Purification and characterization of cell wall-bound peroxidase from vanilla bean

A cold-active ionically bound cell wall peroxidase was purified from a polyvinylpolypyrrolidone extract of mature vanilla beans by ultra filtration of 10 kDa and gel filtration chromatography on Sephacryl S-200. The M_r was 46.5 kDa determined by electrophoresis on SDS-PAGE, while native gel filtration confirmed tetramer enzyme form of approximately 186 kDa. The optimum pH and temperature were 3.8 and 16 °C, respectively, as determined with guaiacol as the substrate (K_m 3.8 mmol/L). The pI was approximately 7.7. The POD was inhibited by 1,4-dithiothreitol, β-mercaptoethanol and sodium azide. The POD showed decreasing activity in the presence of ascorbic acid, NaEDTA and sodium dodecyl sulfate at 1 mmol/L. The enzyme lost 80% of its activity in the presence of 20% ethanol. These results will permit better understanding of POD role in vanilla curing process.     

New insight on the genesis and fate of odor-active compounds in vanilla beans (Vanilla planifolia G. Jackson) during traditional curing

Green vanilla beans were subjected to the traditional curing process in Mexico. Odor-active potential from beans including 23 compounds was monitored through the analysis of both free and glucosidically bound volatiles. 8 of them were aliphatic aldehydes, acids, alcohol and ketone. 15 molecules were shikimate derivatives in which 13 were detected in glucosylated form. Some glucosides were efficiently hydrolyzed while some others partly or not at all after 90 days of curing. Moreover kinetics of hydrolysis of glucosides were not the same. A major part of glucovanillin was hydrolyzed at the first stages of curing while some other glucosides at the advanced stages. Data support hypothesis that hydrolysis of glucosides during curing is rather enzymatic origin than chemical one. Free shikimate derivatives or those liberated from glucosides are prone to chemical or enzymatic interconversions leading to a significant change in the aroma profile of cured vanilla.     

Exploration of Vanilla pompona from the Peruvian Amazon as a potential source of vanilla essence: Quantification of phenolics by HPLC-DAD

This study provides the first chemical investigation of wild-harvested fruits of Vanilla pompona ssp. grandiflora (Lindl.) Soto-Arenas developed in their natural habitat in the Peruvian Amazon. Flowers were hand-pollinated and the resulting fruits were analysed at different developmental stages using an HPLC-DAD method validated for the quantification of glucovanillin and seven other compounds. The method showed satisfactory linearity (r² > 0.9969), precision (coefficient of variation <2%), recoveries (70–100%), limit of detection (0.008–0.212 μg/ml), and limit of quantification (0.027–0.707 μg/ml). The evaluation of crude and enzyme-hydrolyzed Soxhlet-extracted samples confirmed the leading role of glucosides in fruit development. LC–ESI-MS studies corroborated the identities of four glucosides and seven aglycones, among them vanillin (5.7/100 g), 4-hydroxybenzyl alcohol (3.6/100 g), and anisyl alcohol (7.1/100 g) were found in high concentrations. The attractive flavor/aroma profile exhibited by wild V. pompona fruits supports studies focused on the development of this species as a specialty crop.     

Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12–24 hour intervals during 120 h of fermentation. Yeast isolates were grouped by (GTG)₅-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12 h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12 h of fermentation whilst PFGE showed that ~ 88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed.     

Hydrolysis of glycosidically bound flavour compounds from oak wood by Oenococcus oeni

Malolactic fermentation (MLF), which is conducted by lactic acid bacteria (LAB), has a significant influence on the stability and organoleptic quality of wine. Recent studies have shown that when MLF is carried out in oak wood barrels, LAB were also able to interact with wood and increase volatile compound contents such as vanillin during MLF. The release of these compounds indicates that LAB may convert vanillin precursors present in oak wood. In this work, the effect of commercial glycosidases on the released vanillin was firstly studied. This aldehyde is present in wood extracts in monoglycosidic forms where the major glycones are arabinose and xylose. Other aglycons released during MLF in barrels, syringaldehyde and whisky-lactones, can be considered as other sources of aroma. Secondly, strains selected with high activities toward glycoside substrates could hydrolyse vanillin glycoside precursors from oak wood with the same efficiency as commercial enzymes.     

