Fermented Plant Material Research Articles

Genome and functional diversity of Leuconostoc mesenteroides from different habitats and geographic locations

Leuconostoc mesenteroides is widely used in the food industry because of its excellent exopolysaccharide and bacteriocin production capacity. In this study, 178 isolates from dairy products, fermented plant material and the human gut were analyzed by comparative genomics. Firstly, from the perspective of genomic characteristics analysis, we found that the isolates from fermented plant materials have large genomes. In addition, the genomic characteristics of European starter strains are highly diverse, which indicates that gene simplification or decline has occurred in a specific environment. Secondly, the population genetic diversity of Leu. mesenteroides was analyzed based on the whole genome, and it was found that the flora structure had obvious separation source and regional correlation. Leu. mesenteroides in China, Sichuan and Gansu are closely related, and the genetic distance of starter cultures in Europe is close. The ANOVA analysis of the genomic characteristics of the strain and environmental factors showed that the genomic characteristics of the strain were significantly different in different environments, that is, Leu. mesenteroides had habitat adaptability. Finally, through functional annotation, it was found that the environment also affected the functional genome of the isolated strain. Isolates from fermented plant material had unique genes related to α-1, 3-galactosyltransferase and β-galactosidase. Dairy isolates from China had more α-1, 6-mannosyltransferase, while those from northern Europe lacked genes related to maltose phosphorylase. Furthermore, we found that 85% of isolates had bacteriocin-producing genes. This study shows that Leu. mesenteroides has great genomic plasticity and high versatility, and provides a solid genetic foundation for its subsequent development and use.

Superwetting membrane-based strategy for high-flux enrichment of ethanol from ethanol/water mixture.

Ethanol, which can be scalable produced from fermented plant materials, is a promising candidate to gasoline as the next-generation liquid fuel. As an energy-efficient alternative to distillation, membrane-based strategies including pervaporation and reverse osmosis have been developed to recover ethanol from fermentation broths. However, these approaches suffer the drawback of low separation flux. Herein, we report a superwetting membrane system to enrich ethanol from water in a high-flux manner. By synergistically regulating surface energy of the solid porous membrane and hydration between an additive inorganic potassium salt and water, concentrated ethanol can rapidly wetting and permeate the porous membrane, with the salt solution being blocked. Using this newly developed superwetting membrane system, we can achieve fast enrichment of ethanol from water, with flux of two orders magnitude higher than that of pervaporation and reverse osmosis membranes.

BACTERIOCIN ASSOCIATED GENES IN FRENCH AND UKRAINIAN FERMENTED VEGETABLE ISOLATES OF LACTIPLANTIBACILLUS PLANTARUM

Lactiplantibacillus plantarum species is known for the high diversity of the loci encoding bacteriocin (plantaricin) production. The aim of this work was to study the distribution of pln genes in French and Ukrainian isolates of L. plantarum originated from fermented plant material. PCR with the primers to plnA, plnB, plnC, plnD, plnEF, plnI, plnK, plnJ, plnG, plnN, plnW genes was performed to find out the peculiarities of L. plantarum strains. French and Ukrainian isolates shared none of the identical combinations of genes from plantaricin regulon indicating the diversity of pln loci in L. plantarum of fermented plant origin from the distant geographical regions. Certain combinations of genes from plantaricin regulon could be proposed as indicators marking the geographical origin of L. plantarum strains. Among the studied isolates, some resembled the described in the literature L. plantarum strains, and others contained unique combinations of pln genes.

