Unusual sesquiterpene lactones with a new carbon skeleton and new acetylenes from Ajania przewalskii

Four new compounds, a noreudesmanolide (1), a guaianolide (4), and acetylenes (7–8), together with 16 other known compounds, were isolated from Ajania przewalskii. The novel sesquiterpenolide (1) possesses a rare carbon skeleton. Acetylation of 1 gave 1a. New compounds were elucidated as 1β-hydroxyl-2-noreudesm-4(15)-en-5α,6β,7α,11αH-12,6-olide (1), 1β-acetoxyoxyl-2-noreudesm-4(15)-en-5α,6β,7α,11αH-12,6-olide (1a), 8α-angeloyloxyl-3α,4α-dihydroxyguaia-1,9,11(13)-trien-6,12-olide (4), (E)-3β,4α-dihydroxyl-2-(2′,4′-hexadiynylidene)-1,6-dioxaspiro[4,5]decane (7) and 2-hydroxyl-2-[(E)-1α,2β,3-trihydroxyl-3-nonaene-5,7-diyne]-4H-pyran (8), by chemical and spectroscopic methods, including HRESIMS, 1D and 2D NMR. Cytotoxicity of nine compounds was assessed by their effects on selected cancer cell lines, K562, K562/ADM, BGC-823, and Hep-G2 cells. Phenolic acid 12 exhibited strong activity against K562 and K562/ADM cells and sesquiterpenolide 3 strong activity against K562/ADM cells. Radical-scavenging activities of 12 compounds, the mixed solvent extract (petrol ether–ether–methanol = 1:1:1), and the n-butanol extract were determined by ABTS and DPPH radical-scavenging assays. Phenols and coumarins (11–16), MSE, and n-BE displayed significant antioxidation (IC₅₀ < 20 μg/ml).     

In vitro evaluation of red and green lettuce (Lactuca sativa) for functional food properties

Lettuce (Lactuca sativa) is an important leafy vegetable consumed fresh or in salad mixes. We have compared the functional food properties of selected commercial red and green lettuce varieties grown under field conditions. Both lettuce cultivars were extracted with water at biological (38 °C) and room temperatures (22 °C) at pH 2. The residues from each extraction were further extracted, sequentially with methanol and ethyl acetate. The extracts were evaluated for their in vitro lipid peroxidation (LPO) and cyclooxygenase enzyme (COX) inhibitory activities. Amongst the extracts tested, all three extracts of red lettuce showed higher LPO and COX-1 and -2 enzyme inhibitory activities than did the green lettuce extracts. Red lettuce contained a single anthocyanin, cyanidin-3-O-(6″-malonyl-β-glucopyranoside) (1), which immediately converted to cyanidin-3-O-(6″-malonyl-β-glucopyranoside methyl ester) (2) and cyanidin-3-O-β-glucopyranoside (3) under laboratory conditions. Anthocyanins 1 and 2 inhibited LPO by 88% and 91.5%, respectively, at 0.25 μM concentration. Also, they inhibited COX-2 enzyme by 78.9% and 84.3% and COX-1 by 64% and 65.8%, respectively, at 5 μM. The chicoric acid (4), amongst other phenolics, such as quercetin glucoside, ferulic and caffeic acids, isolated from both green and red lettuce, showed 85.6%, 45.6% and 94% of LPO, COX-1 and -2 enzyme inhibitions at 50 μM, respectively. This is the first report of the LPO, COX-1 and -2 enzyme inhibitory activities of compounds 1, 2 and 4. The variation of phenolics in the red and green lettuces, and specifically the lack of anthocyanins in green lettuce, might account for the higher biological activity obtained with the red variety in our study.     

