Analysis by HPLC and LC/MS of pungent piperamides in commercial black, white, green, and red whole and ground peppercorns

Pepper plants accumulate pungent bioactive alkaloids called piperamides. To facilitate studies in this area, high-performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry methods were developed and used to measure the following piperamides in 10 commercial whole (peppercorns) and in 10 ground, black, white, green, and red peppers: piperanine, piperdardine, piperine, piperlonguminine, and piperettine. Structural identification of individual compounds in extracts was performed by associating the HPLC peak of each compound with the corresponding mass spectrum. The piperanine content of the peppers (in mg/g piperine equivalents) ranged from 0.3 for the ground white pepper to 1.4 in black peppercorns. The corresponding range for piperdardine was from 0.0 for seven samples to 1.8 in black peppercorns; for four isomeric piperines, from 0.7 for red to 129 in green peppercorns; for piperlonguminine, from 0.0 in red peppercorns to 1.0 in black peppercorns; and for piperyline, from 0.9 in ground black pepper to 5.9 for red peppercorn. Four well-separated stereoisomeric forms of piperettine with the same molecular weight were present in 19 peppers. The sums of the piperamides ranged from 6.6 for red to 153 for green peppercorns. In contrast to large differences in absolute concentrations among the peppers, the ratios of piperines to total piperamide were quite narrow, ranging from 0.76 for black to 0.90 for white peppercorns, with an average value of 0.84 +/- 0.04 ( n = 19). Thus, on average, the total piperamide content of the peppers consists of 84% piperines and 16% other piperamides. These results demonstrate the utility of the described extraction and analytical methods used to determine the wide-ranging individual and total piperamide contents of widely consumed peppers.     

UV spectrophotometric determination of piperine in pepper preparations: collaborative study

Eight collaborating laboratories performed replicate analyses for piperine on 5 samples representing pepper raw spice, oleoresins, and soluble seasonings. Piperine is extracted into ethylene dichloride and measured at maximal absorbance 342-345 nm with a UV light source. Piperine content is calculated using an absorbance factor derived from piperine. Intralaboratory coefficients of variation (CVo) ranged from 0.5 to 3.1%; interlaboratory coefficients of variation (CVx) ranged from 3.0 to 5.8%. The method has been adopted as an official method of the American Spice Trade Association and as an official first action method by AOAC.     

Physicochemical and microbiological qualities of steamed and irradiated ground black pepper (Piper nigrum L.)

The effects of steam and irradiation treatments on the physicochemical properties (moisture content, pH, extractable yield, reducing sugar, soluble pigment, antioxidant activity, piperine, Hunter's color, and sensory attributes) and microbiological quality (total aerobic bacteria, coliforms, and yeasts and molds) of ground black pepper stored at refrigerated and room temperatures for 6 months were compared and evaluated. Irradiation resulted in a higher microbial reduction in pepper, with minimal effects on the proximate composition, functional components, color, and sensory attributes of the spice. Steamed peppers appeared darker, and a considerable decrease in the piperine content was observed after treatment and storage. This study illustrates that irradiation is a better decontamination method than steam treatment in eliminating microorganisms without apparently affecting the quality of the powdered spice. Storage at 4 degrees C enhanced the microbial quality and minimized the loss of piperine content in ground black peppers.     

Effect of storage on the essential oil composition of Piper nigrum L. fruits of different ripening states

The qualitative and quantitative composition of the essential oil from black, green, and white pepper was determined by using a simultaneous distillation and extraction micromethod for oil isolation and gas chromatography (GC)/flame ionization detection (FID) and GC/mass spectrometry (MS) analysis techniques. The most abundant compounds in pepper oils were (E)-beta-caryophyllene (1.4-70.4%), limonene (2.9-38.4%), beta-pinene (0.7-25.6%), Delta-3-carene (1.7-19.0%), sabinene (0-12.2%), alpha-pinene (0.3-10.4%), eugenol (0.1-41.0%), terpinen-4-ol (0-13.2%), hedycaryol (0-9.1%), beta-eudesmol (0-9.7%), and caryophyllene oxide (0.1-7.2%). Green pepper corn obtained by a sublimation drying method gave more oil (12.1 mg/g) and a much higher content of monoterpenes (84.2%) in the oil than air-dried green pepper corn (0.8 mg/g and 26.8%, respectively). The oil from ground black pepper contained more monoterpenes and less sesquiterprnes and oxygenated terpenoids as compared to green and white pepper oils. After 1 year of storage of pepper samples in a glass vessel at room temperature, the amount of the oils isolated decreased, the content of terpenes decreased, and the amount of oxygenated terpenoids increased. Differently from other pepper samples, 1 year storage of green pepper corn raised the oil amount more than twice of both drying methods.     