Skin Barrier-enhancing, Antiwrinkle, and Antimelanogenic Effects of Probiotic Lysates Composed of Nucleotides

Several previous studies have investigated the skin aging prevention effects of ceramide, hyaluronic acid, and natural or fermented plant materials. Recently, oral administration and dermal application of probiotics or probiotic lysates have shown antiaging effects. The purpose of this study is to optimize the preparation of probiotic lysates with a high concentration of nucleotides and to confirm the effects of probiotic lysates on the skin. Probiotic lysates were prepared by heating at 121°C for various periods with adding of sodium hyaluronic acid. Probiotic lysates of <i>Bifidobacterium longum</i> HDB7072, <i>Lactobacillus paracasei</i> HDB1196, and <i>Lactobacillus acidophilus</i> HDB1014 were applied to normal human epidermal keratinocytes (NHEKs), fibroblast cells, and B16F1 cells, respectively. Cell viability, antioxidant effects, and mRNA expression were evaluated by using MTT assays, DPPH assays, and qRT-PCR. Probiotic lysates prepared by heating the culture medium at 121°C for 2 h with 0.5% sodium hyaluronic acid showed the highest nucleotide concentration. In the three tested skin cells, the cell viability of filtered lysates was similar or higher to that of unfiltered lysates. HDB7072 lysates increased filaggrin expression in NHEKs. HDB1196 lysates showed DPPH radical-scavenging and antiwrinkle effects through the downregulation of matrix metalloproteinase-1 and upregulation of collagen type 1 in fibroblasts. HDB1014 lysates had antioxidant and antimelanogenic effects in B16F1 cells. Cell wall-removed probiotic lysates could be used as novel ingredients to improve skin aging and skin barrier issues.

High-temperature oxidation reduces the bitterness of honeybush infusions depending on changes in phenolic composition

Honeybush tea infusions, especially those prepared from Cyclopia genistoides, can be unacceptably bitter, given the association of this herbal tea with sweet taste. Infusions prepared from both ‘fermented’ (high-temperature oxidised) Cyclopia longifolia and C. genistoides contain high levels of the bitter xanthone, mangiferin, however, C. longifolia is generally less bitter than C. genistoides. The effect of phenolic changes during fermentation on the bitterness of infusions was determined for both species. Bitterness was reduced by 34–68% for C. genistoides and 55–86% for C. longifolia. Fermentation affected the phenolic composition of the two species differently, notably the ratio of mangiferin to isomangiferin remained higher for C. genistoides. Bitterness prediction models for two data sets of infusions (126 fermented and green plant material; 122 fermented plant material) including variable selection based on phenolic content gave good performance (RMSECV < 5). Nine compounds, depending on the data set, were determined to be important for the models, including xanthones, benzophenones, flavones and flavanones. Mangiferin, two tetrahydroxyxanthone-di-O,C-hexose isomers, 3-β-d-glucopyranosyliriflophenone, vicenin-2 and scolymoside were common to both models. Future application of the model would be to screen large numbers of genotypes for the breeding of selections with low bitterness potential based on phenolic composition.

Genome-based reclassification of Weissella jogaejeotgali as a later heterotypic synonym of Weissella thailandensis.

Members of the genus Weissella are mostly found in fermented plant material. Among the Weissella species, two species, Weissella thailandensis and Weissella jogaejeotgali, were isolated from foods fermented from marine animals. The two species showed a high level of 16S rRNA gene similarity (99.39 %), whereas they exhibited a moderate level of DNA-DNA hybridization relatedness (63.9 %) in an earlier study. In this study, we determined the whole genome sequence of W. thailandensis KCTC 3751T and compared it to those of W. jogaejeotgali FOL01T and other related species. The average nucleotide identity value between the type strains of W. thailandensis and W. jogaejeotgali was 96.4 %, which is clearly higher than the cut-off proposed for bacterial species. We, therefore, propose to reclassify W. jogaejeotgali as a later heterotypic synonym of W. thailandensis.

Antibiotic Resistance of LACTOBACILLUS Strains.