Phytochemical,sensory attributes and aroma stability of dense phase carbon dioxide processed Hibiscus sabdariffa beverage during storage

The effect of dense phase carbon dioxide (DPCD) processing (34.5 MPa, 8% CO₂, 6.5 min, and 40 °C) on phytochemical, sensory and aroma compounds of hibiscus beverage was compared to a conventional thermal process (HTST) (75 °C for 15 s) and a control (untreated beverage) during refrigerated storage (4 °C). The overall likeability of the hibiscus beverage for all treatments was not affected by storage up to week 5. DPCD process retained more aroma volatiles as compared to HTST. Aroma profiles in the beverages were mainly composed of alcohols and aldehydes with 1-octen-3-ol, decanal, octanal, 1-hexanol, and nonanal as the compounds with the highest relative percentage peak areas. A loss of only 9% anthocyanins was observed for the DPCD processed hibiscus beverage. Phytochemical profiles in the hibiscus beverage included caffeoylquinic acids, anthocyanins, and flavonols. No major changes in total phenolics and antioxidant capacity occurred during the 14 weeks of storage.     

Characterisation of bound volatile compounds of a low flavour kiwifruit species: Actinidia eriantha

Aroma compounds in fruit are known to occur in free and glycosidically bound forms. The bound volatile fraction of a low flavour kiwifruit species, Actinidia eriantha, was studied. The fruit have a bland and grassy flavour. Glycosidic precursors were isolated from juice by adsorption onto an Amberlite XAD-2 column. After enzymatic hydrolysis with Rapidase AR2000, the released aglycones were analysed by GC–MS. Alcohols, terpenoids and phenolics were the most numerously represented compound classes. Alcohols, benzenoids and phenolics showed the highest concentrations. Major compounds were 2-phenylethanol, furfuryl alcohol, (Z)-3-hexen-1-ol, coniferyl alcohol, isoamyl alcohol and linolenic acid. Several of the bound compounds found, including linoleic, linolenic and benzoic acids and coniferyl alcohol, are precursors of odorous volatiles. Many compounds detected as bound volatiles have not been previously reported as free volatiles in A. eriantha. The bound volatile composition of A. eriantha also showed differences with those of other kiwifruit species.     

Identification and sensory evaluation of flavour enhancers in Japanese traditional dried herring (Clupea pallasii) fillet

Flavour-enhancing components of dried herring fillet (migaki-nishin in Japanese) were isolated and evaluated for their effects on sensory perception. Sensory evaluation revealed that addition of dried herring fillet water-soluble extracts to Japanese noodle soup significantly (P < 0.05) enhanced soup flavour characters, such as thickness, mouthfulness and continuity. The extracts were fractionated by dialysis and chromatography. Fractions containing flavour enhancers were isolated by sensory perception. Results from instrumental analyses showed that the kokumi flavour enhancers in dried herring fillet were creatine and creatinine.     

Antioxidative properties of Pandanus amaryllifolius leaf extracts in accelerated oxidation and deep frying studies

The potential uses of Pandanus amaryllifolius leaf extract as a natural antioxidant were evaluated in refined, bleached and deodorized (RBD) palm olein, using accelerated oxidation and deep frying studies at 180 °C from 0 to 40 h. The extracts (optimum concentration 0.2%) significantly retarded oil oxidation and deterioration (P < 0.05), comparably to 0.02% BHT in tests such as peroxide value, anisidine value, iodine value, free fatty acid, oxidative stability index (OSI), polar and polymer compound contents. In sensory evaluation studies, different batches of French fries were not significantly different (P < 0.05) from one another for oiliness, crispiness, taste and overall acceptability when the same oil was used for up to the 40th hour of frying. P. amaryllifolius leaf extract, which had a polyphenol content of 102 mg/g, exhibited an excellent heat-stable antioxidant property and may be a good natural alternative to existing synthetic antioxidants in the food industry.     

