Satyendra Gautam

Free phenolics and polyphenol oxidase (PPO): the factors affecting post-cut browning in eggplant (Solanum melongena).

Polyphenol oxidase (PPO) catalyses oxidation of phenolics, which results in instant but differential browning in many cut fruits and vegetables, including eggplant. Eight cultivars of eggplant were characterised by their PPO specific activity, phenolic content, browning index, and PPO polymorphism. In fresh eggplant, browning was found to be dependent on both the phenolic content and PPO specific activity, whereas, total phenolic content played a major role in browning of stored fruits. Interestingly, although browning index increased in stored eggplant fruits, PPO activity reduced in four out of eight cultivars studied. Phenolic level was found to increase in all these cultivars during storage. Although a significant level of homology was observed in PPO nucleotide and conceptually translated protein sequence, two cultivars, which displayed highest PPO specific activity, differed in the 38 amino acid stretch in the peptide region 301-338.

Involvement of caspase-3-like protein in rapid cell death of Xanthomonas

Xanthomonas campestris pv. glycines strain AM2 (XcgAM2), the aetiological agent of bacterial pustule disease of soybean, as well as some other strains of Xanthomonas including X. campestris pv. malvacearum NCIM 2310 and X. campestris NCIM 2961, exhibited post‐exponential rapid cell death (RCD) in Luria–Bertani (LB) medium. RCD was not displayed by Xanthomonas strains while growing in starch medium. Addition of starch to LB culture of XcgAM2 at any point of incubation during the exponential growth was found to arrest the onset of RCD. RCD in this organism was found to be associated with the synthesis of an endogenous enzyme similar to human caspase‐3, a known marker of apoptosis in eukaryotes. On sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS‐PAGE) the XcgAM2 caspase appeared to run along a 55 kDa protein molecular weight marker. The caspase‐3‐like protein was detected in all Xanthomonas strains tested. RCD was not detected in Escherichia coli cultures in LB medium. The caspase‐3‐like enzyme activity or pro‐tein was also found to be absent in this bacterium. Caspase‐3‐like protein or Xanthomonas caspase was detected only in the cells of XcgAM2 growing in LB medium and not in those growing in starch medium. The Xanthomonas caspase protein appeared in cells at around 4 h of incubation, and peaked at around 24 h, before finally disappearing at around 54 h of incubation. However, caspase enzyme activity was detected only 12–13 h after incubation and peaked around 18–20 h. Addition of starch at the beginning or during the period of exponential growth in LB cultures of XcgAM2 terminated the synthesis of this protein. It is presumed that starch acted as the repressor of biosynthesis of the Xanthomonas caspase, thereby preventing the organism from undergoing RCD. The cells undergoing RCD also displayed the other markers of eukaryotic apoptosis. These included binding of annexin V to plasma membrane of cells undergoing RCD and the presence of nicked DNA in culture supernatant as evidenced by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick‐end labelling) assay. Caspase‐negative mutants of XcgAM2 did not display post‐exponential RCD. The importance of RCD in Xanthomonas life cycle is not yet clear, however the phenomenon appears to have similarities with eukaryotic apoptosis.

Purification and characterisation of polyphenol oxidase (PPO) from eggplant (Solanum melongena)

Eggplant (Solanum melongena) is a very rich source of polyphenol oxidase (PPO), which negatively affects its quality upon cutting and postharvest processing due to enzymatic browning. PPO inhibitors, from natural or synthetic sources, are used to tackle this problem. One isoform of PPO was 259-fold purified using standard chromatographic procedures. The PPO was found to be a 112 kDa homodimer. The enzyme showed very low K(m) (0.34 mM) and high catalytic efficiency (3.3×10(6)) with 4-methyl catechol. The substrate specificity was in the order: 4-methyl catechol>tert-butylcatechol>dihydrocaffeic acid>pyrocatechol. Cysteine hydrochloride, potassium metabilsulphite, ascorbic acid, erythorbic acid, resorcylic acid and kojic acid showed competitive inhibition, whereas, citric acid and sodium azide showed mixed inhibition of PPO activity. Cysteine hydrochloride was found to be an excellent inhibitor with the low inhibitor constant of 1.8 μM.