The study provides phenotypic and molecular analyses of the antibiotic resistance in 20 Lactobacillus strains including 11 strains newly isolated from fermented plant material. According to the results of disc diffusion method, 90% of tested lactobacilli demonstrated sensitivity to clindamycin and 95% of strains were susceptible to tetracycline, erythromycin, and rifampicin. Ampicillin and chloramphenicol were found to inhibit all bacteria used in this study. The vast majority of tested strains revealed phenotypic resistance to vancomycin, ciprofloxacin, and aminoglycosides. Most of Lactobacillus strains showed high minimum inhibitory concentrations (MICs) of cefotaxime, ceftriaxone, and cefazolin and therefore were considered resistant to cephalosporins. All the strains exhibited multidrug resistance. The occurrence of resistance genes was associated with phenotypic resistance, with the exception of phenotypically susceptible strains that contained genes for tetracycline (tetK, tetL) and erythromycin (ermB, mefA) resistance. The vanX gene for vancomycin resistance was among the most frequently identified among the lactobacilli (75% of strains), but the occurrence of the parC gene for ciprofloxacin resistance was sporadic (20% of strains). Our results mainly evidence the intrinsic nature of the resistance to aminoglycosides in lactobacilli, though genes for enzymatic modification of streptomycin aadA and aadE were found in 20% of tested strains. The occurrence of extended spectrum beta-lactamases (ESBL) was unknown in Lactobacillus, but our results revealed the blaTEM gene in 80% of strains, whereas blaSHV and blaOXA-1 genes were less frequent (20% and 15% of strains, respectively).

Partial purification and characterization of a thermostable mushroom tannase induced during solid state fermentation of Toxicodendron vernicifluum stem bark by Fomitella fraxinea

A thermostable extracellular tannase induced during solid state fermentation of the stem bark of Toxicodendron vernicifluum (Stokes) F.A. Barkley (Anacardiaceae) with Fomitella fraxinea was homogeneously isolated through ammonium sulfate precipitation and purified by DEAE-cellulose, and Sephadex G‒100 gel chromatography. It was then characterized for optimal reaction temperature and pH, thermal and pH stability, effects of metal ions as well as its hydrolysis patterns using 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG) as a substrate. The enzyme was purified 103.9-fold with 2.92% recovery and a single protein band corresponding to it was observed at 44.9 kDa during SDS-PAGE analysis. Optimal temperature and pH were found to be 50–70 °C and 5.5, respectively. This enzyme was substantially stable at temperatures below 70 °C and pH values between 5.0 and 7.0. Its activity was inhibited by Fe2+ ion (66%) and Cu2+ (39%) at a concentration of 5 mM. It also showed a different hydrolysis pattern from that of commercial Aspergillus oryzae tannase when PGG was used as a substrate. The tannase purified from the fermented plant material in this study unusually produced oligomeric galloylglucoses such as digalloylglucose (DGG), trigalloylglucose (TGG) and tetragalloylglucose (TeGG) from PGG while the Asp. oryzae tannase produced gallic acid and methyl gallate as final products. Therefore, this enzyme can be more preferably applied for production of bioactive oligomeric galloylglucoses such as DGG, TGG and TeGG from PGG or tannic acid.

Impact of lactic acid fermentation on acids, sugars, and phenolic compounds in black chokeberry and sea buckthorn juices

The aim of this research was to study the potential of malolactic fermentation to modify the composition of the juices of sea buckthorn, chokeberry and lingonberry. Juices were prepared with and without pectinolytic enzyme treatment, followed by fermentation with commercially available strains of Lactobacillus plantarum, originally isolated from fermented plant materials. The juices before and after the fermentation were analyzed with GC-FID, HPLC-DAD, and HPLC-MS. Enzyme treatment significantly increased the phenolic content in the juices by 11–50%. None of strains showed ability to ferment lingonberry juice. On the other hand, L. plantarum DSM 10492 and DSM 20174 converted all malic acid to lactic acid in sea buckthorn and chokeberry juices, respectively. Fermentation with DSM 10492 reduced the content of flavonols by 9–14% and hydroxycinnamic acids by 20–24% in chokeberry juice. Flavonol glycosides and sugars in sea buckthorn as well as anthocyanins in chokeberry remained unaffected by the fermentation.