Behavior of Salmonella during fermentation,drying and storage of cocoa beans

Due to cocoa being considered a possible source of Salmonella contamination in chocolate, the behavior of Salmonella during some cocoa pre-processing stages (fermentation, drying and storage) was investigated. The fermentation process was carried out on a pilot scale (2 kg beans/box) for 7 days. Every day a fermentation box was inoculated with a Salmonella pool (ca. 4 log MPN/g). The results showed that Salmonella did not affect (P > 0.05) the growth of the main microorganism groups involved in cocoa fermentation. On the other hand, the pathogen was influenced (P < 0.05) by yeast, acetic acid bacteria and pH. In spite of Salmonella showing counts ≤ 1 log MPN/g in the first days, at the end of fermentation it grew in all samples, reaching counts as high as 7.49 log MPN/g. For drying and storage, cocoa beans were inoculated during the fermentation (experiment A) or during the drying (experiment B). In these stages the decline of the water activity affected the pathogen behavior. In experiment A during the drying, Salmonella count increased in most of the samples. In experiment B either a slight growth or no growth in the samples inoculated up to 48 h was observed, whereas the other samples showed reductions from the initial count. After 30 days of storage at room temperature, the water activity decreased to 0.68, and reductions of Salmonella ranged from 0.93 to 2.52 log MPN/g. Despite the reductions observed during the storage, the pathogen was detected even after 120 days. Therefore, the results showed that Salmonella growth or survival depends on when the contamination occurs.     

Relation among taste-related compounds (phenolics and caffeine) and sensory profile of erva-mate (Ilex paraguariensis)

Erva-mate (Ilex paraguariensis St. Hil.) is a native species from temperate regions of South America, such as Brazil, Paraguay, and Argentina, that is consumed as a beverage known as mate. The objective of this research was to determine the content of caffeic acid, catechin, chlorogenic acid, caffeine, and gallic acid in mate to explain their influence in beverage taste and sensory differences between native and reforested plants, as well as between beverages from plants of different regions of Brazil (Santa Catarina and Rio Grande do Sul states). Compounds were determined by HPLC and results were related to a sensory evaluation performed by trained tasters. Tasters considered the beverage from reforested plants to be more bitter than the beverage from native plants. Beverages from reforested plants had significantly higher caffeic acid and lower catechin, chlorogenic acid, caffeine, and gallic acid content than native plants. Beverages from plants of Santa Catarina state had significantly higher catechin, caffeine, and gallic acid content than plants from Rio Grande do Sul state.     

Effect of pork lard content on the chemical, microbiological and sensory properties of a typical fermented meat product (Pitina) obtained from Alpagota sheep

The aim was to investigate the physicochemical, microbiological and sensory properties of Pitina, a typical fermented meat product and evaluate the effect of two levels of pork lard content (Low Fat, LF, 10% vs. High Fat, HF, 30%) on its attributes. HF attained lower pH than LF Pitina, which reached lower water activity. LAB comprised the major flora with substantial counts of micrococci, enterococci and mould and yeast. Gram negative Enterobacteria were recovered as coliforms and faecal coliforms. Listeria monocytogenes was also isolated. The lard level influenced the count of micrococci and some sensory attributes. LF attained higher scores for both hardness and cohesiveness and differed from HF in having a more marked odour of ewe and smoke and sweeter taste. HF had a more pronounced odour and taste of garlic and mould than LF.     

Profile of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus

This study investigated the formation and utilization of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus. Time-course papaya juice fermentations were carried out using pure cultures of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 and a mixed culture of the two yeasts at a ratio of 1:1000 (R2:NCYC2251). Changes in S. cerevisiae cell population, Brix, sugar consumption and pH were similar in the mixed culture and in the S. cerevisiae monoculture. There was an early growth arrest of W. saturnus in the mixed culture fermentation. A range of volatile compounds were produced during fermentation including fatty acids, alcohols, aldehydes and esters and some volatile compounds including those initially present in the juice were utilized. The mixed culture fermentation of S. cerevisiae and W. saturnus benefited from the presence of both yeasts, with more esters being produced than the S. cerevisiae monoculture and more alcohols being formed than the W. saturnus monoculture. The study suggests that papaya juice fermentation with a mixed culture of S. cerevisiae and W. saturnus may be able to result in the formation of more complex aroma compounds and higher ethanol level than those using single yeasts.     