Gamma irradiation effect on shelf-life, texture, polyphenol oxidase and microflora of mushroom (Agaricus bisporus)

An enhancement in the shelf-life of button mushroom (Agaricus bisporus) up to a period of 10 days could be achieved by the application of a gamma ray dose of 2kGy and storage at 10°C. A study of the quality parameters of the mushroom including cap opening, stipe elongation, weight loss, surface microbial load, and polyphenol oxidase activity indicated that the irradiated commodity retained the quality attributes required for its acceptability. The irradiated mushroom showed less brown discoloration compared to non-irradiated controls. The polyphenol oxidase, responsible for causing browning in stored mushroom, was found to have reduced activity in irradiated mushroom.

Shelf Life Extension of Sugarcane Juice Using Preservatives and Gamma Radiation Processing

Preserving raw sugarcane juice is a challenging problem. Sugarcane juice turns brown soon after its extraction and gets spoiled due to fermentation within hours. A combination of gamma radiation (5 kGy) with permitted preservatives and low temperature storage (10 °C) could preserve raw sugarcane juice for more than a month. The preservatives used were citric acid (0.3%), sodium benzoate (0.015%), potassium sorbate (0.025%), and sucrose (10%). The treatment helped in extending the shelf life to 15 d at ambient temperature (26 ± 2 °C) and 35 d at 10 °C. The microbial load was found to be below detectable limit within this period. The biochemicals like phenolics and flavonoids were not found to be affected by addition of these preservatives. The antioxidant activities including free radical scavenging activity, nitrite scavenging activity, and reducing power were also not significantly affected. The sensory evaluation scores showed that the juice with this combination treatment was highly acceptable.

Antimutagenic and antioxidant properties of plumbagin and other naphthoquinones.

The structure-function relationships of the naphthoquinone phytochemicals, plumbagin, juglone, and menadione, have been studied with regard to antimutagenic and antioxidant activities. Antimutagenicity of these compounds was assessed by the Ames test and RNA polymerase B (rpoB)-based rifampicin resistance assay. Antioxidant potential was evaluated by radical scavenging assays and reducing power measurement. Protection of cells and DNA against gamma radiation-induced oxidative damage was assayed by survival analysis and gel electrophoresis profiling, respectively. On the 1,4-naphthoquinone nucleus, plumbagin possesses 5-hydroxyl and 2-methyl functional groups, whereas juglone has only the 5-hydroxyl and menadione only the 2-methyl group. Plumbagin showed strong antimutagenic (against ultraviolet and ethyl methanesulfonate) and antioxidant activities, whereas juglone displayed only strong antimutagenic, and menadione only strong antioxidant activities. Thus, these two functional groups (5-OH/2-CH3) play important roles in the differential bioactivity of naphthoquinones. Escherichia coli, microarray analysis showed upregulation of the genes rep (replication/repair), ybaK (tRNA editing), speE (spermidine synthesis), and yjfC (glutathionyl spermidine synthesis) by plumbagin or juglone, and sodC (superoxide dismutase), xthA (oxidative repair), hycB (electron carrier between hydrogenase 3 and fumarate dehydrogenase), and ligA (formation of phosphodiester bond in DNA) by plumbagin or menadione. Studies with E. coli single-gene knockouts showed that ybaK and speE, reported to prevent mistranslation, are likely to be involved in the antimutagenicity displayed by juglone, and sodC to be involved in the antioxidant activity of menadione.

Induction of apoptosis in human cancer cells by a Bacillus lipopeptide bacillomycin D

A newly isolated and characterized Bacillus amyloliquefaciens strain fiply 3A has been found to produce an extracellular cyclic lipopeptide which structurally resembled bacillomycin D, earlier reported to be produced by Bacillus subtilis. The lipopeptide showed a dose dependent killing of three different human cancer cell lines viz. A549 (alveolar adenocarcinoma), A498 (renal carcinoma) and HCT-15 (colon adenocarcinoma), while not affecting the normal cell line L-132 (pulmonary epithelial cells) when analyzed using MTT assay and FACS analysis. Staining the cells with H2-DCFDA showed an increase in reactive oxygen species (ROS) formation in the lipopeptide treated cell population. Hoechst 33342 staining of nuclei further indicated apoptosis as a major mechanism of cell death in lipopeptide treated cells and the typical symptoms of apoptosis including cell shrinkage, nuclear condensation and fragmentation of nuclei were observed. Lipopeptide treatment induced extensive DNA damage in the treated cells, which was indicated by a TUNEL assay. Flow cytometric analysis exhibited lipopeptide concentration dependent apoptosis which was further confirmed during clonogenic assay of the lipopeptide treated cells.