Phenotypic and genotypic diversity of Lactobacillus buchneri strains isolated from spoiled, fermented cucumber

Lactobacillus buchneri is a Gram-positive, obligate heterofermentative, facultative anaerobe commonly affiliated with spoilage of food products. Notably, L. buchneri is able to metabolize lactic acid into acetic acid and 1,2-propanediol. Although beneficial to the silage industry, this metabolic capability is detrimental to preservation of cucumbers by fermentation. The objective of this study was to characterize isolates of L. buchneri purified from both industrial and experimental fermented cucumber after the onset of secondary fermentation. Genotypic and phenotypic characterization included 16S rRNA sequencing, DiversiLab® rep-PCR, colony morphology, API 50 CH carbohydrate analysis, and ability to degrade lactic acid in modified MRS and fermented cucumber media. Distinct groups of isolates were identified with differing colony morphologies that varied in color (translucent white to opaque yellow), diameter (1 mm–11 mm), and shape (umbonate, flat, circular or irregular). Growth rates in MRS revealed strain differences, and a wide spectrum of carbon source utilization was observed. Some strains were able to ferment as many as 21 of 49 tested carbon sources, including inulin, fucose, gentiobiose, lactose, mannitol, potassium ketogluconate, saccharose, raffinose, galactose, and xylose, while others metabolized as few as eight carbohydrates as the sole source of carbon. All isolates degraded lactic acid in both fermented cucumber medium and modified MRS, but exhibited differences in the rate and extent of lactate degradation. Isolates clustered into eight distinct groups based on rep-PCR fingerprints with 20 of 36 of the isolates exhibiting >97% similarity. Although isolated from similar environmental niches, significant phenotypic and genotypic diversity was found among the L. buchneri cultures. A collection of unique L. buchneri strains was identified and characterized, providing the basis for further analysis of metabolic and genomic capabilities of this species to enable control of lactic acid degradation in fermented plant materials.

Untargeted analysis of chromatographic data for green and fermented rooibos: Problem with size effect removal

While analyzing chromatographic data, it is necessary to preprocess it properly before exploration and/or supervised modeling. To make chromatographic signals comparable, it is crucial to remove the scaling effect, caused by differences in overall sample concentrations. One of the efficient methods of signal scaling is Probabilistic Quotient Normalization (PQN) [1]. However, it can be applied only to data for which the majority of features do not vary systematically among the studied classes of signals. When studying the influence of the traditional “fermentation” (oxidation) process on the concentration of 56 individual peaks detected in rooibos plant material, this assumption is not fulfilled. In this case, the only possible solution is the analysis of pairwise log-ratios, which are not influenced by the scaling constant. To estimate significant features, i.e., peaks differentiating the studied classes of samples (green and fermented rooibos plant material), we propose the application of rPLR (robust pair-wise log-ratios) as proposed by Walach et al. [2]. It allows for fast computation and identification of the significant features in terms of original variables (peaks) which is problematic, while working with the unfolded pair-wise log ratios. As demonstrated, it can be applied to designed data sets and in the case of contaminated data, it allows proper conclusions.

Improved HPLC method for rooibos phenolics targeting changes due to fermentation

The phenolic composition of rooibos (Aspalathus linearis) herbal tea is inherently linked to its health-promoting properties. “Fermentation” (oxidation) develops the characteristic flavour and colour of the herbal tea product. Changes to the phenolic composition during fermentation include conversion of aspalathin, a dihydrochalcone unique to rooibos and the major bioactive compound, to eriodictyol-glucopyranoside isomers. These compounds have not been quantified in rooibos plant material to date, due to separation challenges. In this study an improved HPLC method using a core shell column was developed to provide effective separation of a phenolic precursor (enolic phenylpyruvic acid-2-O-glucopyranoside, PPAG) and 15 rooibos tea phenolics (aspalathin, four eriodictyol-glucopyranoside isomers and 11 other major phenolic compounds) in a reasonable time. Compounds were tentatively identified using authentic reference standards and/or tandem mass spectrometry. The HPLC method utilising diode-array detection for quantitation was validated. After evaluation of different extraction solvents, a 40% aqueous acetonitrile solution was deemed optimal for extraction of phenolic compounds from rooibos plant material. Green, semi-fermented and fermented plant material from 10 sub-divided rooibos bushes were extracted and analysed. Water extracts, representing food ingredient extracts, were also analysed. The largest decreases with fermentation were observed for the dihydrochalcones, with moderate to small decreases for flavonols. Eriodictyol-glucopyranoside isomer concentrations increased following fermentation.

