical studies, and probably the future of black pepper research should be based on discovering the most effective way to use the active compounds of pepper for the development of herbal drugs with fewer contraindications than standard drugs. Further preclinical and clinical studies are needed to prepare and apply phytoformulations based on the black pepper isolates.

Black pepper has a scientific name of‘piper nigrum’is an unripe fruit of peppercorn which is dried, it is a flavor enhancer in food, besides also has health benefits, it contains bioactive compound piperine which adds a spicy taste to it being an antioxidant it reduces risk of various life-threatening illnesses (atherosclerosis, cardiovascular disease and neurological conditions) and also increases bioavailability of various nutrients, piperine at any given dose weakens memory impairment, in low dose piperine potentially increases neuronal density in hippocampus. Black pepper boosts HCL in stomach and has antispasmodic properties. Active compounds present play a significance in stimulating leucocytes increasing immunity against various pathogens. It is given along with iron supplements to improve its bioavailability without any adverse reaction. Piper Nigrum given in significant amount enhances learning and memory inadequacy related to AlCl, also exhibits anticholinesterase activity preventing nerve degeneration. In the ongoing COVID scenario biologically active compounds in piper nigrum: piperdardiine and piperanine are markedly effective against COVID‐19, hence is preferred as a medication. Black pepper rectifies the lipid profile, along with degree of total cholesterol, LDL and triglyceride in patients with cardiovascular diseases, piper nigrum being prosperous in having constituents of vanadium in it contributes in boosting and recovering fron cardiac function after a myocardial infarction and dealing with pressure overload–induced hypertrophy by inhibiting protein tyrosine phosphatases. Piper nigrum can boost melanocyte proliferation and bring back pigmentation to depigmented skin, therefore this could possibly be the treatment for vitiligo. Alkaloid constituent of black pepper, bring to bear anticancer properties in a wide range of malignancies.