Autohydrolysed <Emphasis Type="Italic">Tilapia nilotica</Emphasis> Fish Viscera as a Peptone Source in Bacteriocin Production

Fish processing generates large amounts of solid and liquid wastes. Many different by-products have been produced from fish processing wastes. Studies on solubilization of Bolti fish (Tilapia nilotica) viscera by endogenous enzymes at different pHs are described. Hydrolysis reactions were conducted with freshly thawed viscera utilizing an initial temperature gradient and terminated at various time points by heat inactivation of the enzymes. Various peptones obtained from hydrolysed visceral homogenates of Bolti fish residues showed their suitability for promoting the growth of lactic acid bacteria (mainly Lactobacillus sake Lb 706), microorganisms with particularly complex nutritional requirements especially peptidic sources. The assay of several treatments with L. sakei Lb 706, producer of the bacteriocin sakacin A, demonstrated that optimum conditions for biomass and bacteriocin production only imply a brief autohydrolysis at room temperature. The results showed that the Bolti fish hydrolysates gave remarkable results to those found in costly commercial media, specifically recommended for culturing and large-scale production of lactic acid bacteria.     

Growth of Lactobacillus plantarum in media containing hydrolysates of fish viscera

AIMS: To compare growth of Lactobacillus plantarum on media containing hydrolysates (peptones) from cod viscera with growth on commercial media. METHODS AND RESULTS: Growth of Lact. plantarum on various fish peptones and commercial peptones/extracts was evaluated using both a Bioscreen apparatus (microtiter plates, no pH control) and fermentors (with pH control). Generally, the performance of the fish peptones was good and only beaten by the performance of yeast extract. Replacement of the 22 g l(-1) complex nitrogen source in standard MRS medium with only 5 g l(-1) fish peptone reduced the biomass yield with only 10%, whereas replacement with a mixture of 2.5 g l(-1) fish peptone and 2.5 g l(-1) yeast extract increased the biomass yield by 10%. CONCLUSIONS: Peptones derived from cod viscera support excellent growth of Lact. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: We show that peptones derived from cod viscera are promising constituents of growth media for fastidious food bacteria such as lactobacilli. Media containing these peptones show excellent performance while problems associated with the use of meat-derived peptones (BSE, kosher status) or plant-derived peptones (genetically modified organisms) are avoided.     

Shrimp processing by-products protein hydrolysates: Evaluation of antioxidant activity and application in biomass and proteases production

Protein hydrolysates were prepared from shrimp processing by-products (SPBP) using five proteolytic enzymes: trypsin, Alcalase®, crude enzyme extract from sardinelle (Sardinella aurita) viscera and enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1. The obtained hydrolysates exhibited different degrees of antioxidant activities evaluated through three main tests: 1,1-diphenyl-2-picrylhydrazyl (DPPH)-scavenging activity, reducing power and β-carotene bleaching assays. Hydrolysates were also tested as nitrogen source for microbial growth and proteases production by Escherichia coli, Saccharomyces cerevisiae, B. mojavensis A21 and B. subtilis A26. The reached results showed that the SPBP protein hydrolysates (SPBPPHs) could be a promising alternative to currently available commercial nitrogen sources of other origins.     

Hydrolysates from Atlantic cod (Gadus morhua L.) viscera as components of microbial growth media

Three hydrolysates made from cod viscera by different enzymatic hydrolysis procedures were tested as a combined source for nitrogen, amino acids and vitamins in microbial growth media. Using a panel of five different microbes: Escherichia coli, Bacillus subtilis, Lactobacillus sakei, Saccharomyces cerevisiae and Aspergillus niger, the performance of these viscera hydrolysates was compared to the performance of common commercial peptones in an automated growth analyzer (Bioscreen C). The results show that the fish hydrolysates in general are promising alternatives to currently available commercial nitrogen sources of other origins. In the case of the food-grade and nutritionally fastidious L. sakei, two of the fish hydrolysates were clearly superior to all tested commercial peptones. For several microbes, the choice of the proteolytic enzymes used to produce the fish hydrolysate had considerable impact on performance of the resulting hydrolysate, both in terms of maximum growth rate and biomass production. In terms of hydrolysate performance, the generally best enzyme for production of a fish peptone from cod viscera was found to be Alcalase.     

Proteases production by two Vibrio species on residuals marine media

A comparative study was carried out on the growth and production of alkaline proteases by two Vibrio species using different marine peptones from fish viscera residues. The bacteria tested, Vibrio anguillarum and Vibrio splendidus, are producers of high levels of proteolytic enzymes which act as factors of virulence in fish cultures, causing high mortality rates. The kinetic assays and subsequent comparison with the parameters obtained from the adjustment to various mathematical models, highlighted the potential interest of the media formulated, for their possible production on an industrial scale, particularly the production of proteases by V. anguillarum growing in rainbow trout and squid peptones.     