Kinetics of light-induced cis-trans isomerization of four piperines and their levels in ground black peppers as determined by HPLC and LC/MS

The pungent compounds piperine and isomers thereof, secondary metabolites present in black and white pepper fruit, undergo light-induced isomerizations. To facilitate studies in this area, an HPLC method has been developed for analysis and isolation of the following four possible piperine-derived photoinduced isomers: piperine, isopiperine, chavicine, and isochavicine. The limits of detection (LOD) estimated from calibration plots were approximately 15-30 ng for each isomer. Reproducibilities of the analyses were excellent, and recoveries of spiked samples were as follows (average +/- SD; n = 3): chavicine, 98.4 +/- 2.1%; isopiperine, 96.2 +/- 3.2%; piperine, 104 +/- 3.8%; isochavicine, 98.9 +/- 3.0%. To determine the kinetics of these isomerizations, fluorescent light, sunlight, and UV radiation at 254 nm was used to induce cis-trans geometric isomerization as a function of light intensities and time of exposure determined with the aid of high-performance liquid chromatography (HPLC) and liquid chromatography with diode array UV detection-mass spectrometry (LC-DAD/MS). HPLC was also used to determine the distribution of the isomers in four commercial ground black pepper products used as spices in culinary practice. Isomerization increased with light intensities and time of exposure and leveled off at the so-called photostationary phases. The piperine levels of the four products were quite similar, ranging (in wt %) from 10.17 to 11.68. The amounts of the other three isomers ranged from 0.01 to 0.07 of the total for chavicine; from 0.15 to 0.23 for isopiperine; and from 0.37 to 0.42 for isochavicine. The results establish the utility of the HPLC method for simultaneous analysis of the four isomers both in pure form and in black pepper extracts. The dietary significance of the results is discussed.     

Binding of the pepper alkaloid piperine to bovine beta-lactoglobulin: circular dichroism spectroscopy and molecular modeling study

The pepper alkaloid piperine is a nontoxic, natural dietary compound with a broad range of physiological activity. The present work is the first demonstration of its interaction with a mammalian protein. Circular dichroism (CD) spectroscopy was used to reveal and analyze the binding of piperine to a lipocalin protein. Induced CD spectra measured in pH 7.7 phosphate buffer at 37 degrees C demonstrated reversible, non-covalent association of piperine with bovine beta-lactoglobulin (BLG), the major whey protein in milk. The binding parameters (K(a) approximately 8 x 10(4) M(-1), n = 0.8) determined from the CD titration data showed no significant differences between the piperine binding properties of the two main genetic variants of BLG (A and B). The vanishing extrinsic CD signal obtained upon acidification of the piperine-BLG sample solution (Tanford transition) suggested that the ligand binds in the central hydrophobic cavity of the beta-barrel. The cavity binding concept was further supported by a CD displacement experiment using palmitic acid, the well-known hydrophobic ligand of BLG. Molecular docking calculations showed that piperine can be efficiently accommodated within the calyx of BLG. Additional molecular modeling calculations indicated that the beta-barrel of human tear lipocalin, human serum retinol binding protein, and human neutrophil gelatinase associated lipocalin might also accommodate a piperine molecule.     

Characterization of odorants causing an atypical aroma in white pepper powder (Piper nigrum L.) based on quantitative measurements and orthonasal breakthrough thresholds

