Treatment of soy milk with Debaryomyces hansenii cells immobilised in alginate

Whole cells of Debaryomyces hansenii UFV-1 were permeabilised with ethanol and immobilised in calcium alginate hydrogel. The optimum pH and temperature for α-galactosidase activities of permeabilised free (PFC) and permeabilised immobilised cells (PIC) were 4.5 and 60 °C; and 4.0 and 70 °C, respectively. PIC α-galactosidase was more stable than that of PFC. The incubation of PIC at 60 and 70 °C promoted an increase in α-galactosidase activity. The α-galactosidase activity was maintained when PIC was used in three repeated batches. The K_m values for PIC and PFC α-galactosidases, with ρNPαGal, were 0.82 mM and 0.30 mM, respectively. Soy milk treatment with PIC for 6 h at 60 °C promoted 100% hydrolysis of raffinose oligosaccharides.     

Effect of selected strains of Debaryomyces hansenii on the volatile compound production of dry fermented sausage “salchichón”

Different biotypes of Debaryomyces hansenii, characterized by mitochondrial DNA (mtDNA) restriction analysis, were inoculated in dry fermented sausages to evaluate their influence as single starter culture on volatile compound generation throughout the ripening process. Similar evolution of physicochemical parameters and microbial population was observed in both uninoculated and inoculated sausages. The tested biotypes modified the volatile compound profile of sausages specially in esters, branched alcohols and aldehydes. The biotype of D. hansenii with the E mtDNA restriction pattern is the most suitable to be used as starter culture since it produced volatile compounds involved in flavour development of dry-cured meat products such as 3-methylbutanol, 3-methylbutanal and 2-propanone. Moreover, the use of D. hansenii strains with the B, C2 and E mtDNA restriction patterns, as a mixed starter culture, should be also considered to generate low amount of sulphur compounds in dry-cured meat products.     

Comparison of molecular and metabolomic methods as characterization tools of Debaryomyces hansenii cheese isolates

Debaryomyces hansenii is one of the yeast species most frequently isolated from cheese and salty foods, however little is known about the phenotypic and molecular variability of its strains. In order to explore the possibilities of a large study on its biodiversity, some D. hansenii strains were selectively isolated from pecorino cheese sampled in ten different Italian regions. All isolates were identified as D. hansenii on the basis of conventional and molecular taxonomic analysis. The D1/D2 domain sequences of the 26S-rDNA did not show any variation, confirming the extreme homogeneity of this species. PCR-duplex-RAPD banding patterns analyzed with PCoA showed interesting clustering related to the geographic areas of isolation, although some overlapping between strains derived from different districts could be observed. A FTIR (Fourier Transform Infrared Spectroscopy) metabolomic fingerprint produced groupings weakly related to those observed with RAPD and less associated with the isolation locales. The discriminatory power of metabolomic fingerprint was able to discriminate strains otherwise considered identical. This preliminary study showed that, in spite of the homogeneity at the 26S-rDNA level, the D. hansenii strains exhibit high molecular and metabolomic variability somehow linked to the places of isolation. Further studies will be necessary to better investigate on the link between terroir and strain variability, as well as on the relation between genotypic and metabolomic fingerprints.     

The Influence of Debaryomyces hansenii, Candida deformans and Candida zeylanoides on the aroma formation of dry-cured “lacón”