The non-nutritional performance characteristics of peptones made from rendered protein

Economic considerations require the use of inexpensive feedstocks for the fermentative production of moderate-value products. Our previous work has shown that peptones capable of supporting the growth of various microorganisms can be produced from inexpensive animal proteins, including meat and bone meal, feather meal, and blood meal, through alkaline or enzymatic hydrolysis. In this work, we explore how these experimental peptones compare to commercial peptones in terms of performance characteristics other than chemical make-up; these characteristics can impact fermentation operating cost. It is shown that experimental peptone powders produced through enzymatic hydrolysis are highly hygroscopic and that their physical form is not stable to humid storage conditions; those produced through alkaline hydrolysis and commercial peptones are less hygroscopic. When used in growth medium, all peptones contribute haze to the solution; experiments show that the source of haze is different when using enzyme- versus alkali-hydrolyzed peptones. Alkali-hydrolyzed peptones and all peptones made from blood meal are stronger promoters of media foaming than the commercial peptones; some enzyme-hydrolyzed peptones support very little foam formation and are superior to the commercial peptones in this sense. Alkali-hydrolyzed peptones are roughly equivalent to commercial peptones in the coloration they contribute to media, while enzyme-hydrolyzed peptones contribute intense coloration to media. No peptone caused a significant change in the viscosity of media. The experimental peptones studied here may be acceptable low-cost substitutes for commercial peptones, but none is equivalent to the commercial products in all respects.     

Regeneration of activated charcoal used in decolorization and purification of crude protease from Rhizopus oryzae

Activated charcoal, used for decolorization and purification of crude protease, was regenerated by treatment six times with acetone/water (40:60 v/v), followed by drying. This multistage leaching followed the leaching equation adapted to multistage leaching. The regenerated charcoal was nearly as effective as fresh charcoal in decolorization and purification of crude protease, but only after drying. List of symbols: c ^*, equilibrium solute concentration (w/v) in liquid; c _s, solute concentration in solid phase (w/w); F, interfacial area between solid and liquid phases; k, rate constant for leaching equation; v, volume of liquid per unit weight of adsorbent (solid phase).     

Evaluation of peptones from chicken waste as a nitrogen source for micro-organisms

In this study, chicken peptone was produced by hydrolysing inedible parts derived from chickens using endo-protease and exo-protease. The usefulness of chicken peptone as a nutrient source for bacteria was evaluated in comparison with other commercially produced peptones (animal, soy and casein-derived peptone). Escherichia coli and Bacillus subtilis were used as test strains to determine the effect of peptones from different sources on their growth ability. Both bacteria were successfully cultured in chicken peptone solution, which is similar to peptone solution containing commercial peptones apart from animal peptone. In chemical analysis, chicken peptone contained 12·0% nitrogen; this was similar to the nitrogen content from other commercial peptone sources, except for the 9·0% nitrogen found in soy peptones. The molecular weight of the peptone was determined by gel filtration chromatography, and those of all peptone, except animal-derived peptone, were found to be <5000 Da. In addition, when B. subtilis was cultured in a medium containing chicken peptone, it was shown that the protease activity was highest as compared with other commercial peptones. From these results, it is suggested that chicken peptone can be utilized for microbial culture, and this is an effective method to reuse chicken waste.     

Enhancement of the gas-to-water ethene transfer coefficient by a dispersed water-immiscible solvent: effect of the cells

  For a mass-transfer-limited system, it was demonstrated that the volumetric ethene transfer coefficient (k _l a) from gas to water could be enhanced by dispersing adequate amounts of a water-immiscible organic liquid, namely the perfluorocarbon FC40, in the aqueous phase. When 26% (v/v) FC40 was dispersed in a culture of Mycobacterium parafortuitum an enhancement of k _l a, calculated on a total liquid volume basis, of 1.8 times was found. Steady-state experiments in the absence of microorganisms, however, showed a 1.2-fold enhancement of k _l a at 18.5% (v/v) FC40. At all FC40 volume fractions tested, enhancement factors with cells were higher than enhancements without cells; apparently the microorganisms or their excretion products affected the interfacial areas or characteristic phase dimensions. Received: 4 December 1995 / Received revision: 7 June 1996 / Accepted: 10 June 1996     

Isolation and selection of native microorganisms for the aerobic treatment of simulated dairy wastewaters