Antimicrobial and antioxidant activities of different solvent extracts from fermented and green honeybush (Cyclopia intermedia) plant material

Ethnopharmacological relevanceCyclopia intermedia is indigenous to South Africa and used to prepare honeybush herbal tea. This aromatic herbal tea has been associated with numerous health benefits, mostly based on anecdotal evidence and with very few studies reporting on the antimicrobial activities. AimThe inhibitory effect on the growth of important nosocomial microorganisms and possible association with the antioxidant capacity/content of various solvent extracts of green/unfermented and fermented honeybush plant material were determined in the current study. Materials and methodsThe agar disk diffusion assay was used as a screening assay for the antimicrobial activity of the various honeybush extracts, while the minimal inhibitory concentration (MIC) values, using the broth micro-dilution method, were determined against Streptococcus pyogenes, Staphylococcus aureus and Candida albicans. Active antimicrobial compounds were then shown using thin layer chromatography bioautography. Total antioxidant capacities and -content were also determined for each extract, while the main phenolic compounds were quantified using HPLC. ResultsSix of the eight solvent extracts of honeybush showed antimicrobial activity, with the fermented and green methanol extracts being most effective against S. aureus and C. albicans respectively, whilst the green chloroform extract was most potent against S. pyogenes. Thin layer chromatography-bioautography acknowledged the existence of active antimicrobial fractions within these different solvent honeybush extracts. Regardless of the assay, the green honeybush extracts generally exhibited the highest antioxidant capacity when compared to the fermented honeybush. The total polyphenols were also observed to be highest in green extracts when using water and methanol as solvents. In general the mangiferin and hesperidin contents were higher in the green than the fermented extracts of honeybush plant material. ConclusionsThe inhibitory activity of the various extracts against specific microorganisms was observed to be linearly proportional to the extract concentration. Although the different solvent extracts can only be considered weak antimicrobial agents, three compounds showed specific activity and should be further elucidated in future. It appears the antioxidant capacity of the various solvent herbal extracts did not relate to the antimicrobial activities; however, further work will be required to ascertain this observation. The current data also suggested that the various extracts of honeybush can be considered a good source of a unique blend of natural phytochemical antioxidants and antimicrobial compounds and should be further elucidated.

Bioactivity guided isolation of phytoestrogenic compounds from Cyclopia genistoides by the pER8:GUS reporter system

The popular South African herbal tea, honeybush, is made from several Cyclopia species (family: Fabaceae), amongst them Cyclopia genistoides. Phytoestrogenic potential of C. genistoides. has been recently reported, however bioactivity-guided isolation of compounds with estrogenic activity has not yet been performed.A transgenic plant system, Arabidopsis thaliana pER8:GUS, was used to assay the estrogen-like activity of C. genistoides. The quantitative determination of the active compounds in the fermented and non-fermented plant material was performed by HPLC. Subsequent bioactivity-guided fractionation led to the isolation of genistein, naringenin, isoliquiritigenin, luteolin, helichrysin B and 5,7,3′,5′-tetrahydroxyflavanone, four of them first reported in the genus.Helichrysin B, naringenin and 5,7,3′,5′-tetrahydroxyflavanone differed in quantity in the fermented and unfermented herbs, the fermented plant material contained two compounds with substantial estrogenic-like activity in higher concentration (naringenin and 5,7,3′,5′-tetrahydroxyflavanone), whereas the less active helichrysin B was more abundant in the unfermented herb. The fractions as well as compounds inhibited the growth of human cancer cell lines A2780 and T47D.These results underline the phytoestrogenic activity of C. genistoides and support the rationale to the fermentation process.