The volatile profile of dry-cured “lacón” that has been inoculated with three different yeasts were determined and compared with a non-inoculated dry-cured “lacón”. Yeasts (Debaryomyces hansenii, Candida deformans and Candida zeylanoides) that were used as starter cultures in the present study were selected among yeasts that were isolated from native dry-cured “lacón” at different stages of ripening process. These starters were spread on dry-cured “lacón” surface in order to test their capacity to contribute on the generation of volatile compounds. A total of forty two volatile compounds were detected by dynamic headspace sampling followed by gas chromatography–mass spectrometry analysis. Significant differences (P < 0.001) on the volatile profiles of different batches were found in comparison with non-inoculated samples, showing the highest total area values for the inoculated ones. Esters were the most abundant chemical family in all batches studied except for C. zeylanoides batch, which showed greater amount of hydrocarbons than esters. The second more abundant family was hydrocarbons for control and C. deformans batches (147.6 and 445.24 × 10⁶ area units, respectively), alcohols for D. hansenii (363.77 × 10⁶ area units) and esters for C. zeylanoides (248.33 × 10⁶ area units). However, the aldehyde compound group in control batch samples was found to be significantly higher than in the inoculated ones (P < 0.001). Among inoculated batches, D. hansenii batch showed the lowest hexanal content (14.42 × 10⁶ area units) in comparison with non-inoculated batch (105.99 × 10⁶ area units). Among all batches studied, D. hansenii batch presented the highest area values for esters, alcohols, linear hydrocarbons, ketones, acids and furans; control batch for aldehydes and C. zeylanoides batch for branched hydrocarbons. Therefore, the study showed that every yeast strain produced a specific volatile profile which was also different from that of the control dry-cured “lacón”.     

Impact of Debaryomyces hansenii strains inoculation on the quality of slow dry-cured fermented sausages

Debaryomyces hansenii strains, M4 and P2, isolated from natural fermented sausages were inoculated in slow fermented sausages to study their effect on processing parameters, microbial population, volatile compound and sensory characteristics. The inoculation of D. hansenii strains, M4 and P2, did not affect the ripening process as no differences in pH and Aw were detected. The dominance of the inoculated yeast strains along the process was followed by RAPDs of M13 minisatellite. The inoculated yeasts, P2 and M4, were recovered at the end of the ripening process although P2 appeared in higher counts than M4. The sausages inoculated with P2 resulted in a decrease in lipid oxidation values (TBARS) and a reduction of lipid-oxidation derived aldehydes in addition to a highest acid compound abundance. M4 inoculated sausages resulted in highest sulphur containing compound abundance. However, no differences in consumer acceptance were detected. Moreover, both yeast strains were responsible for the generation of ethyl methyl-branched ester compounds in the dry-cured sausages.     

Hydrolysis of oligosaccharides in soybean products by Debaryomyces hansenii UFV-1 α-galactosidases

α-Galactosides are abundant sugars found in legumes such as soybean. Since humans and monogastric animals lack α-galactosidase in the digestive tract, they are unable to digest these sugars, which induce flatulence. The use of α-galactosidases is promising as a means to overcome this problem, and to increase the consumption of soy products. Immobilized α-galactosidase, derived from Debaryomyces hansenii UFV-1, exhibited an activity of 40 U per g of silica and an activity yield of 50%. The optimum pH of free and immobilized α-galactosidase was 5.0 and the optima temperatures were 60 and 80 °C, respectively. The soymilk stachyose was completely hydrolyzed by different enzyme forms after incubation for 4 h at 60 °C, while raffinose was reduced by 100%, 25% and 68% by free, immobilized enzymes and permeabilized cells, respectively. The soy molasses treatment with free enzyme for 6 h promoted reduction in stachyose and raffinose contents by 100% and 50%, respectively.     

Ammonia production and its possible role as a mediator of communication for Debaryomyces hansenii and other cheese-relevant yeast species

Ammonia production by yeasts may contribute to an increase in pH during the ripening of surface-ripened cheeses. The increase in pH has a stimulatory effect on the growth of secondary bacterial flora. Ammonia production of single colonies of Debaryomyces hansenii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Geotrichum candidum was determined on glycerol medium (GM) agar and cheese agar. The ammonia production was found to vary, especially among yeast species, but also within strains of D. hansenii. In addition, variations in ammonia production were found between GM agar and cheese agar. Ammonia production was positively correlated to pH measured around colonies, which suggests ammonia production as an additional technological parameter for selection of secondary starter cultures for cheese ripening. Furthermore, ammonia appeared to act as a signaling molecule in D. hansenii as reported for other yeasts. On GM agar and cheese agar, D. hansenii showed ammonia production oriented toward neighboring colonies when colonies were grown close to other colonies of the same species; however, the time to oriented ammonia production differed among strains and media. In addition, an increase of ammonia production was determined for double colonies compared with single colonies of D. hansenii on GM agar. In general, similar levels of ammonia production were determined for both single and double colonies of D. hansenii on cheese agar.     

