Dark chocolates supplemented with <Emphasis Type="Italic">Lactobacillus</Emphasis> strains

Dark chocolate masses and chocolates were supplemented with viable cells of two bacterial strains Lactobacillus caseii and Lactobacillus paracasei with potential probiotic properties, which were lyophilized in milk. Total number of live bacteria in the lyophilizate was 7.9×10⁹ cfu/g. Sucrose or isomalt and aspartame were used as bulking substances and sweeteners. Sensory attributes of these chocolates were not different from that of traditional chocolates. Calorie value of sucrose-free chocolate was lower by approximately 11.1–14.6% (dependent on their formulation) relative to chocolate sweetened with sucrose. Chocolate, which contains isomalt and aspartame can be consumed by diabetics. Numbers of live L. casei and L. paracasei cells in the examined batches of chocolate were very high and approached 10⁶–10⁷ cfu/g after 12 months of keeping at 4 and 18 °C. Neither the texture nor the total and volatile acidity of chocolate masses were changed by addition of the lyophilized preparation of Lactobacillus cells. Casson yield values of dark sucrose-free chocolate masses supplemented with this lyophilizate were decreased by approximately 3–55% (dependently on fat contents in these masses) as compared to that of analogous chocolate masses sweetened with sucrose.     

Volatile flavor composition of <Emphasis Type="Italic">gamguk</Emphasis> (<Emphasis Type="Italic">Chrysanthemum indicum</Emphasis>) flower essential oils

The volatile composition of the essential oil from fresh gamguk (Chrysanthemum indicum) flowers was investigated. The volatile constituents were extracted by the hydro distillation method. Volatile compositional changes of gamguk prepared via different drying methods (shade- and freeze-drying methods) were also determined. Total 36, 63, and 55 volatiles constituents were confirmed in the essential oil from fresh and shade-, and freeze-dried flowers. Ketones were predominant in the volatiles of gamguk flowers (%): fresh, 43.8; shade dried, 30.3; and freeze dried, 36.1. Camphor was the most abundant volatile component in all samples, and the content of borneol was also remarkable. The content of camphor was higher in fresh sample than those of dried samples while borneol concentration was significantly increased in the dried samples.     

Characterization of moisture content and specific gravity of branchwood and stemwood of <Emphasis Type="Italic">Aningeria robusta</Emphasis> and <Emphasis Type="Italic">Terminalia ivorensis</Emphasis>

Aningeria robusta and Terminalia ivorensis of diameters ranging from 10 cm to 25 cm were examined. The following results were obtained: The overall (sapwood and heartwood combined) moisture contents of the branchwood of both Aningeria robusta and Terminalia ivorensis were significantly greater than those of their corresponding stemwood, with the branchwood of Terminalia ivorensis being the highest. It was also observed that the overall specific gravity of the branchwoods of both species was higher than that of the corresponding stemwood, with the branchwood of Aningeria robusta exhibiting the highest specific gravity. Detailed analyses of moisture content distribution and specific gravity of the heartwood and sapwood of Aningeria robusta and Terminalia ivorensis are also presented.     

Effect of ionic liquid-containing system on betulinic acid production from betulin biotransformation by cultured <Emphasis Type="Italic">Armillaria luteo</Emphasis>-<Emphasis Type="Italic">virens</Emphasis> Sacc cells

The aim of this study is to develop an ionic liquid-containing system for efficient production of betulinic acid by cultured cells Armillaria luteo-virens Sacc ZJUQH100-6. Several parameters affecting betulinic acid formation in the IL-containing system were investigated. The addition of [EMIM][BF₄] in hexane-containing reaction medium gave rise to better betulinic acid formation in comparison with other ILs used. The optimal concentration of IL in IL-containing co-solvent system is 50% (v/v). As a co-substrate, butanol is found to be useful for intracellular betulin-28-monooxygenase synthesis during the whole phase. The concentrations of substrate and resting cells have been found to exert a significant effect on betulinic acid. Moreover, the reaction time in this IL-containing system was less than that in the conventional one. The effect of the constructed IL-containing system on cell membrane structure was comparatively observed. Under the developed IL-containing system, the highest yield of product observed was 11.14% at 18 h, higher than that in monophase aqueous one (P < 0.05), whereas the activity of monooxygenase showed the same variation as betulinic acid formation.     