Application of the aroma extract dilution analysis on an extract of white pepper powder showing an intense fecal, cowshed-like off-flavor revealed 3-methylindole (fecal, swine-manure) and 4-methylphenol (fecal, horse-like) with the highest flavor dilution (FD) factors among the 22 odor-active compounds detected. In addition, high FD factors and/or undesirable odor qualities suggested 3-methylphenol (phenolic), butanoic acid (cheese-like), and 2- and 3-methylbutanoic acid (cheese-like) as well as pentanoic acid and hexanoic acid (cheese-like odors) as contributors to the malodor. Although the intensities of the off-note were clearly different in 50 commercial samples of white pepper, quantitation of 3-methylindole and 3- and 4-methylphenol as well as of the five short-chain acids by means of stable isotope dilution assays showed similar concentrations in most of the samples. Storage of a freshly ground white pepper powder for up to 7 months revealed a significant decrease in the typical odor qualities of white pepper and an increase in the fecal odor note with storage time. Because the concentrations of the odorants mentioned above were not much changed during storage, possibly very volatile odorants, such as alpha-pinene, which are able to mask the malodor, are lost during storage of, in particular, pepper powders. On the basis of odor activity values, which were calculated using breakthrough thresholds, in particular, 3-methylindole, 4-methylphenol, 3-methylphenol, and butanoic acid could be suggested as the main sources of the fecal off-flavor.     

Structural and sensory characterization of key pungent and tingling compounds from black pepper (Piper nigrum L.)

To gain a more comprehensive knowledge on whether, besides the well-known piperine, other compounds are responsible for the pungent and tingling oral impression imparted by black pepper, an ethanol extract prepared from black pepper (Piper nigrum L.) was screened for its key sensory-active nonvolatiles by application of taste dilution analysis (TDA). Purification of the compounds perceived with the highest sensory impact, followed by LC-MS and 1D/2D NMR experiments as well as synthesis, led to the structure determination of 25 key pungent and tingling phytochemicals, among which the eight amides 1-(octadeca-2E,4E,13Z-trienyl)piperidine, 1-(octadeca-2E,4E,13Z-trienyl)pyrrolidine, (2E,4E,13Z)-N-isobutyl-octadeca-2,4,13-trienamide, 1-(octadeca-2E,4E,12Z-trienoyl)-pyrrolidine, 1-(eicosa-2E,4E,15Z-trienyl)piperidine, 1-(eicosa-2E,4E,15Z-trienyl)pyrrolidine, (2E,4E,15Z)-N-isobutyl-eicosa-2,4,15-trienamide, and 1-(eicosa-2E,4E,14Z-trienoyl)-pyrrolidine were not yet reported in literature. Sensory studies by means of a modified half-tongue test revealed recognition thresholds ranging from 3.0 to 1150.2 nmol/cm2 for pungency and from 520.6 to 2162.1 nmol/cm2 for the tingling orosensation depending on their chemical structure.     

临夏花椒主要病虫害及综合防控措施

针对临夏地区花椒病虫害呈现出多发、突发、频发、局部暴发等特点,为明确临夏地区花椒病虫害的发生规律,从而建立和推广病虫害绿色防控技术体系,以期为临夏花椒病虫害诊断和绿色防控技术提供参考和科学指导,推进花椒产业绿色可持续发展。调研总结了临夏花椒黑胫病、干腐病、炭疽病、煤污病、黑斑病、木腐病、锈病、白粉病等8种主要病害的病原及症状、传播和发病条件,以及棉蚜、山楂叶螨、铜色跳甲、红胫跳甲、瘿蚊、蚱蝉等6种主要虫害的形态特征、生活习性以及对枝梢、果实等的为害症状,提出了临夏花椒病虫害综合防控措施。     

Role of the fermentation process in off-odorant formation in white pepper: on-site trial in Thailand

In white pepper samples, directly taken from a retting batch at a pepper production plant in Thailand, 3-methylindole, 4-methylphenol, 3-methylphenol, and butanoic acid, recently confirmed to be responsible for the characteristic fecal off-odor frequently detected in white pepper powders, were quantified by stable isotope dilution analyses. The results clearly showed that, in particular, 3-methylindole (fecal, swine-manure-like), 4-methylphenol (fecal, horse-like), and butanoic acid (cheese-like) were biochemically formed during retting, thus indicating that fermentation is the crucial step for off-odorant formation during white pepper processing. Model fermentation experiments performed with different manufacturing regimens revealed that white pepper, containing no substantial amounts of these odorants, can be produced from ripe starting material by a short fermentation under water and with frequent exchange of water. The overall aroma of such pepper was superior as compared to the aroma of white pepper produced according to the traditional procedure.     