Effect of Zataria multiflora Boiss. essential oil and nisin on Salmonella typhimurium and Staphylococcus aureus in a food model system and on the bacterial cell membranes

The effects of different concentrations of Zataria multiflora Boiss. essential oil (EO: 0, 5, 15 and 30 μl 100 ml⁻¹) and nisin (N: 0, 0.25 and 0.5 μg ml⁻¹), temperatures (T: 25 and 8 °C), and storage times (up to 21 days) on growth of Salmonella typhimurium and Staphylococcus aureus in a commercial barley soup were evaluated in a factorial design study. The growth of S. typhimurium was significantly (P < 0.05) decreased by EO concentrations and their combinations with N concentrations at 8 °C. For S. aureus, the viable count was significantly (P < 0.05) inhibited by EO and N concentrations and their combinations, incubated at both storage temperatures. The mechanism of the antimicrobial action of EO, N, and their combinations against cell membranes of the tested organisms were also studied by measurement of the release of cell constituents and by the electronic microscopy observations of the cells. The significant increase of the cell constituents’ release of both organisms was observed as a result of treatments with EO and EO in combination with N. Electronic microscopy observations revealed that the cell membranes of S. typhimurium treated by EO and EO in combination with N were significantly damaged, while cells treated with only N looked similar to untreated cells. The electron micrographs of treated cells of S. aureus with EO, N, and their combination also showed important morphological damages and disrupted membranes.     

Bioactive dammarane-type triterpenoids derived from the acid hydrolysate of Gynostemma pentaphyllum saponins

From an acid hydrolysate of the crude saponins of Gynostemma pentaphyllum, three triterpenes, including a new compound (23S)-3β-hydroxydammar-20,24-dien-21-oic acid 21,23-lactone (1) and two known aglycons (20S, 23R)-3β,20β-dihydroxydamma-24-dien-21-oic acid 21,23-lactone (2) and (20S, 24S)-20,24-epoxydammarane-3β,12β,25-triol (3), were isolated. Their structures were established on the basis of extensive spectral evidence (HR-ESI-MS, IR, 1D and 2D-NMR experiments). In bioactive assays in vitro, compound 1 was found to have potent cytotoxicity against the human breast cancer cells MDA-MB-435, whereas compounds 2 and 3 exhibited modest inhibitory activity toward porcine pancreatic lipase. The results indicated that acid treatment of G. pentaphyllum extract could produce diverse structures with interesting bioactivity.     

Modeling within the Bayesian framework, the inactivation of pectinesterase in gazpacho by pulsed electric fields

The inactivation of pectinesterase (PE) activity in gazpacho (a Spanish ready-to-use cold vegetable soup) under pulsed electric fields (PEFs) was studied. Samples were exposed to 4 μs monopolar or bipolar square-wave pulses at 5–35 kV cm⁻¹ electric field intensity for up to 1500 μs and 200 Hz (temperature below 40 °C). Inactivation of PE was greater when treatment time and electric field intensity increased, and bipolar pulses were more effective inactivating PE than monopolar ones. Estimation of parameters and quantities involved in the tested models were performed within the Bayesian framework. The kinetic evolution of the enzyme was explained using a 3-parameter biexponential model based on a mechanism involving two irreversible consecutive steps. Rate constant k₁ was not dependent on neither electric field intensity nor pulse polarity. Rate constant k₂ and ratio between the activities of intermediate forms and the native ones of the enzyme Λ were affected by those conditions.     

Microbial profiles of frozen trimmings and silver sides prepared at Indian buffalo meat packing plants

To assess microbiological quality of buffalo meat trimmings (TT = 114) and silver sides (SS = 41), samples were collected from four different Indian meat packing plants. The aim of this study was: (i) to evaluate standard plate count (SPC), psychrotrophic count (PTC), Enterococcus feacalis count (EFC), Staphylococcus aureus count (SAC) and Escherichia coli count (ECC) and the presence of Salmonella spp. and Listeria monocytogenes; and (ii) also to determine vero toxic E. coli (VTEC) by polymerase chain reaction (PCR). TT samples had significantly higher (P < 0.001) SPC, PTC, EFC, and SAC than SS, while across the meat types there was no difference (P > 0.05) in ECC. E. coli was recovered from 32.4% TT and 19.5% SS samples. The prevalence rate of Salmonella spp. and L. monocytogenes in TT was 1.75% and 0.87%, respectively. But no SS sample was found to be positive for any of these two pathogens. VTEC was found in 2.58% of all the tested samples. This finding suggests that TT contain higher microbes but only small numbers of pathogens of latent zoonotic importance. The present study confirmed the importance of maintaining good process hygiene at meat plants for microbiological status of buffalo meat.     