The ability of <Emphasis Type="Italic">Lactobacillus</Emphasis> adhesin EF-Tu to interfere with pathogen adhesion

Probiotic bacteria play an important role in preventing widespread colonization of enteropathogens in human gastrointestinal tract. Lactobacillus plantarum CS24.2, isolated from child fecal sample, and L. rhamnosus GG, a widely accepted commercial probiotic strain, were examined in vitro for their ability to inhibit the colonization of enteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhi (S. Typhi) on human intestinal epithelial cell line (Caco-2). Different adhesion assays such as competitive inhibition, adhesion inhibition, and displacement were carried out for the assessment of antagonistic activity of probiotic bacteria toward adhesion of enteropathogens to Caco-2 cells. Both the probiotic bacteria were able to inhibit pathogen colonization between 30–90% under different assay conditions. The gene coding for the known Lactobacillus adhesion factor—elongation factor Tu (EF-Tu)—was amplified from L. plantarum CS24.2 and cloned into E. coli expression vector, pET30(a). The partially purified recombinant EF-Tu was able to inhibit 50% adhesion of L. plantarum CS24.2 to Caco-2 cells but there was no effect on L. rhamnosus GG adhesion under competitive inhibition assay. To investigate the functional role of lactobacilli EF-Tu in pathogen inhibition to Caco-2 cells, competitive adhesion assay was carried out and there was significant reduction in the adhesion of E. coli (35.3%) and S. Typhi (47.7%) to Caco-2 cells.     

Resistance of Microencapsulated <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> LA1 to Processing Treatments and Simulated Gut Conditions

This investigation reports the effect of microencapsulation using sodium alginate and starch on the tolerance of probiotic Lactobacillus acidophilus LA1 to selected processing conditions and simulated gastrointestinal environments. The organism survived better in the protected form at high temperatures (72, 85, and 90 °C) and at high salt concentrations (1%, 1.5%, and 2%). The free cells were completely destroyed at 90 °C whereas the microencapsulated cells reduced by 4.14 log cycles. The log cycle reduction was 5.67 and 2.30, respectively, in free and protected cells when incubated for 3 h with 2% (w/v) NaCl. Homogenization did not affect the viability of the cells but led to the disruption of the protective encapsulating material around the cells. Microencapsulation provided better protection at simulated conditions of gastric pH (1.0, 1.5, and 2.0) and at high bile salt concentrations (1.0%, 1.5%, and 2.0%). The free and protected cells registered 5.47 and 2.16 log cycle reduction, respectively, after 3-h incubation at 2% bile salt (w/v). The release of the microencapsulated organisms in simulated colonic pH required 2.5 h. These studies demonstrated that microencapsulation of probiotic L. acidophilus LA1 in sodium alginate is an effective technique of protection against extreme processing conditions and under simulated gastrointestinal environment.     

Disinfection effect and its mechanism of electrolyzed oxidizing water on spores of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> var. <Emphasis Type="Italic">niger</Emphasis>

A study was carried out on the disinfection efficiency of electrolyzed oxidizing water (EOW) on spores of Bacillus subtilis var. niger. The results showed a remarkable fungicidal rate of 100% after 20 min duration of 191 mg/L active available chlorine (ACC). The disinfection effect was improved with increased ACC or prolonged disinfection time, while organic interferents exerted a strong concentration-dependent inhibition against the disinfection. The disinfection mechanism was also investigated at bio-molecular level. EOW decreased dehydrogenase activity, intensified membrane permeability, elevated suspension conductivity, and caused leakage of intracellular K⁺, proteins, and DNA, indicating a damage of cell walls and membranes. Effects of EOW on microbiological ultra-structures were also verified by transmission electronic microscopy (TEM) images, showing that EOW destroyed protective barriers of the microbe and imposed some damages upon the nucleus area.     

Purification of <Emphasis Type="Italic">S</Emphasis>-Alk(en)yl alka/enethiosulfinates of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.) by using recycling preparative HPLC

S-Alk(en)yl alka/enethiosulfinates formed in crushed garlic were purified by using recycling preparative HPLC. Allyl 2-propenethiosulfinate and methyl methanethiosulfinate were effective separated because no other thiosulfinates were coeluted with them. Allyl methanethiosulfinate and methyl 2-propenethiosulfinate, trans-1-propenyl methanethiosulfinate and methyl trans-1-propenethiosulfinate, (cis)- and (trans)-1-propenyl 2-propenethiosulfinate were eluted as 1 peak, respectively, and further separated by using additional kinds of column. Since the quantity of trans-1-propenyl trans-1-propenethiosulfinate (PPTHS) was small in garlic, PPTHS was isolated from a mixture of blanched onion homogenate and garlic alliinase and purified. Purified thiosulfinates can be employed for the study of antimicrobial activity, flavor, and greening of garlic.     