Microbial succession of Debaryomyces hansenii strains during the production of Danish surfaced-ripened cheeses

Surface-ripened cheeses of the Danbo type were analyzed for the presence of yeasts with special emphasis on Debaryomyces hansenii. Samples were taken from pasteurized milk, brine, and inoculation slurries and from cheese surfaces during ripening at a Danish dairy. D. hansenii was found to be the dominant yeast species throughout the ripening period, whereas other yeast species such as Trichosporon spp., Rhodotorula spp., and Candida spp. were found in minor concentrations during early stages of cheese ripening. Mitochondrial DNA RFLP was used to show that several strains of D. hansenii were present from the onset of ripening. Thereafter, a microbial succession among the strains took place during the ripening. After 3 d of ripening, only one strain was found. This particular strain was found to be dominant in 16 additional batches of surface-ripened cheeses. We investigated the cause of the observed microbial succession by determining the variation in strains with regard to their ability to grow on lactate and at different pH and NaCl concentrations. The strains were shown to vary in their ability to grow on lactate. In a full factorial design at three levels with factor levels close to the actual levels on the cheese surface, differences in pH and NaCl tolerances were observed. The dominant strain was found to be better adapted than other strains to the environmental conditions existing in surface-ripened cheeses during production [e.g., lactate as the main carbon source, pH 5.5 to 6.0 and NaCl concentrations of 7 to 10% (wt/vol)].     

Hollow fibre-based liquid phase microextraction combined with high-performance liquid chromatography for the analysis of flavonoids in Echinophora platyloba DC. and Mentha piperita

A simple, inexpensive and efficient three phase hollow fibre liquid phase microextraction (HF-LPME) technique combined with HPLC was used for the simultaneous determination of flavonoids in Echinophora platyloba DC. and Mentha piperita. Different factors affecting the HF-LPME procedure were investigated and optimised. The optimised extraction conditions were as follows: 1-octanol as an organic solvent, pH_donor = 2, pH_acceptor = 9.75, stirring rate of 1000 rpm, extraction time of 80 min, without addition of salt. Under these conditions, the enrichment factors ranged between 146 and 311. The values of intra and inter-day relative standard deviations (RSD) were in the range of 3.18–6.00% and 7.25–11.00%, respectively. The limits of detection (LODs) ranged between 0.5 and 7.0 ng mL⁻¹. Among the investigated flavonoids quercetin was found in E. platyloba DC. and luteolin was found in M. piperita. Concentration of quercetin and luteolin was 0.015 and 0.025 mg g⁻¹ respectively.     

Comparison of volatile compounds produced in model cheese medium deacidified by Debaryomyces hansenii or Kluyveromyces marxianus

The aroma of a deacidified cheese medium is the result of the overall perception of a large number of molecules belonging to different classes. The volatile compound composition of (60%) cheese medium (pH 5.8) deacidified by Debaryomyces hansenii (DCM(Dh)) was compared with the one deacidified by Kluyveromyces marxianus (DCM(Km)). It was determined by dynamic headspace extraction, followed by gas chromatography separation and quantification as well as by mass spectrometry identification. Whatever the media tested, a first class of volatile compounds can be represented by the ones not produced by any of the yeasts, but some of them are affected by K. marxianus or by D. hansenii. A second class of volatile compounds can be represented by the ones produced by K. marxianus, which were essentially esters. Their concentrations were generally higher than their thresholds, explaining the DCM(Km) global fruity odor. A third class can be represented by the ones generated by D. hansenii, which were essentially methyl ketones with fruity, floral (rose), moldy, cheesy, or wine odor plus 2-phenylethanol with a faded-rose odor. The impact of methyl ketones on the DCMDh global flavor was lower than the impact of 2-phenylethanol and even negligible. Therefore, the global faded-rose odor of D. hansenii DCM can be explained by a high concentration of 2-phenylethanol.     

