Keiko Shirai

Pilot scale lactic acid fermentation of shrimp wastes for chitin recovery

Abstract Lactic fermentation of shrimp waste on solid substrates was studied as a means of preservation for chitin recovery. Shrimp wastes were fermented in 100-g flasks with varying levels of inoculation with lactobacilli as well as different types and levels of carbohydrate. Sucrose was selected as the carbohydrate source in further experimental work due to its better acid production potential as compared to lactose and milk whey powder. Lactic acid fermentation was scaled-up from 2 to 30 kg in column reactors using geometric similarity as the scale-up criterion. The pH rapidly decreased to less than 5.0, allowing preservation of wastes for at least 3 months. During ensilation, deproteinisation and demineralisation were observed. Chitin obtained from the silage was treated with acid and alkali for mineral and protein removal.

Chitosan selectivity for removing cadmium (II), copper (II), and lead (II) from aqueous phase: pH and organic matter effect

The aim of this study was to investigate the selectivity of chitosan for cadmium, copper and lead in the presence and absence of natural organic matter (NOM) in different pH solutions. Adsorption isotherms of one and three adsorbates at initial concentration of 5-100mg/L were carried out in batch reactors at pH 4, 5, or 7 and 25 degrees C in reactive and clarified water. The chitosan employed had a MW of 107.8 x 10(3)g/mol and degree of acetylation (DA) of 33.7%. The chitosan adsorption capacity at pH 4 in reactive water was 0.036, 0.016, 0.010mmol/g for Pb(2+), Cd(2+), and Cu(2+), respectively, and it decreased for Pb(2+) and Cd(2+) in clarified water. Conversely, experiments carried out in clarified water showed that the cadmium adsorption capacity of chitosan was enhanced about three times by the presence of NOM at pH 7: an adsorption mechanism was proposed. Furthermore, it was found that the biosorbent selectivity, in both reactive and clarified water at pH 4, was as follows Cu(2+)>Cd(2+)>Pb(2+). Finally, the preliminary desorption experiments of Cd(2+) conducted at pH 2 and 3 reported 68 and 44.8% of metal desorbed, which indicated that the adsorption mechanism occurred by electrostatic interactions and covalent bonds.

Effect of initial glucose concentration and inoculation level of lactic acid bacteria in shrimp waste ensilation

Fermentation conditions and microorganisms were determined, based on acid production, glucose concentration as carbohydrate source. Inoculation levels to obtain a stable shrimp waste silage were also determined. Shrimp waste ensilation was an efficient method of preservation, allowing the recovery of chitin and another added-value products such as pigments, proteins and enzymes. From the various lactic acid bacteria tested, Lactobacillus pentosus and Lactobacillus sp. (B2) were the best lactic acid producers, although small quantities of acetic acid were detected in samples inoculated with Lactobacillus pentosus. Therefore B2 was chosen for the analysis of glucose consumption as well as for the determination of optimum inoculation levels. The best results were obtained at 10% (w/w wet basis) and 5% (v/w wet basis) respectively. Presence of starters and initial glucose concentration were critical factors in the fermentation of shrimp waste. High initial glucose and starter concentrations reduced the time and increased the amount of lactic acid produced. The fermentation pattern changed during ensilation from hetero to homofermentative. Shrimp waste ensilation prevented the growth of spoilage microorganisms keeping their microbial counts steady and pH values within the acid region.

Structural Characterization of Chitin and Chitosan Obtained by Biological and Chemical Methods

Chitin production was biologically achieved by lactic acid fermentation (LAF) of shrimp waste (Litopenaeus vannameii) in a packed bed column reactor with maximal percentages of demineralization (D(MIN)) and deproteinization (D(PROT)) after 96 h of 92 and 94%, respectively. This procedure also afforded high free astaxanthin recovery with up to 2400 μg per gram of silage. Chitin product was also obtained from the shrimp waste by a chemical method using acid and alkali for comparison. The biologically obtained chitin (BIO-C) showed higher M(w) (1200 kDa) and crystallinity index (I(CR)) (86%) than the chemically extracted chitin (CH-C). A multistep freeze-pump-thaw (FPT) methodology was applied to obtain medium M(w) chitosan (400 kDa) with degree of acetylation (DA) ca. 10% from BIO-C, which was higher than that from CH-C. Additionally, I(CR) values showed the preservation of crystalline chitin structure in BIO-C derivatives at low DA (40-25%). Moreover, the FPT deacetylation of the attained BIO-C produced chitosans with bloc copolymer structure inherited from a coarse chitin crystalline morphology. Therefore, our LAF method combined with FPT proved to be an affective biological method to avoid excessive depolymerization and loss of crystallinity during chitosan production, which offers new perspective applications for this material.