Antioxidant activity of half <Emphasis Type="Italic">N</Emphasis>-acetylated water-soluble chitosan in vitro

Different molecular weight water-soluble chitosan (half N-acetylated chitosan) was prepared and the structure of water-soluble chitosan was characterized by FT-IR. The pH dependence of water solubility of water-soluble chitosan was evaluated from turbidity. Total antioxidant activity, reducing power, superoxide anion radical and hydroxyl radical quenching assay, metal chelating activity, and H₂O₂ scavenging activity were used for the evaluation of different molecular weight half N-acetylated chitosan in vitro. Low-molecular weight water-soluble chitosan (WSC4) exhibited high reductive capacity and expressed good inhibition of linoleic acid peroxidation in the linoleic acid model system. WSC4 (0.25 mg/mL) scavenged 78.8% of superoxide radical. At 5 mg/mL, scavenging percentage of WSC1, WSC2, WSC3, and WSC4 against hydroxyl radical was 49.3%, 66.8%, 77.1%, and 83.7%, respectively. These results indicate that water-soluble chitosan is an ideally natural antioxidant, and its antioxidant activity depends on its molecular weight.     

Cold-adapted structural properties of trypsins from walleye pollock (<Emphasis Type="Italic">Theragra chalcogramma</Emphasis>) and Arctic cod (<Emphasis Type="Italic">Boreogadus saida</Emphasis>)

Complementary DNA clones encoding trypsins were isolated from pyloric ceca of cold-adapted fish, walleye pollock (Theragra chalcogramma) (WP-T) and Arctic cod (Boreogadus saida) (AC-T). The isolated full-length cDNA clones of WP-T and AC-T were 852 and 860 bp, respectively, and both cDNAs were contained an open reading frame of 726 bp. WP-T and AC-T seemed to be synthesized as preproenzyme that contains a signal peptide, an activation peptide, and a mature trypsin. Although the amino acid sequence identities of WP-T and AC-T to that of bovine trypsin were 64 and 63%, respectively, they completely conserved the structural features for catalytic function of trypsin. On the other hand, WP-T and AC-T possessed the four Met residues (Met135, Met145, Met175 and Met242) in their molecules and the deletion of Tyr151 and substitution of Pro152 for Gly in their autolysis loops when aligned with the sequences of tropical-zone fish and bovine trypsins. In addition, the contents of charged amino acid residues at the N-terminal regions (positions 20–50) of WP-T and AC-T were extremely higher than those of other fish and bovine trypsins. Moreover, one amino acid (Asn72) and two amino acids (Asn72 and Val75) coordinating with Ca²⁺ in bovine trypsin were exchanged for another amino acids in WP-T (His) and AC-T (His and Glu), respectively, and the contents of negative charged amino acids at their Ca²⁺-binding regions were lower than those of tropical-zone fish and bovine trypsins. Therefore, it was considered that these structural characteristics of WP-T and AC-T are closely related to their lower thermostability.     

<Emphasis Type="Italic">In silico</Emphasis> phylogenetic analysis of lactic acid bacteria and new primer set for identification of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> in food samples

Lactic acid bacteria (LAB) include species very closely related both physiologically and genotypically. Therefore, the identification of this bacteria group using conventional phenotypic methods is ambiguous and cumbersome. In this study, we have analyzed a recA gene fragment from 30 bacteria, including LAB and species common in the human gastrointestinal tract, aiming to evaluate the gene conservation among them and the development of primers and PCR conditions able to discriminate Lactobacillus plantarum strains from LAB closely related. The fragment with 995 bp of recA gene has grouped LAB, enterobacteria and bifidobacteria, in different clusters. A novel primer pair, LPrecAF and LPrecAR with 23 and 18 bp, respectively, has allowed the single amplification of a 108 bp fragment of L. plantarum strains contained in culture broth and fermented dairy samples. The observed detection limit for food samples and for cultures broth were 1 × 10³ and 7 × 10² CFU mL⁻¹, respectively. This approach proved to be a simple and efficient method for the identification and monitoring of L. plantarum in food, feeds, and culture broth. Moreover, the assay could be used in the studies from human or environmental microbiota.     