Increased flavour diversity of Chardonnay wines by spontaneous fermentation and co-fermentation with Hanseniaspora vineae

Discovery, characterisation and use of novel yeast strains for winemaking is increasingly regarded as a way for improving quality and to provide variation, including subtle characteristic differences in fine wines. The objective of this work was to evaluate the use of a native apiculate strain, selected from grapes, Hanseniaspora vineae (H. vineae) 02/5A. Fermentations were done in triplicate, working with 225 L oak barrels, using a Chardonnay grape must. Three yeast fermentation strategies were compared: conventional inoculation with a commercial Saccharomyces cerevisiae strain, ALG 804, sequential inoculation with H. vineae and then strain ALG 804 and spontaneous fermentation. Yeast strain identification was performed during fermentation, in which the apiculate strain was found to be active, until 9% of alcohol in volume, for the co-fermentation and the spontaneous fermentation was completed by three native S. cerevisiae strains. Basic winemaking parameters and some key chemical analysis, such as concentration of glycerol, biogenic amines, organic acids, and aroma compounds were analysed. Sensory analysis was done using a trained panel and further evaluated with professional winemakers. Sequential inoculation with H. vineae followed by S. cerevisiae resulted in relatively dry wines, with increased aroma and flavour diversity compared with wines resulting from inoculation with S. cerevisiae alone. Wines produced from sequential inoculations were considered, by a winemaker’s panel, to have an increased palate length and body. Characteristics of wines derived from sequential inoculation could be explained due to significant increases in glycerol and acetyl and ethyl ester flavour compounds and relative decreases in alcohols and fatty acids. Aroma sensory analysis of wine character and flavour, attributed to winemaking using H. vineae, indicated a significant increase in fruit intensity described as banana, pear, apple, citric fruits and guava. GC analysis of the relative accumulation of 23 compounds to significantly different concentrations for the three fermentation strategies is discussed in relation to aroma compound composition.     

Toxic compounds in essential oils of coriander, caraway and basil active against stored rice pests

Essential oils, distilled from seeds of Coriander sativum and Carum carvii and from leaves of five different varieties of Ocimum basilicum, were fractionated by column chromatography and tested in the laboratory for volatile toxicity against three stored rice pests (Sitophilus oryzae, Rhyzopertha dominica and Cryptolestes pusillus). The active fractions were analyzed by GC-MS. Coriander contained linalool (1617 ppm of the oil) as the main product active against the three pests. Camphor-rich fractions (over 400 ppm) were very toxic to R. dominica and C. pusillus. The caraway profile included carvone and limonene as expected but (E)-anethole, generally regarded as a minor product in the essential oil of this species, was also a major component, being present at 365 ppm. Carvone was the most effective (972 ppm) monoterpenoid against S. oryzae. In addition, (E)-anethole at 880 ppm was toxic to R. dominica while vapors of limonene (1416 ppm) and fenchone-rich (554 ppm) fractions killed adults of C. pusillus only. Three major essential oil profiles were present in the five varieties of O. basilicum analyzed: methyl eugenol/estragole, estragole and estragole/linalool chemotypes. The abundance of components had a strong influence on the outcome of the bioassays. Fractions, where combinations of products occurred with or without other minor compounds, were often more toxic than any one compound alone.     

Influence of processing conditions on flavour compounds of custard apple (Annona squamosa L.)

Changes in volatile compounds of fruit pulp of Annona squamosa, as influenced by the conditions of processing, were studied. Sweet and pleasant flavored pulp from mature ripe fruits was subjected to treatments such as frozen and stored (for 12 months), heated to 55 °C (critical temperature) and 85 °C (pasteurization temperature) for 20 min each, and spray dried with skim and whole milk powders. Volatiles from these samples were extracted into dichloromethane and n-pentane (1:1), and were subjected to gas chromatograph (GC) and gas chromatograph-mass spectrometer (GC-MS) analysis for identification and quantification of chemical constituents. Terpenes such as α-pinene, β-pinene, linalool, germacrene-d and spathulenol, esters like sec-butylbutanoate, and methyllinolenate, along with benzyl alcohol and two oxygenated sesquiterpenes were found to be the major volatiles of the fresh pulp. The 12-month-stored frozen pulp did not differ from the fresh pulp in the flavour spectrum. Heating fresh pulp at 55 and 85 °C, tended to produce increased flavour spectrum, the compounds relatively being more at 85 °C. At 55 °C, significant increase in the quantities of α-pinene, β-pinene, linalool, germacrene and spathulenol were observed; higher quantities of cineole, limonene, α-cubebene and α-copaene, caryophyllene, α-farnecene and δ-cadenene were formed, while these were totally absent in fresh pulp. Significant increase in quantities of α-pinene, β-pinene, 1,8-cineole, limonene, aromadendrene, α-farnecene, γ-cadenene, δ-cadenene and spathulenol were found by heating pulp at 85 °C. Spray-dried samples, showed increased flavor note with the use of whole milk powder as compared to the skim milk powder.     