Analysis of ginsenosides in Panax ginseng in high pressure microwave-assisted extraction

High pressure microwave assisted extraction (HPMAE) was applied to extract the ginsenosides from Panax ginseng root. The influences of extraction solvent, extraction pressure and extraction time were individually investigated. HPMAE has been compared with other extraction methods, including Soxhlet extraction, ultrasound-assisted extraction and heat reflux extraction. The determination of ginsenosides was performed by HPLC–ESI-MS. The results indicated that the HPMAE not only took a shorter time but also afforded higher extraction yields of ginsenosides, especially ginsenoside Rb₁, Rc, Rb₂ and Rd. Furthermore, the neutral ginsenosides and malonyl ginsenosides in Panax ginseng root extracts by HPMAE were investigated. The malonyl ginsenoside m-Rb₁, m-Rc, m-Rb₂ and m-Rd degraded in HPMAE at 400 kPa (109–112 °C) in 70% (v/v) ethanol–water and at 600 kPa (112–115 °C) in methanol, and transformed into corresponding neutral ginsenoside Rb₁, Rc, Rb₂ and Rd. Using water as extraction solution, the neutral ginsenosides degraded under HPMAE at 400 kPa (135–140 °C), and transformed into less polarity rare ginsenosides.     

Determination of hyperoside and 2″-acetylhyperoside from Ligularia fischeri by high performance liquid chromatography

Ligularia fischeri and its main flavonoids, hyperoside and 2″-acetylhyperoside, posses antioxidant properties. This study was carried out to investigate the contents of hyperoside and 2″-acetylhyperoside in L. fischeri by using high performance liquid chromatography (HPLC). An HPLC–photodiode array (PDA) detection method was established for the simultaneous determination of hyperoside and 2″-acetylhyperoside in L. fischeri. Two flavonoids were successfully separated in less than 20 min using an YMC RP 18 column. The mobile phase was composed of water (A) and acetonitrile (B) with isocratic elution system (23% B) at a flow rate of 1 mL/min. Their calibration curves showed good linear regression (r > 0.9992) within the test ranges. The method was validated for specificity, accuracy, precision, and limits of detection. The determined two compounds were well separated with a linear range of 18–180 μg/mL. The contents of hyperoside and 2″-acetylhyperoside were 0.387 ± 0.002 and 0.526 ± 0.006 mg/g in L. fischeri, respectively.     

Inactivation kinetics of pulsed electric field-resistant strains of Listeria monocytogenes and Staphylococcus aureus in media of different pH

A study of the effect of pulsed electric fields (PEF) on the inactivation of Listeria monocytogenes STCC 5672 and Staphylococcus aureus STCC 4459 in McIlvaine buffer covering a range from pH 3.5 to 7.0 was conducted. Mathematical models based on the Weibull distribution were developed to describe the influence of the electric field strength, treatment time and pH of the treatment medium on the lethality of both Gram positive pathogenic bacteria after PEF treatments. Both microorganisms were more sensitive to PEF in media of low pH, although the influence of the pH on the PEF resistance was more significant in S. aureus. In the best cases scenario, the highest inactivation levels achieved were 3.3 and 6.1 log₁₀ cycles for L. monocytogenes and S. aureus respectively in pH 3.5 after 500 μs of 35 kV/cm. Based on these results and those observed in literature, L. monocytogenes STCC 5672 at any pH investigated has been shown as one of the most PEF resistant microorganism. Therefore, this microorganism should be considered as a possible target microorganism to define process criterion for PEF pasteurization.     