Antioxidant synergism between fruit juice and <Emphasis Type="Italic">α</Emphasis>-tocopherol. A comparison between high phenolic black chokeberry (<Emphasis Type="Italic">Aronia melanocarpa</Emphasis>) and high ascorbic blackcurrant (<Emphasis Type="Italic">Ribes nigrum</Emphasis>)

Black chokeberry juice (Aronia melanocarpa, Elliot), blackcurrant juice (Ribes nigrum, Ben Lomond) and α-tocopherol were found to protect phosphatidyl choline against oxidation in a peroxidating liposome system as evidenced by lag phases for formation of conjugated dienes. When present together, black chokeberry juice and α-tocopherol showed a clear synergistic effect on the length of the lag phase, while effects of blackcurrant juice and α-tocopherol were additive. The concentration of total phenolics in black chokeberry juice was six times higher than in blackcurrant juice (gallic acid equivalents). Ascorbic acid corresponded to 1% of total phenolics in black chokeberry juice and 10% in blackcurrant juice. Based on the length of the lag phase, the phenolics present in black chokeberry were on an average, twice efficient as scavengers of lipid peroxyl radicals as phenolics in blackcurrant. Black chokeberry was by HPLC analysis of peroxidating liposomes, in contrast to blackcurrant, found to protect α-tocopherol efficiently against oxidation to the end of the lag phase. The phenolics present in black chokeberry juice were concluded to be more efficient in regenerating or protecting α-tocopherol than ascorbic acid or the phenolics in blackcurrant. As for the phenolics, this was further evidenced by ranking of their radical scavenging activity as studied by ESR-spectroscopy.     

Evaluation of <Emphasis Type="Italic">flaA</Emphasis> Short Variable Region Sequencing,Multilocus Sequence Typing,and Fourier Transform Infrared Spectroscopy for Discrimination between <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> Strains

Discriminatory and robust typing methods are needed to improve the understanding of the dynamics of food-borne Campylobacter infections and epidemiology in primary animal production. To evaluate the strain discriminatory potential of typing methods, flaA short variable region (SVR) sequencing and Fourier transform infrared (FTIR) spectroscopy were applied on a collection of 102 epidemiologically related and unrelated Campylobacter jejuni strains. Previous application of FTIR spectroscopy for subtyping of Campylobacter has been limited. A subset of isolates, initially discriminated by flaA SVR sequencing, were further subjected to multilocus sequence typing (MLST). It was found that flaA SVR sequencing had a slightly higher discriminatory power than FTIR spectroscopy, based on the Simpson diversity index. The clustering of strains indicated that FTIR spectroscopy is indeed a suitable method for discrimination of Campylobacter. The isolates were assigned to six clusters based on flaA SVR sequences and nine clusters based on the FTIR spectroscopy profiles. Furthermore, the cluster analysis of flaA SVR sequences, MLST, and FTIR spectroscopy profiles showed a high degree of congruence, assigning the isolates to similar cluster structures. In conclusion, FTIR spectroscopy can be applied for subtyping of Campylobacter, and the high discriminatory potential of both flaA SVR sequencing and FTIR spectroscopy render them suitable screening methods for large numbers of strains.     

Iron(II)-induced isomerization of (<Emphasis Type="Italic">all</Emphasis>-<Emphasis Type="Italic">E</Emphasis>)-xanthophyll pigments lutein,zeaxanthin, and β cryptoxanthin in acetone

Lutein, zeaxanthin, and β-cryptoxanthin are xanthophyll pigments that own conjugated double bonds; some factors can convert (all-E)-xanthophylls to their (Z)-isomers such as high temperature, illumination, and oxidants. In the present work, the Fe(II)-induced isomerization of (all-E)-lutein, (all-E)-zeaxanthin, and (all-E)-β-cryptoxanthin in acetone were analyzed by HPLC coupled with DAD and APcI-MS. The three (all-E)-xanthophylls were baseline separated and their (Z)-isomers were identified by the chromatographic retention, UV/vis spectra and positive mass spectrometry with reference values reported in the literature. The results showed that Fe(II) exhibited a rapidly isomerization induction of (all-E)-xanthophylls to their (Z)-isomers exceeding a 8/1 mass ratio of Fe(II)/pigments. The (13-Z)-lutein and (13′-Z)-lutein were identified as the major (Z)-isomers of (all-E)-lutein treated with FeSO₄·7H₂O, and (13-Z)-zeaxanthin was identified as the major isomerization product of (all-E)-zeaxanthin. Furthermore, a type of (Z)-β-cryptoxanthin was also detected. Increasing either mass ratio of Fe(II)/pigments or the incubation time of FeSO₄·7H₂O and (all-E)-xanthophylls may affect on the stabilities of (Z)-isomers derived from (all-E)-xanthophylls.     