Influence of colored plastic mulch on soil temperature,growth, nutrimental status,and yield of bell pepper under shade house conditions

The aim of this study was to determine if plastic mulch films may have positive effects on bell pepper plants grown under shade house conditions. The experimental design was split-plot, where large plots contained four plastic mulch films (black, aluminum, silver, and white) compared to bare soil and small plots contained two bell pepper cultivars. Plant shoot dry weight was higher in plastic mulch treatments than in bare soil. The net photosynthesis rate in the cultivar SWG-46 exceeded in net photosynthesis with respect to the cultivar SWG-42 in one of three sampling dates. Nutrients in leaf showed a similar behavior in plastic mulch treatments than the control but the total content of nitrogen (N), potassium (K), and sulfur (S) was increased. The total yield was higher in black plastic mulch than in the other plastic mulch colors and lowest in the control.     

Antioxidant systems and their relationship with the response of pepper fruits to storage at 20 degrees C

Fresh peppers (Capsicum annuum L., variety California) in their green and red ripe stages were stored at 20 degrees C for 7 and 19 days to determine the effects of storage on whole fruit antioxidant capacity (TAA) and ascorbate (ASC) content, as well as on some antioxidant enzyme activities, such as catalase (CAT), superoxide dismutase (SOD), and those of the ASC-glutathione cycle. At least one Mn-SOD, two Fe-SODs, and three CuZn-SODs were detected in the fruit extract after native polyacrylamide gel electrophoresis. All of the SOD isozymes and glutathione reductase had higher activity levels in the red control fruits than in the green fruits, whereas the activities of monodehydroascorbate and dehydroascorbate reductase were higher in green fruits. Ascorbate peroxidase (APX) was found to be similar in both fruits. SODs, CAT, and APX seem to be involved in pepper fruit ripening and senescence during storage at 20 degrees C, perhaps influencing the active oxygen species levels in the fruit. TAA, as well as the ASC content, was higher in red peppers than in green, and storage increased the ASC in both green and red fruits.     

Rapid spectrophotometric determination of red and yellow isochromic carotenoid fractions in paprika and red pepper oleoresins

A rapid method has been developed for the determination of the red (R) and yellow (Y) isochromic carotenoid pigments fractions in paprika and oleoresins, based on UV-visible spectrophotometric measurement at two characteristic wavelengths and application of the Lambert-Beer law for multicomponent mixtures. The wavelengths 472 and 508 nm were selected as the most appropriate for simultaneous quantification of these fractions in the acetone extract of pigments. Experimental determination of the specific absorption coefficients (epsilon) for the two pigment fractions (R, Y) at 472 and 508 nm yielded equations to calculate the concentration of the two fractions, the total pigment content, and the ratio between the two fractions. The error in the determination of the isochromic fractions by the proposed spectrophotometric method was <5% when the results were compared with those obtained by HPLC analysis. The method can be applied to the direct extract of pigments, thereby avoiding saponification and minimizing errors from pigment degradation and sample manipulation as well as shortening the time of analysis (5 min in the case of oleoresins).     

Hydrophobic grid membrane filter/MUG method for total coliform and Escherichia coli enumeration in foods: collaborative study

Twenty-four laboratories participated in a collaborative study to validate a hydrophobic grid membrane filter (HGMF) method incorporating the use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) for enumeration of total coliform and Escherichia coli bacteria in foods by comparing its performance against the AOAC 3-tube MPN method (46.013-46.016). Raw milk, raw ground poultry, whole egg powder, cheese powder, and ground black pepper were included in the study. The total coliform methods did not differ significantly, except that the 3-tube method detected a significantly higher level of total coliforms than did the HGMF method in the ground black pepper. Conversely, the HGMF/MUG E. coli method detected significantly higher numbers of E. coli present in the egg powder, cheese powder, and ground black pepper samples, while not differing significantly from the 3-tube method for the raw milk and raw ground poultry samples. The overall confirmation rate of MUG-positive colonies isolated using the HGMF method was 99.5%. The hydrophobic grid membrane filter/MUG method has been adopted official first action as an additional method to AOAC official final action method 46.030-46.034.     