Milk fat/protein degrading microorganisms were isolated from different locations of a dairy wastewater treatment system with the goal of developing an inoculum for bioaugmentation strategies. Eight isolates, identified by 16S rRNA gene sequence analysis as belonging to the genera Bacillus, Pseudomonas, and Acinetobacter, were tested for their ability to remove COD and protein from a milk-based medium (3000 mg/L COD) and compared to a commercial bioaugmentation inoculum. The Acinetobacter isolate exhibited a pellet-type growth in liquid culture, a property that could potentially aid in the separation of microbes and liquid phase following treatment. Based on the individual degradation capacity and growth behavior of the isolates, three microorganisms were further selected and tested together. This consortium exhibited a COD removal similar to the commercial inoculum (57% and 63%, respectively), but higher protein (consortium: 93%; commercial inoculum: 54%), and fat removals (consortium: 75%; commercial inoculum: 38%).     

Peptones from diverse sources: pivotal determinants of bacterial growth dynamics

Aims: In view of the major problems encountered by microbiologists in obtaining reproducible data on growth dynamics in complex media, we studied the effects of different peptones made from different biological sources and produced by numerous manufacturers. Methods and Results: Peptones (including casein, gelatin, meat, soy and yeast) were assessed as a constituent of the pre‐enrichment broth buffered peptone water (BPW). Generation times (g) and yields of Salmonella serovar Typhimurium were significantly affected by the type of peptone employed with yeast peptones generating yields of 7·04 × 10⁹ CFU ml^?1 and gelatin peptones producing 0·81 × 10⁹ CFU ml^?1. Medium sterilization was also found to have significant effects (P = 0·000) upon subsequent bacterial growth. Filter sterilization of BPW media produced lower generation times compared with those obtained after sterilization by autoclaving. Finally, it was observed that some peptones which produced good growth when inoculated with healthy organisms, showed relatively poor growth when inocula were sublethally injured by heating. Conclusions: Variation in peptone as a constituent of BPW has a significant effect on growth and enumeration of bacteria. Significance and Impact of the Study: Increased consideration with respect to culture media may significantly improve bacterial growth and experimental reproducibility.     

Enzymatic degradation of the antibiotic sulfamethazine by using crude extracts of different halophytic plants

Abstract

The biodegradation of the antibiotic sulfamethazine (SMT) by using different crude extracts of halophytes was investigated. For this purpose, crude water extracts of the halophytes Chenopodium quinoa, Sesuvium portulacastrum, and Tripolium pannonicum were prepared. Different amounts of SMT were added to the different extracts (final concentration of 1, 2, and 5?mg L^?1) and incubated at 37?°C. Crude extracts of T. pannonicum were further used to evaluate the degradation rate over time. In order to evaluate the influence of endophytic or naturally plant-associated microorganisms on the biodegradation of SMT, extracts from plants grown in sterile and non-sterile conditions were compared. SMT was analyzed by liquid chromatography coupled to positive ion electrospray mass spectrometry (ESI LC-MS). Based on the findings, crude extracts of T. pannonicum have a high potential to biodegrade SMT with a decrease up to 85.4% (4.27?±?0.10?mg L^?1) from an initial concentration of 5?mg L^?1. The lowest activity was obtained using extracts of C. quinoa with degradation of 4.5%. Extracts of plants cultivated under sterile and non-sterile conditions do not have any significant difference in SMT degradation. Therefore, microorganisms and their enzymatic activities do not seem to play a significant role during this process.     

Use of hydrolysates from Atlantic cod (Gadus morhua L.) viscera as a complex nitrogen source for lactic acid bacteria

Hydrolysates of cod viscera were tested as an alternative to commonly used complex nitrogen sources (peptones and/or extracts) for the type strains of the lactic acid bacteria Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus sakei and Pediococcus pentosaceus. Comparative studies with MRS-like media containing different nitrogen sources showed that all the fish hydrolysates performed equally well or better than commercial extracts/peptones for all selected lactic acid bacteria.     

Simultaneous hydrocarbon biodegradation and biosurfactant production by oilfield-selected bacteria