Investigation of dyes degradation intermediates with <Emphasis Type="Italic">Scytalidium thermophilum</Emphasis> laccase

Scytalidium thermophilum laccase was able to successfully decolourise Congo Red, Bromo-Cresol Green, Malachite Green, Phenol Red and Indigo Carmine under optimised conditions. The cited dyes belonging to three different classes were named azo, triarylmethane and indigoid. The decolourisation rates were 100, 95, 76, 57 and 22 mg h⁻¹ U⁻¹ for Indigo Carmine, Malachite Green, Bromo-Cresol Green, Congo Red and Phenol Red, respectively. The degradation products were characterised by UV–vis and FT-IR techniques, and their cytotoxicity was monitored. UV–visible absorption spectra and FT-IR analysis showed a complete degradation of Congo Red, Bromo-Cresol Green and Malachite Green, a partial degradation of Phenol Red and a transformation of Indigo Carmine. Toxicity study revealed that most of the treated dyes were less toxic than those before treatment, especially for Malachite Green. In fact, Scytalidium thermophilum laccase degraded Malachite Green into non-toxic products. Scytalidium thermophilum laccase constitutes a powerful tool for effective bioremediation of rich-dye textile effluents and was, therefore, found worthy of investigation for potential applications in restoration work and other biotechnological uses.     

Volatile compounds of traditional and virus-resistant breeding lines of <Emphasis Type="Italic">Muchamiel</Emphasis> tomatoes

Currently, sensory quality is the primary objective for almost all tomato breeding programs. In this study, postharvest behaviour of a breeding line with genetic resistance to important viruses (ToMV, TSWV, and TYLCV) has been compared with the original traditional landrace (Muchamiel) from an aromatic point of view. The breeding line has been obtained by backcrossing, introgressing three resistance genes but trying to keep the aromatic characteristics of the traditional variety. The main aim was to obtain qualitative (solid phase microextraction, SPME) and semi-quantitative (simultaneous steam-distillation extraction, SDE, and hydrodistillation, HD) volatile compositions of Muchamiel tomatoes. Fruits were randomly picked at a green-immature stage and stored at 10 °C during 13 days. Volatile compositions of tomatoes in two stages of ripening (green-immature and red-mature) were obtained by three different extraction methods: SDE, HD, and SPME. Besides, sensory evaluation with a trained panel was carried out on both types of tomatoes at two ripening stages. Main fresh tomato aroma compounds (cis-3-hexenal, hexanal, cis-3-hexanol, 6-methyl-5-hepten-2-one, 2-isobutylthiazole and β-ionone) were found at similar concentrations in both types of samples, confirming the success of the breeding program. Finally, SPME could be considered as a useful tool to get realistic values on fresh tomato odour, while HD and SDE results correlated better with fresh tomato aroma. Although a long time has been required to develop the breeding line, results indicate that sensory quality has been recovered through backcrossing, confirming the success of the breeding program.     

Chelating,antioxidant and antiproliferative activity of <Emphasis Type="Italic">Vicia sativa</Emphasis> polyphenol extracts

The metal chelating activity, antioxidant properties, and the effect on cell growth of a polyphenol extract from Vicia sativa have been investigated, and compared to those of soybean. The extracts from V. sativa seeds contained three and five times more polyphenols and flavonoids than soybean, respectively. The soybean polyphenol extracts showed higher copper and iron chelating activity than those from V. sativa, although polyphenols from V. sativa were more effective in preventing β-carotene oxidation and showed higher reducing power and scavenging activity than soybean polyphenols. In addition, V. sativa polyphenols were toxic to THP-1 leukemic cells, as opposed to polyphenols extracted from soybean that did not show any antiproliferative activity at similar concentrations. In conclusion, V. sativa polyphenol extracts show promising antioxidant and antiproliferative activities that may be of interest from a functional point of view and for the revalorization of this ancient crop.     

Optimization of carotenoids by <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis>

Response surface methodology was applied to maximize the yield and productivity of carotenoids by Rhodotorula glutinis strain 1151 using supplemented tomato waste based medium. Higher concentration of tomato waste extract and yeast extract favored the production of carotenoids. In contrast to carotenogenesis higher concentration of yeast extract negatively affected the formation of biomass whereas higher amount of glucose in the medium favored biomass indicating that carotenogenesis is not correlated to biomass. The optimal concentration of medium components for maximum total carotenoids and corresponding biomass production as obtained from model were calculated to be as 660 mL/L, 1.5, 4.5, 7.4, and 10 g/L for tomato extract, malt extract, yeast extract, peptone, and glucose, respectively.