Water-extractable phytochemicals from Phyllanthus niruri exhibit distinct in vitro antioxidant and in vivo hepatoprotective activity against paracetamol-induced liver damage in mice

The antioxidative and hepatoprotective potential of Phyllanthus niruri, a widely used medicinal plant in Brazil, were investigated. The aqueous extracts of leaves showed inhibition against thiobarbituric acid-reactive species (TBARS), induced by different pro-oxidants (10 μM FeSO₄ and 5 μM sodium nitroprusside) in rat liver, brain and kidney homogenates. The extracts also lowered the formation of TBARS in phospholipids extracted from egg yolk. The plant exhibited strong antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl free radical (IC₅₀, 43.4 ± 1.45 μg/ml) and iron chelation assay. The hepatoprotective activity of the extracts were also demonstrated in vivo against paracetamol-induced liver damage, as evidenced by the decrease in serum glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT), and increased catalase activity in the liver in treatment groups, compared to the control. The results of the present study suggest the potential use of P. niruri in the treatment of various diseases, among which liver disease is the most important, due to its ability to act as an antioxidant. Furthermore, since the treatment of human intoxications with paracetamol is always limited to the administration of N-acetyl-cysteine, additional studies would be important to determine whether aqueous extracts of P. niruri could increase the efficacy of N-acetyl-cysteine against paracetamol acute hepatotoxicity.     

Optimisation of antioxidant activity of grape cane extracts using response surface methodology

Solid–liquid extraction and response surface methodology were used to optimise conditions for the antioxidant activity of grape cane extracts. The independent processing variables were ethanol concentration, temperature and solvent to solid ratio. Ethanol concentration and temperature significantly affected antioxidant activity measured by the Trolox equivalent antioxidant capacity (TEAC) assay and the oxygen radical absorbance capacity using fluorescein (ORAC_FL) method (p  0.01), whereas the solvent to solid ratio did not significantly affect the activity (p > 0.05). Antioxidant activity of the extracts, determined by the TEAC assay, varied from 85.6 to 238.6 μmol Trolox equivalents/g of dry sample. ORAC_FL values ranged from 308.4 to 1302.7 μmol Trolox equivalents/g of dry sample. Ethanol concentrations of 40.4% and 55.4% were optimal for the highest antioxidant activities measured by the TEAC assay and the ORAC_FL method, respectively. The optimal temperature was 83.6 °C. Antioxidant activity correlates with total phenolic content of extracts.     

Improving the enrichment procedure for Enterobacteriaceae detection

The current ISO standard method for detection of Enterobacteriaceae (21528-1:2004) includes enrichment in EE broth which has been shown to be inhibitory to some members of this family, notably Cronobacter spp. A shortened procedure omitting the EE broth has been proposed, however competition from Gram-positive flora may be detrimental to the effective recovery of low levels of target organisms in some sample matrices. In this study we investigated novel cost effective modifications, designed to improve ISO 21528-1:2004 for the detection of Enterobacteriaceae. Initial experiments used a worse-case scenario involving stressed Enterobacteriaceae strains known to grow poorly in laboratory media as well as representative background competitors from powdered milk. The interaction between the Enterobacteriaceae and their competitors was characterised and additives to enhance the growth of target strains over non-target strains were investigated.Supplementation of BPW with 40 μM 8-hydroxyquinoline, 0.5 g L⁻¹ ammonium iron(III) citrate, 0.1 g L⁻¹ sodium deoxycholate and 0.1 g L⁻¹ sodium pyruvate (BPW-S) improved the recovery of Enterobacteriaceae from artificially and naturally contaminated samples. This improvement of the pre-enrichment broth may also be of interest for methods designed to detect specific foodborne pathogens belonging to the Enterobacteriaceae (e.g. Salmonella spp., Cronobacter spp.) that require a pre-enrichment step in BPW.     