Aims: To study the bacterial diversity associated with hydrocarbon biodegradation potentiality and biosurfactant production of Tunisian oilfields bacteria. Methods and Results: Eight Tunisian hydrocarbonoclastic oilfields bacteria have been isolated and selected for further characterization studies. Phylogenetic analysis revealed that three thermophilic strains belonged to the genera Geobacillus, Bacillus and Brevibacillus, and that five mesophilic strains belonged to the genera Pseudomonas, Lysinibacillus, Achromobacter and Halomonas. The bacterial strains were cultivated on crude oil as sole carbon and energy sources, in the presence of different NaCl concentrations (1, 5 and 10%, w/v), and at 37 or 55°C. The hydrocarbon biodegradation potential of each strain was quantified by GC–MS. Strain C450R, phylogenetically related to the species Pseudomonas aeruginosa, showed the maximum crude oil degradation potentiality. During the growth of strain C450R on crude oil (2%, v/v), the emulsifying activity (E24) and glycoside content increased and reached values of 77 and 1·33 g l^?1, respectively. In addition, the surface tension (ST) decreased from 68 to 35·1 mN m^?1, suggesting the production of a rhamnolipid biosurfactant. Crude biosurfactant had been partially purified and characterized. It showed interest stability against temperature and salinity increasing and important emulsifying activity against oils and hydrocarbons. Conclusions: The results of this study showed the presence of diverse aerobic bacteria in Tunisian oilfields including mesophilic, thermophilic and halotolerant strains with interesting aliphatic hydrocarbon degradation potentiality, mainly for the most biosurfactant produced strains. Significance and Impact of the Study: It may be suggested that the bacterial isolates are suitable candidates for practical field application for effective in situ bioremediation of hydrocarbon‐contaminated sites.     

Biosynthesis of protease by Pseudomonas aeruginosa MN7 grown on fish substrate

Fish powders and fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were prepared and tested as growth media for alkaline protease production by Pseudomonas aeruginosa MN7. Cultivated in fish substrate as carbon source, the strain exhibited a slightly greater protease production (about 7800 U ml^–1) than that obtained with commercial peptones (about 7222 U ml^–1). Furthermore, P. aeruginosa MN7 produced the same amount of protease when cultivated in medium containing only fish substrate or that containing all ingredients, indicating that the strain can obtain its carbon and nitrogen requirements directly from whole fish proteins. Moreover, it was found that extensive hydrolysis of fish proteins did not increase protease formation. Protease production in media containing only FPH prepared by Alcalase was about 70% of those obtained with MN7 protease digest of fish protein or with meat-fish powder. These results indicate that sardinella substrates are an excellent carbon and nitrogen source for the growth of P. aeruginosa MN7 and the production of protease.     

Growth of Escherichia coli, Pichia pastoris and Bacillus cereus in the Presence of the Ionic Liquids [BMIM][BF4] and [BMIM][PF6] and Organic Solvents

The influence of the two most commonly used ionic liquids (1-butyl-3-methyl imidazolium tetrafluoroborate, [BMIM][BF₄], 1-butyl-3-methyl imidazolium hexafluorophosphate, [BMIM][PF₆]) and three selected organic solvents (dimethylsulfoxide, ethanol, methanol) on the growth of Escherichia coli, Pichia pastoris and Bacillus cereus was investigated. [BMIM][BF₄] was toxic at 1% (v/v) on all three microorganisms. The minimal inhibitory concentration (MIC) of [BMIM][BF₄] on E. coli growth was between 0.7 and 1% (v/v). In contrast, [BMIM][PF₆] was less toxic for P. pastoris and B. cereus, whereas E. coli was not able to tolerate [BMIM][PF₆] (MIC value: 0.3–0.7% v/v). Growth of P. pastoris was unaffected by [BMIM][PF₆] at 10% (v/v). Similar results were found for dimethylsulfoxide. Thus, ionic liquids (ILs) can have substantial inhibitory effects on the growth of microorganisms, which should be taken into account for environmental reasons as well as for the use of ILs as co-solvents in biotransformations. Revisions requested 2 November 2005; Revisions received 20 December 2005     

Degradation of starchy substrates by a crude enzyme preparation and utilization of the hydrolysates for lactic fermentation

An enzyme preparation obtained from Aspergillus ustus, possessing cellulase, α-amylase, amyloglucosidase, proteinase and d-xylanase activities, was used along with commercial bacterial α-amylase and amyloglucosidase for the degradation of ragi (Eleusine coracana) flour and wheat (Triticum vulgare) bran. Lactic acid yield from ragi hydrolysate, adjusted to 5% reducing sugars (w/v), was 25% when fermented with Lactobacillus plantarum. The yields increased to 78% and 94% when the ragi hydrolysate was fortified with 20% and 60% (v/v) wheat bran hydrolysate, respectively. When commercial α-amylase and amyloglucosidase were used for the hydrolysis of ragi and wheat bran and L. plantarum was employed to ferment the hydrolysates containing 5% reducing sugars (w/v), lactic acid yields were 10% in ragi hydrolysate and 57% and 90% when the ragi hydrolysate was fortified with 20% and 60% (v/v) of wheat bran hydrolysate, respectively. α-Amylase and amyloglucosidase hydrolysed wheat bran added at 20% (v/v) as the sole source of nutrient to soluble starch hydrolysate (5% reducing sugars) gave 22% yield of lactic acid. The yield increased to 55% by the utilization of A. ustus enzyme preparation in addition to α-amylase and amyloglucosidase for wheat bran hydrolysis.     