Influence of postharvest UV-B treatment and fermentation on secondary plant compounds in white cabbage leaves

The influence of postharvest UV-B on its own and in combination with fermentation (e.g. sauerkraut production) on formation and degradation of bioactive compounds was investigated in white cabbage, processed according to traditional Chinese fermentation methods. The pattern of polyphenols was affected by postharvest UV-B: Newly formed coumaroylglycoside, feruloylglycoside, caffeoylglycoside (up to 1mg/g dry matter; 4days) and quercetintriglycoside (0.4–0.5mg/gdm; 4days) might be related to postharvest increase in enzyme activity in the biosynthesis. Decreasing contents were observed for the glucosinolates glucobrassicin and 4-methoxyglucobrassicin, but the formation of the degradation products dihydroascorbigen and dihydro-4-methoxyascorbigen, which might be related to cell shrinking as mechanical damage. Fermentation resulted in deglycosidation of hydroxycinnamic acids. Newly generated cinnamic acid from coumaric acid aglycone was detected in fermented plant material combined with UV-B (50μg/g). Glucosinolates and dihydroascorbigens were completely degraded. This study shows exemplary UV-B as a supplemental step to improve the nutritional quality of processed plants.

Lactococcus lactis metabolism and gene expression during growth on plant tissues.

Lactic acid bacteria have been isolated from living, harvested, and fermented plant materials; however, the adaptations these bacteria possess for growth on plant tissues are largely unknown. In this study, we investigated plant habitat-specific traits of Lactococcus lactis during growth in an Arabidopsis thaliana leaf tissue lysate (ATL). L. lactis KF147, a strain originally isolated from plants, exhibited a higher growth rate and reached 7.9-fold-greater cell densities during growth in ATL than the dairy-associated strain L. lactis IL1403. Transcriptome profiling (RNA-seq) of KF147 identified 853 induced and 264 repressed genes during growth in ATL compared to that in GM17 laboratory culture medium. Genes induced in ATL included those involved in the arginine deiminase pathway and a total of 140 carbohydrate transport and metabolism genes, many of which are involved in xylose, arabinose, cellobiose, and hemicellulose metabolism. The induction of those genes corresponded with L. lactis KF147 nutrient consumption and production of metabolic end products in ATL as measured by gas chromatography-time of flight mass spectrometry (GC-TOF/MS) untargeted metabolomic profiling. To assess the importance of specific plant-inducible genes for L. lactis growth in ATL, xylose metabolism was targeted for gene knockout mutagenesis. Wild-type L. lactis strain KF147 but not an xylA deletion mutant was able to grow using xylose as the sole carbon source. However, both strains grew to similarly high levels in ATL, indicating redundancy in L. lactis carbohydrate metabolism on plant tissues. These findings show that certain strains of L. lactis are well adapted for growth on plants and possess specific traits relevant for plant-based food, fuel, and feed fermentations.

High-dimensional nested analysis of variance to assess the effect of production season, quality grade and steam pasteurization on the phenolic composition of fermented rooibos herbal tea

A nested analysis of variance combined with simultaneous component analysis, ASCA, was proposed to model high-dimensional chromatographic data. The data were obtained from an experiment designed to investigate the effect of production season, quality grade and post-production processing (steam pasteurization) on the phenolic content of the infusion of the popular herbal tea, rooibos, at ‘cup-of-tea’ strength. Specifically, a four-way analysis of variance where the experimental design involves nesting in two of the three crossed factors was considered.For the purpose of the study, batches of fermented rooibos plant material were sampled from each of four quality grades during three production seasons (2009, 2010 and 2011) and a sub-sample of each batch was steam-pasteurized. The phenolic content of each rooibos infusion was characterized by high performance liquid chromatography (HPLC)-diode array detection (DAD). In contrast to previous studies, the complete HPLC-DAD signals were used in the chemometric analysis in order to take into account the entire phenolic profile.All factors had a significant effect on the phenolic content of a ‘cup-of-tea’ strength rooibos infusion. In particular, infusions prepared from the grade A (highest quality) samples contained a higher content of almost all phenolic compounds than the lower quality plant material. The variations of the content of isoorientin and orientin in the different quality grade infusions over production seasons are larger than the variations in the content of aspalathin and quercetin-3-O-robinobioside. Ferulic acid can be used as an indicator of the quality of rooibos tea as its content generally decreases with increasing tea quality. Steam pasteurization decreased the content of the majority of phenolic compounds in a ‘cup-of-tea’ strength rooibos infusion.