Repellent activity of essential oils from seven aromatic plants grown in Colombia against Sitophilus zeamais Motschulsky (Coleoptera)

Essential oils isolated from seven aromatic plants grown in Colombia were analyzed by gas chromatography–mass spectrometry (GC–MS) and evaluated for repellent activity against Sitophilus zeamais (Coleoptera: Curculionidae) using the area preference method. Most oil components were oxygenated monoterpenoids or phenolic compounds. Six oils were repellent, with Lippia origanoides the most active. Eucalyptus citriodora and Tagetes lucida were also repellant at doses between 0.063 and 0.503 μL/cm².     

Consumer acceptance and steak cutting yields of beef top sirloin and knuckle subprimals

Beef knuckles (n = 150) and center-cut top sirloin butts (n = 150) were used to determine portion-controlled steak cutting yields, palatability characteristics, and consumer acceptance of rectus femoris (RF), vastus lateralis (VL), and gluteus medius (GM) steaks. Steak yields were higher (P < 0.05) for top sirloins than knuckles. Trained sensory panel ratings for overall tenderness, juiciness, and flavor were similar between RF and GM. Consumer panel ratings for tenderness and juiciness were higher (P < 0.05) for GM than RF; however, consumer perceptions of overall like and flavor were similar for GM and RF. Vastus lateralis received lower (P < 0.05) trained panel and consumer ratings for all traits than either RF or GM. Palatability of VL will need improvement to be a viable foodservice offering. Yet, these data suggest that RF would amply substitute for GM in foodservice settings, and that knuckle steak yields would be adequate for foodservice applications.     

Inhibitory effect of Antrodia camphorata constituents on the Helicobacter pylori-associated gastric inflammation

Helicobacter pylori infection plays a crucial role in the pathogenesis of peptic ulcer, and gastric cancer. Among the 13 chemical constituents isolated from fruiting bodies of Antrodia camphorata, methyl antcinate B (4), antcins K (10) and A (12) displayed potential anti-H. pylori activity with inhibition zones of 13, 12 and 10 mm, respectively, at a concentration of 0.2 mM. The isolates 4 and 10 exhibited a dose response inhibition of H. pylori adhesion and invasion to AGS cells in a range of concentrations between 0.005 and 0.02 mM, while 12 has moderate effect at relatively higher concentration. Furthermore, at these concentrations (0.005–0.02 mM) the isolates 4 and 10 also inhibited the H. pylori-induced nuclear factor (NF)-кB activation, and the subsequent release of interleukin (IL)-8 in AGS cells. These results open the possibility of considering A. camphorata a chemopreventive agent for peptic ulcer or gastric cancer, but this bioactivity should be confirmed in vivo in the future.     

Bioactivity of Ocimum gratissimum L. oil and two of its constituents against five insect pests attacking stored food products

The fumigant and repellent effects of Ocimum gratissimum L. oil and its constituents, β-(Z)-ocimene and eugenol, were evaluated against adults of Sitophilus oryzae (L.), Tribolium castaneum (Herbst), Oryzaephilus surinamensis (L.), Rhyzopertha dominica (F.) and Callosobruchus chinensis (L.). The fumigant toxicities of the oil and two of its constituents were assessed at four rates (0, 1, 5 and10 μL/L air) in space fumigation, whereas repellence of the oil and eugenol in acetone was evaluated in choice bioassays at five rates (0, 1.0, 2.0, 3.0 and 4.0 μL oil/2 g grain). Results showed that fumigant toxicity and repellence of the oil and its constituents were significantly (P<0.0001) influenced by concentration and time after treatment. At 1 μL/L air, the oil caused 98%, 99% and 100% mortality of R. dominica, O. surinamensis and C. chinensis, respectively, 24 h after treatment, whereas eugenol achieved 79%, 61% and 100% kill of the same insects. β-(Z)-ocimene produced a weaker toxicity with 8%, 11% and 59% mortality of R. dominica, O. surinamensis and C. chinensis, respectively. Except for T. castaneum which was more tolerant, LC₅₀ values for tested insects ranged from 0.20 to 14, 0.01 to 17 and 0.80 to 23 μL/L air 24 h after treatment for O. gratissimum oil, eugenol and β-(Z)-ocimene, respectively. All test insects had percentage repellence (PR) values which ranged from 37.5% to 100% and 45% to 100% for the oil and eugenol, respectively. However, C. chinensis showed a dose-dependent decrease in PR values in the eugenol bioassay with a corresponding dose-dependent mortality on treated grains. Ocimum gratissimum oil and its constituents are potential alternatives to synthetic fumigants in the treatment of durable agricultural products. Successful adoption of plant oils in the protection of food commodities promises an eco-friendly option compatible with international biosafety regulations.     