Effectiveness of products from four locally grown plants for the management of Acanthoscelides obtectus (Say) and Zabrotes subfasciatus (Boheman) (both Coleoptera: Bruchidae) in stored beans under laboratory and farm conditions in Northern Tanzania

The effectiveness of whole or powdered leaves (botanicals) from four locally grown plant species applied at a rate of 1.5 kg per 100 kg beans (Phaseolus vulgaris) against Acanthoscelides obtectus and Zabrotes subfasciatus was compared under laboratory and farm conditions. In the laboratory, Chenopodium ambrosioides, applied as powder or as whole leaves, was the most effective, with 100% mortality of adult insects in less than three days and no progeny. Less C. ambrosioides (about 200 g per 100 kg beans) still resulted in 100% mortality within 24 h. Tagetes minuta applied as powder also increased mortality and reduced oviposition and progeny production significantly. The other treatments - T. minuta applied as leaves, and Azadirachta indica or Cupressus lusitanica applied as powder or as whole leaves - had no significant effects upon mortalities, oviposition rate, or progeny production compared with control treatments. When the rate of application was increased to about 8.3 kg per 100 kg beans, there was a slight increase in mortality using T. minuta and A. indica, but not with C. lusitanica. An additional trial with C. ambrosioides from different collections and with plants at different stages of development revealed considerable variations in the efficacy of the treatment.In the on-farm trials, A. indica-seed powder was the most effective treatment, followed respectively by leaf powders of C. ambrosioides, C. lusitanica and T. minuta. All treatments were significantly more effective than the control in reducing the numbers of live insects; they also reduced numbers of damaged beans and maintained germination rates after 5 months of storage. The results of evaluations of the treatments made by farmers just after the trials and five years later are reported.     

Thin-layer high-vacuum distillation to isolate volatile flavour compounds of cocoa powder

The volatile fraction of commercial cocoa powders was isolated using thin-layer high-vacuum distillation (TLHVD) of Soxhlet extracts. Calculated and experimental recovery of the internal standard n-undecanoic acid methylester did agree, and a good reproducibility was found for the procedure. Around 70 volatile compounds were identified and semi-quantified using internal standard-based gas chromatography (GC), coupled GC–mass spectrometry (GC–MS) and GC-olfactometry (GC-O). Dehydromevalonic acid lactone, (R)-(−)-pantolactone and two diastereomer solerols were identified for the first time after purification by micropreparative GC and re-analysis using GC–MS and chiral GC. Strong sensory contributions also came from acetic acid, 2/3-methyl butanoic acid, phenylacetaldehyde, furaneol, dihydroxymaltol, vanillin and phenylacetic acid.     

Studies on use of Enteromorpha in snack food

Seaweeds, also known as sea vegetables, are of nutritional interest, as they are rich in vitamins, minerals and dietary fiber. Enteromorpha compressa (Linnaeus), green seaweed (chlorophyta), which is a rich source of iron and dietary fibre was used as an ingredient in the preparation of Pakoda, a common traditional snack food in India. Pakoda samples showed increases in ash, protein and total dietary fibre contents with increase in Enteromorpha level, accompanied by a nearly fivefold increase in iron content (26.4–126 mg/100 g) and fourfold increase in calcium content (30.1–124 mg/100 g). Bioavailability of iron in Enteromorpha, and Pakoda containing 7.5% of Enteromorpha, did not show any difference (55–56%) at pH 7.5 (intestinal condition). But, at pH 1.35 (gastric condition) the bioavailability of iron in Pakoda containing Enteromorpha was found to be slightly higher (27.1%) than that in Enteromorpha. Reducing power (155–222 μg/g) increased as the Enteromorpha level increased from 0% to 15%. But the addition of Enteromorpha was found to decrease free radical-scavenging activity and the total phenol content. Pakoda containing up to 7.5% of Enteromorpha was found to have a sensory quality comparable with that of Pakoda without Enteromorpha.