Stabilization of Hydroxynitrile Lyases from Two Variants of Passion Fruit,Passiflora edulis Sims and Passiflora edulis Forma flavicarpa,by C-Terminal Truncation

Because the synthesis of chiral compounds generally requires a broad range of substrate specificity and stable enzymes, screening for better enzymes and/or improvement of enzyme properties through molecular approaches is necessary for sustainable industrial development. Herein, the discovery of unique hydroxynitrile lyases (HNLs) from two species of passion fruits, Passiflora edulis forma flavicarpa (yellow passion fruit, PeHNL-Ny) and Passiflora edulis Sims (purple passion fruit, PeHNL-Np), isolated and purified from passion fruit leaves is reported. These are the smallest HNLs (comprising 121 amino acids). Amino acid sequences of both enzymes are 99 % identical; there is a difference of one amino acid in a consensus sequence. PeHNL-Np has an Ala residue at position 107 and is nonglycosylated at Asn105. Because it was confirmed that natural and glycosylated PeHNL-Ny showed superior thermostability, pH stability, and organic tolerance to that of PeHNL-Np, it has been speculated that protein engineering around the only glycosylation site, Asn105, located at the C-terminal region of PeHNL-Ny, might contribute to the stabilization of PeHNL. Therefore, the focus is on improved stability of the nonglycosylated PeHNL by truncating its C-terminal region. The C-terminal-truncated PeHNLΔ₁₀₇ was obtained by truncating 15 amino acids from the C terminus followed by expression in Escherichia coli. PeHNLΔ₁₀₇ expressed in E. coli was not glycosylated, and showed improved thermostability, solvent stability, and reusability similar to that of the wild-type glycosylated form of PeHNL expressed in Pichia pastoris. These data reveal that the lack of the high-flexibility region at the C terminus of PeHNL might be a possible reason for improving the stability of PeHNL.     

Quality characteristics of high salt fermented fish sauce (budu) produced using autochthonous Virgibacillus halodenitrificans PS21 and Staphylococcus simulans PMRS35

This study evaluated the effects of autochthonous starters on the quality characteristics of budu. The effects of individual or combined inoculation of Virgibacillus halodenitrificans PS21 and Staphylococcus simulans PMRS35 were compared to those of non-inoculation during 150 days of budu production. The starters directly affected characteristics of budu with browner colour and enhanced proteolysis and lipolysis as indicated by degree of hydrolysis and free fatty acid contents. The combined starter inoculation resulted in a high aspartic acid, glutamic acid and lysine levels. Additionally, key desirable volatiles, including 2-methylbutanal, 3-methylbutanal, benzaldehyde and 2-ethyl furan, were dominant in the starter-inoculated budu. In a sensorial analysis, the highest scores for appearance, colour, taste and flavour were assigned for inoculated budu with combined starters. Therefore, the addition of starters (V. halodenitrificans PS21 and S. simulans PMRS35) in budu production improved the quality characteristics and reduced the fermentation period.     

Effects of Tropical Citrus Essential Oils on Growth, Aflatoxin Production, and Ultrastructure Alterations of Aspergillus flavus and Aspergillus parasiticus

Ethyl acetate extracts and hydrodistillated essential oils from five cultivars of tropical citrus epicarps were evaluated for their inhibitory activities against Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus, and Penicillium sp. using disk diffusion and broth microdilution assays. Essential oils prepared from kaffir lime (Citrus hystrix DC) and acid lime (Citrus aurantifolia Swingle) epicarps exhibited stronger antifungal activity to all fungi than their ethyl acetate extracts with minimum inhibitory concentration and minimum fungicidal concentration values of 0.56 and 1.13 mg/ml (dry matter), respectively, against aflatoxin-producing A. flavus and A. parasiticus. The dominant components of the essential oil from kaffir lime were limonene, citronellol, linalool, o-cymene, and camphene, whereas limonene and p-cymene were major components of acid lime essential oil. Pure limonene, citronellal, and citronellol were five to six times less fungicidal than the natural essential oils, indicating the synergistic activity of many active compounds present in the oils. Kaffir and acid lime essential oils significantly reduced aflatoxin production of A. flavus and A. parasiticus, particularly lime essential oil, which completely inhibited growth and aflatoxin production of A. flavus at the concentration of 2.25 mg/ml. Target cell damage caused by acid lime essential oil was investigated under transmission electron microscopy. Destructive alterations of plasma and nucleus membrane, loss of cytoplasm, vacuole fusion, and detachment of fibrillar layer were clearly exhibited in essential-oil-treated cells.     

Persistence of maternal milk derived Lactobacillus plantarum in the infant feces and its antagonistic activity against Escherichia coli O157:H7

Food Science and Biotechnology - The diversity of lactic acid bacteria (LAB) in maternal milk and feces from Thai mother-infants pairs were revealed through nested PCR-DGGE. LAB species residing in...     

Improvement of Freeze-Dried Lactobacillus Plantarum Survival Using Water Extracts and Crude Fibers from Food Crops

A synbiotic product of combined Lactobacillus plantarum TISTR 875 with water extracts and crude fibers from corn, mungbean, and soybean was formulated to investigate the survival of L. plantarum during freeze-drying and storage. The impacts of those water extracts and crude fibers on probiotic survival were determined in both a cultural medium and a freeze-drying medium. L. plantarum cultivated in de Man, Rogosa, and Sharpe (MRS) broth containing 2 % of water extract from soybean with 2 % mungbean fiber showed only 0.11 log CFU/ml cell reduction. The survival of L. plantarum harvested at the late log phase, mid stationary, phase and late stationary phase did not show statistical significance (P?>?0.05), whereas an initial pH of 6.5 and growth temperature of 37 °C showed greater impact (P?<?0.05). The addition of corn extract to the freeze-drying medium as a cryoprotectant had a similar effect on L. plantarum survival as sucrose, but it was better (P?<?0.05) than fructo-oligosaccharide and exopolysaccharides from Weissella cibaria A2, soybean extract, mungbean extract, soybean, corn, and mungbean fibers. A protective coating of corn extract was revealed and observed using scanning electron microscopy. The freeze-dried L. plantarum, cultivated in MRS broth containing soybean extract and mungbean fiber with corn extract as a cryoprotectant, retained high viability of 7.21 and 6.88 log CFU/ml after 8-week storage in a vacuum-packed aluminum foil-laminated polyethylene sachet and a nitrogen-flushed glass vial, respectively.