Microbial utilization of the industrial wastewater pollutants 2-ethylhexylthioglycolic acid and <Emphasis Type="Italic">iso</Emphasis>-octylthioglycolic acid by aerobic Gram-negative bacteria

Industrial wastewater from the production of sulfur containing esters and the resulting products of this synthesis, 2-ethylhexylthioglycolic acid (EHTG) and iso-octylthioglycolic acid (IOTG), were deployed in this study to enrich novel bacterial strains, since no wastewater and EHTG or IOTG degrading microorganisms were hitherto described or available. In addition, nothing is known about the biodegradation of these thiochemicals. The effect of this specific wastewater on the growth behaviour of microorganisms was investigated using three well-known Gram-negative bacteria (Escherichia coli, Pseudomonas putida, and Ralstonia eutropha). Concentrations of 5% (v/v) wastewater in complex media completely inhibited growth of these three bacterial strains. Six bacterial strains were successfully isolated, characterized and identified by sequencing their 16S rRNA genes. Two isolates referred to as Achromobacter sp. strain MT-E3 and Pseudomonas sp. strain MT-I1 used EHTG or IOTG, respectively, as well as the wastewater as sole source of carbon and energy for weak growth. More notably, both isolates removed these sulfur containing esters in remarkable amounts from the cultures supernatant. One further isolate was referred to as Klebsiella sp. strain 58 and exhibited an unusual high tolerance against the wastewater’s toxicity without utilizing the contaminative compounds. If cultivated with gluconic acid as additional carbon source, the strain grew even in presence of more than 40% (v/v) wastewater. Three other isolates belonging to the genera Bordetella and Pseudomonas tolerated these organic sulfur compounds but showed no degradation abilities.     

Beneficial effects of enhanced aeration using perfluorodecalin in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cultures for lipase production

The inadequate supply of oxygen to biomass is a critical factor to the productivity of most aerobic submerged fermentations. This happens because oxygen is sparingly soluble in the aqueous media. The use of a second liquid phase of perfluorocarbon (PFC), an oxygen-carrying compound, in the culture medium can increase the availability of oxygen to the microorganisms. The effect of perfluorodecalin on Yarrowia lipolytica cultures was investigated in shake-flask cultures. It was found that the specific growth rate of Y. lipolytica, a strictly aerobic yeast, increases with increasing PFC concentration. Extracellular lipase production was increased with 20% (v/v) of PFC and agitation of 250 rev/min. It was shown that the PFC presence benefitted lipase production and not just its secretion to the extracellular medium.     

Growth performance,fatty‐acid composition,lipid deposition and hepatic‐lipid metabolism‐related gene expression in juvenile pond loach Misgurnus anguillicaudatus fed diets with different dietary soybean oil levels

A 10 week feeding trial was conducted to evaluate the effects of different dietary soybean oil (SO) levels on growth performance, fatty‐acid composition and lipid deposition in viscera, histology and histochemistry of liver, intestine and hepatic‐lipid metabolism‐related gene expressions in pond loach Misgurnus anguillicaudatus juveniles. Misgurnus anguillicaudatus (mean ± s.d. mass 0·40 ± 0·01 g) were fed five experimental diets containing SO at different concentrations: 0, 20, 32, 56 and 100% SO and a diet containing 100% fish oil (100% FO). The mass gains and specific growth rates of M. anguillicaudatus fed 20% SO and 100% FO diets were significantly higher than those of the other groups (P < 0·05). The lipid content of viscera and the amount of cytoplasmic vacuolation in the liver increased with incremental dietary SO level. Meanwhile, increasing dietary SO levels up‐regulated the messenger (m)RNA levels of lipogenic genes (such as Δ6fad, scd, pparγ, fas and srebp‐1) and down‐regulated the mRNA levels of the lipolytic genes (such as pparα, cpt1, atgl and hsl) in the liver. The percentage of 20:4n‐6 significantly (P < 0·05) increased with increasing dietary SO level, which might be correlated with the up‐regulation of the mRNA level of Δ6fad. The highest levels of dietary SO, however, had a negative effect on growth performance, lipid deposition of viscera and histology and histochemstry of liver and intestine. The increased lipid accumulation induced by incremental dietary SO level probably occurred through different strategies for lipid metabolism as a result of competition between lipolysis and lipogenesis and between export and import of lipids in this species.