Suppression of error prone pathway is responsible for antimutagenic activity of honey

Honey, both unifloral (Syzygiumcumini) and bifloral, demonstrated strong antimutagenicity against physical (UV, γ) and chemical (ethylmethane sulfonate) mutagens as ascertained by rpoB/RifR and Ames tests. The effect of honey was evaluated in radiation (UV or γ) exposed Escherichia coli cells for SOS response, a well known error prone repair pathway known to significantly contribute to mutagenicity by quantifying LexA repressor level, measuring cell filamentation frequency, and prophage induction by SIVET (Selectable--In-Vivo Expression Technology) assay. LexA was almost completely degraded, phenotypically long filamentous cells (∼30 μm) were formed, and SIVET induction frequency was increased in radiation exposed E. coli cultures, however, these changes were significantly inhibited in presence of honey confirming its strong antimutagenic nature. Further, rpoB/RifR mutation frequency upon UV exposure in E. coli recA- cells was found to be negligible, whereas, E. coliumuC- and umuD- knockouts showed comparatively higher mutation frequency. Honey did not show any effect on mutagenesis in these knockouts, indicating the SOS dependence of the observed mutagenesis. Honey was also found to suppress EMS induced mutagenesis but through SOS independent mechanism. Phenolics present in honey were found to be one of the important factors contributing to the antimutagenicity of honey.

Molecules Involved in the Modulation of Rapid Cell Death in Xanthomonas

In earlier studies from this laboratory, Xanthomonas campestris pv. glycines was found to exhibit a nutrition stress-related postexponential rapid cell death (RCD). The RCD was exhibited in protein-rich media but not in starch or other minimal media. This RCD in X. campestris pv. glycines was found to display features similar to those of the programmed cell death (PCD) of eukaryotes. Results of the present study showed that the observed RCD in this organism is both positively and negatively regulated by small molecules. The amino acids glycine and l-alanine as well as the D isomers of valine, methionine, and threonine were found to induce the synthesis of an active caspase-3-like protein that was associated with the onset of RCD. Addition of pyruvate and citrate to the culture medium induced both the synthesis of active caspase-3-like protein and RCD. Higher levels of intracellular accumulation of pyruvate and citrate were also observed under conditions favoring RCD. On the other hand, dextrin and maltose, the hydrolytic products of starch, inhibited the synthesis of the caspase-3-like protein. Addition of glucose and cyclic AMP (cAMP) to the RCD-favoring medium prevented RCD. Glucose, cAMP, caffeine (a known inhibitor of a phosphodiesterase that breaks down cAMP), and forskolin (from the herb Coleus forskholii, known to activate the enzyme adenylate cyclase that forms cAMP) inhibited the caspase enzyme activity in vivo and consequently the RCD process. The addition of glucose and other inhibitors of RCD enhanced intracellular cAMP accumulation. This is the first report demonstrating the involvement of small molecules in the regulation of nutrition stress-related stationary-phase rapid cell death in X. campestris pv. glycines, which is programmed.

Microbial decontamination of honey of Indian origin using gamma radiation and its biochemical and organoleptic properties.

Gamma radiation is known to inactivate microorganisms in various foods and thus ensures their microbial safety. In the present study, process parameters were standardized for achieving microbial decontamination of honey of Indian origin. Study was also carried out to examine the effect of gamma radiation treatment on the biochemical, antioxidant, antibacterial, and organoleptic attributes of the honey. A 15 kGy dose of gamma radiation was found to be sufficient for complete microbial decontamination of honey including spores, thus improving its microbial safety without affecting the quality attributes.