Production of β-N-acetylhexosaminidase of Verticillium lecanii by solid state and submerged fermentations utilizing shrimp waste silage as substrate and inducer

Abstract The aim of this study was to utilise shrimp waste silage both as substrate and inducer of β-N-acetylhexosaminidase of Verticillium lecanii in submerged (SF) and solid state fermentations (SSF), taking advantage of the abundance and composition of crustacean wastes (main commercial source of chitin). β-N-Acetylhexosaminidase was produced in SF (initial pH 6) inoculated with spores or mycelia, 540 and 965.5 U/g of initial dry substrate (U/g IDS), respectively. SSF were carried out using a mixture of shrimp waste silage and sugar cane pith bagasse at initial pH of 6. The increment of moisture content and mycelia as inoculum in SSF improved the enzyme yield significantly and reduced the lag phase, i.e. 75% of moisture content inoculated with spores or mycelia produced 1016 and 1673 U/g IDS, respectively. SSF produced a higher β-N-acetylhexosaminidase yield than SF, but required a longer time (24 h). Specific activity in SSF was only 40% higher than SF, due to impurities from shrimp waste silage and sugar cane bagasse. Shrimp waste silage was an efficient inducer of the extracellular enzyme, compared with media supplemented with sucrose where enzymic activity was not detected.

Effect of temperature on chitin and astaxanthin recoveries from shrimp waste using lactic acid bacteria.

The chitin and astaxanthin recoveries by lactic acid fermentation of shrimp wastes (Litopenaeus sp) were conducted in bed-column reactors at 15, 20, 25, 30, 35, 40 and 45 degrees C. The response surface methodology showed that the fermentations carried out in the 27-36 degrees C temperature range with lactic acid above 0.319 mmol/g resulted in the highest demineralization. The maximal deproteinizations were attained from 30 to 40 degrees C. The extraction of free-astaxanthin did not present significant differences between 20 and 35 degrees C and the proportion of cis-stereoisomer forms increased with temperature. The growth rates of Lactobacillus plantarum were estimated in the 15-45 degrees C range and analyzed by Arrhenius and square root models. The cardinal values were 3.94 and 51.7 degrees C for minimum and maximum temperatures, respectively, with activation energy of 43.38 Jmol(-1).

Ability of some strains of lactic acid bacteria to degrade phytic acid

Twelve strains of lactic acid bacteria were examined for their ability to degrade phytate. In media in which phytic acid was the source of phosphate, phytate degradation was observed. Phytate disappearance may however not only be due to phytase, as phytic acid coprecipitated with protein as a consequence of a fall in pH during fermentation.

Ultrasonication and steam-explosion as chitin pretreatments for chitin oligosaccharide production by chitinases of Lecanicillium lecanii

In this study, chitin oligosaccharides have been successfully produced using chitinases from submerged fermentation of Lecanicillium lecanii. The highest Hex, Chit and Prot production was 0.14, 0.26 and 2.05 U/mg of protein, respectively, which were attained varying pH from 5 to 8 after 96 h. Culture conditions conducted at constant pH of 6 resulted in significantly lower enzyme production. The crude enzyme was partially purified by salting out with (NH4)2SO4 followed by size exclusion chromatography to isolate the chitinase mixture for further chitin hydrolysis assays. In this regard, chitin substrates were pretreated with sonication and steam explosion prior to enzymatic reaction. Structural changes were observed with steam explosion with 11.28% reduction of the crystallinity index attained with the lowest chitin/water ratio (0.1g/mL). Pretreated chitins reached the highest production of reducing sugars (0.37 mg/mL) and GlcNAc (0.59 mg/mL) in 23.6% yield.

Chitosan-based microcapsules containing grapefruit seed extract grafted onto cellulose fibers by a non-toxic procedure

A novel non-toxic procedure is described for the grafting of chitosan-based microcapsules containing grapefruit seed oil extract onto cellulose. The cellulose was previously UV-irradiated and then functionalized from an aqueous emulsion of the chitosan with the essential oil. The novel materials are readily attained with durable fragrance and enhanced antimicrobial properties. The incorporation of chitosan as determined from the elemental analyses data was 16.08+/-0.29 mg/g of sample. Scanning electron microscopy (SEM) and gas chromatography-mass spectroscopy (GC-MS) provided further evidence for the successful attachment of chitosan microcapsules containing the essential oil to the treated cellulose fibers. The materials thus produced displayed 100% inhibition of Escherichia coli and Staphylococcus epidermidis up to 48 h of incubation. Inhibition of bacteria by the essential oil was also evaluated at several concentrations.

Biocontrol potential and polyphasic characterization of novel native Trichoderma strains against Macrophomina phaseolina isolated from sorghum and common bean.

Native strains of Trichoderma isolated from sorghum and common bean crop soils were investigated to assess their biocontrol potential over the phytopathogenic fungus Macrophomina phaseolina, isolated from diseased plants. The Trichoderma strains were characterized with a polyphasic approach, which combined the analysis of their morphological characteristics, enzymatic activity, macro- and microculture test results, rDNA restriction patterns (AFLP), ITS1–5.8S–ITS2 rDNA sequences, and protein profiles. The integration of these data sets can be used to select new isolates as biological control agents against native fungal phytopathogens. In general, we observed a positive correlation between the secretion of β-1,3-glucanase and N-acetylhexosaminidase, and the biocontrol capacities of all the Trichoderma isolates. Strains with the best hyperparasitic behavior against M. phaseolina isolated from diseased bean and sorghum were Trichoderma sp. (TCBG-2) and Trichoderma koningiopsis (TCBG-8), respectively.