Proteolytic but chitinase-deficient microbial cultures were isolated from shrimp shell waste and characterized. The most efficient isolate was found to be a mixed culture consisting of two Bacillus licheniformis strains, which were first determined microscopically and physiologically. Molecular characterization was carried out by sequencing the 16S rRNA gene of both strains. According to the residual protein and ash content, the chitin obtained by fermentation of such a mixed culture was found to be comparable to a commercially available, chemically processed product. However, the strikingly high viscosity (80 versus 10 mPa of the commercially available sample) indicates its superior quality. The two strains differed in colony morphology and in their secretion capabilities for degradative extracellular enzymes. Sequencing of the loci encoding amylase, cellulase, chitinases, and proteases, as well as the degS/degU operon, which is instrumental in the regulation of degradative enzymes, and the pga operon, which is responsible for polyglutamic acid production, revealed no differences. However, a frameshift mutation in chiA, encoding a chitinase, was validated for both strains, providing an explanation for the ascertained absence of chitinolytic activities and the concomitant possibility of producing highly viscous chitin in a fermentational deproteinization process.Chitin (a polysaccharide consisting of -1,4-linked N-acetyl-D-glucosamine moieties) is the second most abundant biopolymer on earth; thus, it represents a nearly constant source of renewable raw material. Along with its deacetylated derivative chitosan, chitin recently has gained biotechnological significance, not only because of favorable pharmaceutical features, such as antimicrobial, anticholesterol, and antitumor activities, but also because of its potential for wastewater treatment, drug delivery, and wound healing and as a dietary fiber (26).Approximately 50 to 60% of the total weight of shellfish, such as shrimp, crab, and krill, consists of nonedible material, i.e., "heads" and exoskeletons rich in chitin but also protein, which form, on the one hand, major environmental pollutants as a result of uncontrolled dumping (12). On the other hand, however, due to their chemical composition (20 to 30% chitin, 20 to 40% protein, 30 to 60% minerals, and 0 to 14% lipids) and their actual availability from seafood industries, shrimp waste also constitutes the major source for chitin and chitosan production (21). Currently applied methods to purify and modify chitin from such material and to transform it to useful carbohydrate products involve harsh chemical treatments accompanied by uncontrollable hydrolysis and chemical modifications that eventually result in the formation of undesired by-products such as irregularly deacetylated polymers (33).Exploitation limits are mainly set by the purification costs, which mainly arise from removal of proteins and calcium carbonate by alternating acid and alkali treatment, ultimately resulting in large amounts of aque...
The formation of water soluble vitamins (vitamin B12, vitamin B6, riboflavin, thiamine, nicotinic acid and nicotinamide) during the tempe solid substrate fermentation was investigated. The role of several strains of Rhizopus oligosporus, R. arrhizus, and R. stolonifer and the role of several bacteria in the vitamin formation process were checked. All fungal and bacterial strains were isolated from Indonesian tempe and soaking water samples. The Rhizopus strains formed riboflavin, nicotinic acid, nicotinamide and vitamin B6. The final concentrations of these substances depended on the different strains involved and on the fermentation time. Isolates of R. oligosporus were generally the best vitamin formers. The moulds did not produce physiologically active vitamin B12. The thiamine content decreased during fermentation. The addition of bacteria, which had been selected in a screening for vitamin B12 production, resulted in an increase of physiologically active vitamin B12. Citrobacter freundii and Klebsiella pneumoniae showed the best formation capabilities. Furthermore, the bacteria produced riboflavin and vitamin B6 in addition to the moulds. The influence of Rhizopus on the vitamin B12 formation of Cit. freundii was also investigated. The vitamin content of tempe that was fermented with the mould and the bacterium was three times as high as a control fermentation with Cit. freundii only.
The influence of some fermentation parameters on vitamin B12 formation by strains of Citrobacterjfreundii and Klebsiella pneumoniae isolated from Indonesian tempeh samples during tempeh fermentation was investigated. A decrease in fermentation temperature from 32 to 24°C led to a decrease in vitamin B12 formation. Inoculation of soybeans with different numbers of cells of C. freundii at the beginning of solid-substrate fermentation showed that only the velocity of vitamin formation and not the final amount of vitamin formed depended on the number of cells. The addition of cobalt and 5,6-dimethylbenzimidazole increased the vitamin B12 content of tempeh. Nevertheless, levels of incorporation of the two precursors into the vitamin B12 molecule were very low. Neither C. freundii nor K. pneumoniae possessed the genes encoding the enterotoxins Shiga-like toxin SLT IA, heat-labile enterotoxin LT Ih, and heat-stable enterotoxin ST Ih, as indicated by PCR. This result supports the suggested use of these two strains to form vitamin B12 during tempeh fermentation in Indonesia.
Activity-guided fractionation in combination with sensory analytics, LC-TOF-MS, and 1D/2D-NMR spectroscopy enabled the identification of the bitter tasting diarylheptanoids asadanin, giffonin P, and the previously not reported ( E)-7,9,10,13-tetrahydroxy-1,7-bis(2-hydroxyphenyl)hept-9-en-11-one and 4,12,16-trihydroxy-2-oxatricyclo[13.3.1.1]-nonadeca-1(18),3,5,7(20),8,15,17-heptaen as well as the yet unknown astringent compounds 2-(3-hydroxy-2-oxoindolin-3-yl) acetic acid 3- O-6'-galactopyranosyl-2″-(2″oxoindolin-3″yl) acetate and 3-( O-β-d-glycosyl) dioxindole-3-acetic acid in Cimiciato-infected hazelnuts exhibiting a bitter off-taste. Quantitative LC-MS/MS studies, followed by dose/activity considerations confirmed for the first time asadanin to be the key contributor to the bitter taste of Cimiciato-infected hazelnuts. Furthermore, quantitative studies demonstrated that neither the physical damage alone nor a general microbial infection is able to initiate a stress-induced asadanin generation, but most likely either specific Cimiciato-specific microorganisms associated with the bugs or specific chemical stimulants in the bugs' saliva is the cause triggering asadanin biosynthesis. Finally, also germination was found for the first time to activate diarylheptanoid biosynthesis, resulting in higher contents of bitter tasting phytochemicals and development of the bitter off-taste.
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