Spice extracts as dose-modifying factors in radiation inactivation of bacteria

Three spices, chili, black pepper, and turmeric, were tested for the effect of their aqueous extracts on the sensitivity of three bacteria, Escherichia coli, Bacillus megaterium, and Bacillus pumilusspores, to gamma-radiation. It was found that the extracts of the three spices offered protection to these organisms against inactivation by gamma-radiation. These spice extracts were also tested for their protection of naked plasmid DNA. Radiation-induced degradation of plasmid pUC18 DNA was reduced in the presence of the spice extracts. The maximum protection was offered by the chili extract followed by that of black pepper and turmeric. The two known antioxidants, curcumin and piperine from turmeric and black pepper, respectively, were shown to protect the plasmid DNA from the degradation by gamma-radiation. Experiments with the plasmid pUC18 DNA indicated that the spice extracts probably protected microorganisms by protecting their DNA. These studies indicated the importance of spices among ingredients in food as dose-modifying factors during radiation processing.     

双螺旋对辊式辣椒收获装置的设计与试验

针对色素辣椒采收需求大,人工采收困难,采收效率低,破损率高等问题,该研究设计了一种双螺旋对辊式辣椒收获装置。首先通过对辣椒与螺旋钢棒接触点进行受力分析,确定影响采收性能的主要因素,并通过单因素试验确定优化试验中各因素选取范围。并以打完脱叶剂2 d后,辣椒茎秆含水率≤40%的新疆巴州焉耆县色素辣椒为试验对象,以采净率和破损率为试验指标,以工作速度、对辊转速、对辊间距和对辊螺距为试验因素,进行四因素五水平正交中心组合优化试验;运用Design-expert 10软件对试验结果进行参数优化,通过验证试验对优化后的参数进行验证。试验结果表明:当工作速度为2.1 km/h,对辊转速为142 r/min,对辊间距为24.3 mm,对辊螺距为10 cm时,采净率为98.7%,破损率为3.46%,满足色素辣椒收获机田间作业要求。研究结果可为色素辣椒收获机的设计和优化提供参考。     

From wine to pepper: rotundone, an obscure sesquiterpene, is a potent spicy aroma compound

An obscure sesquiterpene, rotundone, has been identified as a hitherto unrecognized important aroma impact compound with a strong spicy, peppercorn aroma. Excellent correlations were observed between the concentration of rotundone and the mean 'black pepper' aroma intensity rated by sensory panels for both grape and wine samples, indicating that rotundone is a major contributor to peppery characters in Shiraz grapes and wine (and to a lesser extent in wine of other varieties). Approximately 80% of a sensory panel were very sensitive to the aroma of rotundone (aroma detection threshold levels of 16 ng/L in red wine and 8 ng/L in water). Above these concentrations, these panelists described the spiked samples as more 'peppery' and 'spicy'. However, approximately 20% of panelists could not detect this compound at the highest concentration tested (4000 ng/L), even in water. Thus, the sensory experiences of two consumers enjoying the same glass of Shiraz wine might be very different. Rotundone was found in much higher amounts in other common herbs and spices, especially black and white peppercorns, where it was present at approximately 10000 times the level found in very 'peppery' wine. Rotundone is the first compound found in black or white peppercorns that has a distinctive peppery aroma. Rotundone has an odor activity value in pepper on the order of 50000-250000 and is, on this criterion, by far the most powerful aroma compound yet found in that most important spice.     

基于机器视觉技术的尖椒冠层SPAD值测定仪的开发

为了快速准确地测量尖椒的冠层植被指数值,该文在理论分析的基础之上,开发了一套基于机器视觉技术的用于测量尖椒冠层SPAD(soil and plant analyzer development,SPAD)值的仪器。该仪器利用步进电机控制载有绿波段和近红外波段两片滤光片的转台,实现滤光片在CCD相机镜头前的切换,从而得到冠层的绿色波段图像和近红外波段图像。制作了近红外反射率在尖椒冠层和土壤背景之间的参照板,并以其为阈值结合冠层的近红外图像来分离尖椒冠层和土壤背景。通过建立图像灰度值和反射率之间的动态模型,计算作物冠层在相应波段的反射率,得到冠层植被指数,将作物冠层的SPAD值作为对比进行分析。经过试验建立该仪器测得的冠层植被指数GNDVI(green normalized difference vegetation index,GNDVI)值和冠层实际SPAD值之间的模型,结果表明两者之间具有较高的相关性,决定系数R2=0.8768。表明该仪器适用于尖椒冠层SPAD值的测定。