Membrane permeabilization and cellular death of Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae as induced by high pressure carbon dioxide treatment

In this study, the relationship between (irreversible) membrane permeabilization and loss of viability in Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae cells subjected to high pressure carbon dioxide (HPCD) treatment at different process conditions including temperature (35–45 °C), pressure (10.5–21.0 MPa) and treatment time (0–60 min) was examined. Loss of membrane integrity was measured as increased uptake of the fluorescent dye propidium iodide (PI) with spectrofluorometry, while cell inactivation was determined by viable cell count. Uptake of PI by all three strains indicated that membrane damage is involved in the mechanism of HPCD inactivation of vegetative cells. The extent of membrane permeabilization and cellular death increased with the severity of the HPCD treatment. The resistance of the three tested organisms to HPCD treatment changed as a function of treatment time, leading to significant tailing in the survival curves, and was dependent on pressure and temperature. The results in this study also indicated a HPCD-induced damage on nucleic acids during cell inactivation. Transmission electron microscopy showed that HPCD treatment had a profound effect on the intracellular organization of the micro-organisms and influenced the permeability of the bacterial cells by introducing pores in the cell wall.     

Simultaneous quantification of major flavonoids in "Bawanghua", the edible flower of Hylocereus undatus using pressurised liquid extraction and high performance liquid chromatography

A pressurised liquid extraction (PLE) and high performance liquid chromatography (HPLC) method was developed for simultaneous quantification of six major flavonoids in edible flower of Hylocereus undatus. In order to achieve the baseline separation of two pairs of isomers, the HPLC conditions were optimised with different kind of reversed phase columns and mobile phase gradient programs. In addition, the solvent concentration, extraction temperature, extraction time and flush cycle for PLE were also optimised. Zorbax SB-C8 (100×2.1 mm, 1.8 μm) column was chosen with acetonitrile and water containing 0.1% trifluoroacetic acid as mobile phase, the six analytes were eluted with baseline separation. The calibration curves showed good linearity (r(2)>0.9994) with LODs and LOQs less than 0.90 and 3.60 ng respectively. The RSDs for intra- and inter-day repeatability was not more than 1.09% and 1.79% respectively. The overall recovery of the assay was 96.9-105.2%. The sample was stable for at least 12 h. The newly established method was successfully applied to quantify six flavonoids in different parts of "Bawanghua", and the commercial samples from different locations.     

A laboratory assessment of the potential of different strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in stored cowpea

Twelve indigenous and exotic isolates of Beauveria bassiana and Metarhizium anisopliae were evaluated for their virulence and their ability to suppress populations of Callosobruchus maculatus in stored cowpea. LT₅₀ values ranged from 3.11 to 6.12 days following immersion in aqueous suspensions containing 1×10⁸ conidia ml⁻¹. Indigenous isolates that had been recovered from C. maculatus were more virulent in laboratory bioassays than exotic isolates from other insects. The two isolates with the shortest LT₅₀ values were compared in dose-response assays by immersion and by exposure to cowpea grains treated with dry conidia. In both assays B. bassiana 0362 was consistently more virulent than M. anisopliae 0351. By immersion, LC₅₀ values on day 6 post-treatment were 9.10×10⁴ and 7.10×10⁵ conidia ml⁻¹ for B. bassiana 0362 and M. anisopliae 0351, respectively. Exposure to treated grains gave LC₅₀ values of 1.15×10⁷ and 4.44×10⁷ conidia g⁻¹ grain for B. bassiana 0362 and M. anisopliae 0351, respectively. In 1 kg batches of cowpea stocked with 50 adult C. maculatus, B. bassiana 0362 at both 1×10⁷ and 1×10⁸ conidia g⁻¹ grain led to significant adult mortality and reduced F₁ emergence relative to untreated populations. At 1×10⁸ conidia g⁻¹ the effect of the fungus persisted into the F₁ generation. The net reproductive rates, R₀, measured 26 days after insects were released were 5.16 and 7.32 for the high and low doses compared to 9.52 for the untreated control.     