Occurrence and sensory perception of Z-2-(β-d-glucopyranosyloxy)-3-phenylpropenoic acid in rooibos (Aspalathus linearis)

Z-2-(β-d-glucopyranosyloxy)-3-phenylpropenoic acid (PPAG), a compound postulated to contribute to the taste and mouthfeel of fermented rooibos tea (Aspalathus linearis), was isolated from unfermented rooibos plant material. Its structure was unequivocally confirmed by LC–MS, –MS2, FT-IR and NMR of the underivatised natural product, and optical rotation measurements of the hydrolysed sugar moiety. A similar compound, postulated to be E-2-(β-d-glucopyranosyloxy)-3-phenylpropenoic acid, was also detected. Analysis of the leaves of a large number of rooibos plants (n=54), sampled at commercial plantations, showed that PPAG is not ubiquitously present in detectable quantities in the leaves of different plants. This leads to large variation in the fermented plant material, infusions and food-grade extracts. PPAG was shown to have a slightly bitter to astringent taste and a detection threshold of 0.4mg/l in water.

Quantitative and Physical Evaluation of Patchouli Essential Oils Obtained from Different Sources of Pogostemon cablin

Patchouli essential oil can be obtained from fresh, dried and fermented plant material. It is a highly valuable product in the fragrance industry and its quality changes depending upon raw material age and oil storage. In this work, patchouli essential oils derived from different treatments have been subjected to GC-FID quantitative analysis using an internal standard (ISTD) method with response factors (RF). Samples were obtained from i) fresh plants; ii) hydrodistillation of one year mature and fermented plants; iii) hydrodistillation of one year mature plants; iv) commercial products from Indonesia and Malaysia. Linear Retention Indices (LRI) for both polar and non-polar GC-MS analyses were also measured as a tool for qualitative analysis towards a homologous series of C7-C30 n-alkanes. The results obtained confirmed that, in all samples, patchouli alcohol was the main volatile constituent, with higher amount in lab-scale produced oils, compared with commercial samples. Other major compounds, in lab oils and commercial samples respectively, were: delta-guaiene, alpha-guaiene, pogostol, seychellene and alpha-patchoulene. Another 36 compounds were also found.

Comprehensive two‐dimensional liquid chromatographic analysis of rooibos (Aspalathus linearis) phenolics

Rooibos tea is an unique beverage prepared from unfermented and fermented plant material of the endemic Cape fynbos plant, Aspalathus linearis. The well-known health-promoting benefits of rooibos are partly attributed to its phenolic composition. Detailed investigation of the minor phenolic constituents of rooibos is, however, hampered by the limitations associated with conventional HPLC methods used for its analysis. In this study, the applicability of comprehensive two-dimensional liquid chromatographic methods for the in-depth analysis of rooibos phenolics was investigated. Phenolic compounds were separated according to polarity by hydrophilic interaction chromatography (HILIC) in the first dimension, whilst reversed-phase liquid chromatography (RP-LC) provided separation according to hydrophobicity in the second dimension. Ultraviolet photodiode array and electrospray ionisation mass spectrometry were used to identify phenolic compounds. Comprehensive HILIC × RP-LC demonstrated its applicability for the analysis of a diverse range of phenolic compounds in unfermented and fermented rooibos samples, in which large qualitative differences in the phenolic composition were established. The combination of these orthogonal separations provided a significant improvement in resolution, as exemplified by practical peak capacities in excess of 2000 and 500 for off-line and on-line methods, respectively.