Biochemical and thermal characterization of crude exo-polygalacturonase produced by Aspergillus sojae

Crude exo-polygalacturonase enzyme (produced by Aspergillus sojae), significant for industrial processes, was characterized with respect to its biochemical and thermal properties. The optimum pH and temperature for maximum crude exo-polygalacturonase activity were pH 5 and 55 °C, respectively. It retained 60–70% of its activity over a broad pH range and 80% of its initial activity at 65 °C for 1 h. The thermal stability study indicated an inactivation energy of E_d = 152 kJ mol⁻¹. The half lives at 75 and 85 °C were estimated as 3.6 and 1.02 h, respectively. Thermodynamic parameters, ΔH^*, ΔS^* and ΔG^*, were determined as a function of temperature. The kinetic constants K_m and V_max, using polygalacturonic acid as substrate, were determined as 0.424 g l⁻¹ and 80 μmol min⁻¹, respectively. SDS-PAGE profiling revealed three major bands with molecular weights of 36, 53 and 68 kDa. This enzyme can be considered as a potential candidate in various applications of waste treatment, in food, paper and textile industries.     

Growth and metabolic behaviour of Lactococcus lactis subsp. lactis IPLA 947 in anaerobic lactose-limited chemostat cultures

Lactococcus lactis subsp. lactis IPLA 947 is a starter strain isolated from a Spanish farmhouse acid-coagulated cheese. To use this strain as starter culture in Afuegal Pitu cheese manufacture, anaerobic lactose-limited chemostat cultures were used to optimise the active biomass production in a simple medium (BRFS). Growth, lactose consumption and metabolite production were measured at different pH values (5.5, 6.0 and 6.5) and dilution rates (0.06 to 0.96 h^–1). The influence of both variables on the quoted parameters was described by means of mathematical models. These provided a satisfactory representation of the experimental data. The bacterial response surface showed a significant increase in the biomass when the pH increased and the dilution rate decreased. As dilution rate increased, biomass yield increased but only at the lowest pH. The lactose consumption and the ratio of metabolic end products were also influenced by both variables. As dilution rate decreased, the residual lactose decreased and increased amounts of metabolites other than lactate (acetate, formate, ethanol) were produced. The minimum lactose consumption was obtained at pH 5.5 and 0.96 h^–1 dilution rate. A significant rise of acetate and formate levels occurred as pH increased. A maximum lactate level, along with an acetate, formate and ethanol production close to the minimum, was predicted by mathematical modelling at 0.5173 h^–1 dilution rate and pH 6.054.

Fractionation and identification of ACE-inhibitory peptides from α-lactalbumin and β-casein produced by thermolysin-catalysed hydrolysis

The thermolysin catalysed hydrolysates of α-lactalbumin and β-casein were fractionated by size-exclusion chromatography (SEC) and reversed-phase high performance liquid chromatography (RP-HPLC) in order to identify the peptides responsible for the high ACE-inhibitory activity of these hydrolysates. The SEC fractionation separated many co-eluting peptides into different fractions allowing individual peptides to be isolated in one or two subsequent semi-preparative RP-HPLC fractionation steps. Five potent ACE-inhibitory peptides from α-lactalbumin were isolated. They all contained the C-terminal sequence -PEW, corresponding to amino acid residues 24–26 in α-lactalbumin, and had IC₅₀ values of 1–5 μm. From one SEC fraction of the β-casein hydrolysate two potent ACE-inhibitory peptides were isolated and identified as f58-76 and f59-76 of β-casein A2. They both contained IPP as the C-terminal sequence and had IC₅₀ values of 4 and 5 μm. From another SEC fraction a new but less ACE-inhibitory peptide from β-casein was identified (f192–196; LYQQP).     