高效液相色谱法测定维生素K胶囊中维生素K2含量不确定度评估

目的评估高效液相色谱法测定维生素K胶囊中的维生素K_2含量的不确定度。方法采用高效液相色谱法测定维生素K胶囊中的维生素K_2含量,建立数学模型,对测定过程的主要不确定度分量进行计算,得出合成不确定度和扩展不确定度。结果维生素K胶囊中维生素K_2的含量为181.2μg/g时,在置信区间95%下,其扩展不确定度为7.5μg/g(k=2)。结论不确定度的主要来源为标准曲线配制的过程,该评估模型为维生素K胶囊中维生素K_2的含量测定的准确性提供了参考依据。     

Production of exopolysaccharides by Lactobacillus and Bifidobacterium strains of human origin, and metabolic activity of the producing bacteria in milk

This work reports on the physicochemical characterization of 21 exopolysaccharides (EPS) produced by Lactobacillus and Bifidobacterium strains isolated from human intestinal microbiota, as well as the growth and metabolic activity of the EPS-producing strains in milk. The strains belong to the species Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus vaginalis, Bifidobacterium animalis, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum. The molar mass distribution of EPS fractions showed 2 peaks of different sizes, which is a feature shared with some EPS from bacteria of food origin. In general, we detected an association between the EPS size distribution and the EPS-producing species, although because of the low numbers of human bacterial EPS tested, we could not conclusively establish a correlation. The main monosaccharide components of the EPS under study were glucose, galactose, and rhamnose, which are the same as those found in food polymers; however, the rhamnose and glucose ratios was generally higher than the galactose ratio in our human bacterial EPS. All EPS-producing strains were able to grow and acidify milk; most lactobacilli produced lactic acid as the main metabolite. The lactic acid-to-acetic acid ratio in bifidobacteria was 0.7, close to the theoretical ratio, indicating that the EPS-producing strains did not produce an excessive amount of acetic acid, which could adversely affect the sensory properties of fermented milks. With respect to their viscosity-intensifying ability, L. plantarum H2 and L. rhamnosus E41 and E43R were able to increase the viscosity of stirred, fermented milks to a similar extent as the EPS-producing Streptococcus thermophilus strain used as a positive control. Therefore, these human EPS-producing bacteria could be used as adjuncts in mixed cultures for the formulation of functional foods if probiotic characteristics could be demonstrated. This is the first article reporting the physicochemical characteristics of EPS isolated from human intestinal microbiota.     

The potential of Cycloclasticus and Altererythrobacter strains for use in bioremediation of petroleum-aromatic-contaminated tropical marine environments

Cycloclasticus sp. A5, which has been suggested to be a major degrader of petroleum aromatics spilled in temperate seas, showed higher degrading activities for petroleum aromatics, at both 25 °C and tropical sea temperature 30 °C, than the novel aromatic-degrading isolates, related to Altererythrobacter epoxidivorans (97.5% similarity in the almost full-length 16S rRNA gene sequence) and Rhodovulum iodosum (96.3% similarity), obtained after enrichment on crude oil in a continuous supply of Indonesian seawater. Cycloclasticus A5 degraded petroleum aromatics at a similar rate or faster at 30 °C as compared to 25 °C, but its growth on acetate was severely inhibited at 30 °C. These results suggest that, although their abundance would be low in tropical seas not contaminated with aromatics, the Cycloclasticus strains could be major degraders of petroleum aromatics spilled in tropical seas. The 16S rRNA gene of the Cycloclasticus strains has been identified from Indonesian seawater, and the gene fragments showed 96.7−96.8% similarities to that of Cycloclasticus A5. Introducing Cycloclasticus A5 may be an ecologically advantageous bioremediation strategy for petroleum-aromatic-contaminated tropical seas because strain A5 would disappear at 30 °C after complete consumption of the aromatics. Altererythrobacter and Rhodovulum-related isolates grew well on pyruvate in 10% strength marine broth at 30 °C whereas Cycloclasticus A5 did not grow well on acetate in the broth at 30 °C. These growth results, along with its petroleum-aromatic-degrading activity, suggest that the Altererythrobacter isolate could be an important petroleum-aromatic degrader in and around nutrient-rich tropical marine environments.