Production and stability of chlorine dioxide in organic acid solutions as affected by pH, type of acid, and concentration of sodium chlorite, and its effectiveness in inactivating Bacillus cereus spores

We studied the production and stability of chlorine dioxide (ClO₂) in organic acid solutions and its effectiveness in killing Bacillus cereus spores. Sodium chlorite (5000, 10,000, or 50,000 μg/ml) was added to 5% acetic, citric, or lactic acid solution, adjusted to pH 3.0, 4.0, 5.0, or 6.0, and held at 21 °C for up to 14 days. The amount of ClO₂ produced was higher as the concentration of sodium chlorite was increased and as the pH of the acid solutions was decreased. However, the stability in production of ClO₂ was enhanced by increasing the pH of the organic acid solutions. To evaluate the lethal activity of ClO₂ produced in various acid solutions as affected by acidulant and pH, suspensions of B. cereus spores were treated at 21 °C for 1, 3, 5, or 10 min in hydrochloric acid or organic acid solutions (pH 3.0, 4.0, 5.0, or 6.0) containing ClO₂ at concentrations of 100, 50, or 25 μg/ml. Populations of viable spores treated with ClO₂ at concentrations of 100 or 50 μg/ml in organic acid solutions decreased more rapidly than populations treated with the same concentrations of ClO₂ in HCl. Rates of inactivation tended to increase with higher pH of ClO₂ solutions. Results show that ClO₂ formed in organic acid solutions has higher stability and is more lethal to B. cereus spores than ClO₂ formed at the same concentration in HCl solution. This finding emphasizes the benefits of using organic acid solutions to prepare ClO₂ intended for use as an antimicrobial.     

Protection of Lactobacillus acidophilus from the low pH of a model gastric juice by incorporation in a W/O/W emulsion

The protective effect of a W/O/W emulsion for Lactobacillus acidophilus from a model gastric juice was investigated in order to develop a method for utilizing the advantages of the probiotics. The bacteria were included in the inner-phase solution of the W/O/W emulsion, and a method for counting the viable-bacteria included in the W/O/W emulsion was developed. The relative viability of the bacteria included in the W/O/W emulsion was 49% at 2 h in the model gastric juice, whereas the viability of the bacteria directly dispersed in the juice declined to 1.3% even at 0.67 h. The relative viabilities of the encapsulated bacteria in the model gastric juice at 2 h were 0.12 and 1.10 for the emulsions having the median diameters of 11.9 and 25.4 μm, prepared with inner-phase volume ratios at 0.03 and 0.45, respectively. The relative viabilities of the bacteria in the W/O/W emulsions with the median diameters of 11 and 27 μm, prepared at the homogenization speed of 2.2×10⁴ and 9.8×10³ rpm, were 0.4 and 0.8, respectively. These results suggested that both the inner-phase volume ratio and the median diameter of oil droplet affected the relative viability of the included bacteria.     

Influence of freeze-drying conditions on survival of Oenococcus oeni for malolactic fermentation

Malolactic fermentation (MLF) is an important process in wine production. Oenococcus oeni is most often responsible for MLF. Starter culture technology, involving the inoculation of O. oeni into wines, has been developed for inducing MLF. In this study, the effects of cell washing, pH of suspension medium, preincubation in sodium glutamate, initial cell concentration and freezing temperature on viability of freeze-dried O. oeni H-2 were investigated. The cell viability of samples without washing with potassium phosphate buffer was significantly lower than those samples undergone washing. When pH of suspension medium was 7.0 the cell survival was the highest. The cell viability was enhanced when the cells were preincubated at 25 °C before freezing. When 2.5% sodium glutamate was used as protective agent in suspension medium, the optimal initial cell concentration was 10⁹ CFU/ml. The cell viability increased by 21.6% as freezing temperature decreased from − 20 °C to − 65 °C. However, when the cells were frozen in liquid nitrogen (− 196 °C), the cell survival significantly decreased.