Evaluation of Natural Pigments Extracted from Tomato Wastes and the Possibility of their Encapsulation

This study aimed to evaluate the best methods from extracting natural pigments from tomato fruit wastes by four techniques used to extract lycopene and β-carotene each of them consisting of three solvents: ethanol, acetone and hexane in the following ratios (1:1:1), (2:1:1), (1:2:1) and (1:1:2) ml respectively. We studied too the possibility of encapsulation by freeze drying with a mixture of gelatin and gum Arabic as a carrier in nine microcapsules differing with respect to the total encapsulant (E) (2.5, 5.0, and 7.5%) and core (C) concentrations, the latter varying in relation to the total weight of encapsulant (25, 50, and 75%). The nine microcapsules were coded as follows: (1) E2.5/C25; (2) E2.5/C50; (3) E2.5/C75; (4) E5.0/C25; (5) E5.0/C50; (6) E5.0/C75; (7) E7.5/C25; (8) E7.5/C50; and (9) E7.5/C75. We found that the best solvent mixture for the extraction process was 1:1:2, and the most efficient microcapsules were E5.0/C75, E5.0/C50 and E2.5/C25. By studying the stability of the best three microcapsules when exposed to different values of heat, light, oxygen and pH, it was found that the most stable of them was E5.0/C75, followed by E5.0/C50 and then E2.5/C25. It was therefore recommended that further future studies are needed to evaluate the potential of this microcapsule as a natural additive in food, pharmaceuticals and cosmetics.

An Implication Of Actinomycetes On Human Well-Being: A Review

This review conceptualizes about the actinomycetes and its contribution to human health by playing a key role as bioactive secondary metabolites, such as enzymes, antibiotics and pigments, leading to their diverse applications and use in various industries. These searches have been uncommonly successful, and around 66% of naturally happening antibiotics, including many medically important, have been isolated from actinomycetes. The speedy occurrence of antimicrobial resistance among pathogens has led to a renewed interest to search for novel antimicrobial agents, but these antibiotics are not enough for the treatment of all diseases because there is a berserk requirement for a novel actinomycetes to combat against the antibiotic-resistant strains of pathogenic microorganisms, which are quickly expanding bit by bit. Actinomycetes are the important providers to the pharmaceutical and other industries and are well known for their capacity to produce secondary metabolites many of which are active against pathogenic microorganisms.

L-DOPA extradiol dioxigenase de amanita muscaria: revisão da sequência codificadora, expressão heteróloga, caracterização funcional e contextualização filogenética / Amanita muscaria L-DOPA extradiol dioxygenase: coding sequence review, heterologous expression, functional characterization, and phylogenetic context

Extradiol dioxigenases são enzimas que catalisam a clivagem oxidativa de ligações C-C entre grupos hidroxila fenólicos adjacentes utilizando catecóis como substratos. Esta classe de enzimas é bem caracterizada em bactérias, onde catalisam a degradação de compostos aromáticos. Na maioria das plantas Caryophyllales, como a beterraba, primavera e a maravilha, L-3,4-diidroxifenilalanina (L-DOPA) extradiol dioxigenases (DODAs) catalisam a clivagem oxidativa de L-DOPA na posição 4,5 gerando o ácido betalâmico, aldeído precursor das betalaínas, uma classe de pigmentos naturais que substitui as antocianinas na pigmentação dessas espécies. Alguns fungos basidiomicetos também produzem betalaínas, como o agário-das-moscas (Amanita muscaria). Nesse organismo, DODA é capaz de catalisar uma clivagem adicional na posição 2,3 da L-DOPA, formando muscaflavina, um isômero do ácido betalâmico que dá origem a uma outra classe de pigmentos naturais: as higroaurinas. Desde a caracterização do gene dodA, o qual codifica para a DODA de A. muscaria (AMAMU), não existem relatos na literatura que explorem a promiscuidade catalítica desta enzima, sua relação com outras linhagens de DODAs e a síntese quimioenzimática de betalaínas a partir desta enzima. Dessa forma, buscamos contextualizar as relações filogenéticas e funcionais entre AMAMU e diferentes linhagens de DODAs, bem como estabelecer um método que viabilize a clonagem, expressão heteróloga e caracterização funcional destaenzima. As análises filogenéticas revelaram que AMAMU possui uma evolução convergente com DODAs de plantas e bactérias e que, apesar de AMAMU ser funcionalmente homóloga à DODA da bactéria Escherichia coli, esta última apresenta homologia com DODAs de plantas. Logo, não há uma relação direta entre a sequência primária de DODAs e sua função. Nós também demonstramos que não há uma relação entre a expressão de transcritos de BvDODA1, e de seu parálogo BvDODA2, e a diferença de pigmentação entre variedades de beterrabas amarelas e vermelhas. A clonagem da sequência codificadora (CDS) publicada para o gene dodA de A. muscaria resultou na retenção do primeiro íntron, o que impedia a sua expressão. Então, uma nova CDS de 558 nucleotídeos foi proposta para este gene, a qual inclui um códon de início da tradução que se mantém na fase de leitura e codifica para uma proteína de 185 resíduos, 43 a menos que AMAMU. A expressão desta CDS resultou na proteína recombinante AmDODA, capaz de catalisar a síntese de ácido betalâmico e muscaflavina a partir de L-DOPA e D-DOPA. AmDODA possui um tamanho aproximado de 22 kDa, com um pH ótimo de atividade de 8,5 e uma constante de Michaelis (KM) de 3,7 ± 0,9 mmol L-1 e de velocidade máxima (Vmax) de 3,3 ± 0,4 µ mol min-1 mg-1. Sua utilização foi demonstrada na síntese quimioenzimática de betalaínas-modelo com potencial aplicação como sondas para microscopia confocal de fluorescência de dois fótons. Neste contexto, esta Tese explora os aspectos moleculares, bioquímicos e biológicos da DODA do fungo A. muscaria e traz importantes contribuições acerca da pigmentação por betalaínas na natureza

Extradiol dioxigenases are enzymes that catalyze the oxidative cleavage of C-C bonds between adjacent phenolic hydroxyl groups using catechols as substrates. This class of enzymes is well characterized in bacteria, where they catalyze the degradation of aromatic compounds. In most plants of the Order Caryophyllales, such as beet, paperflower and four o'clock flower, L-3,4-dihydroxyphenylalanine (L-DOPA) extradiol dioxygenases (DODAs) catalyze the oxidative 4,5-cleavage of L-DOPA generating the betalamic acid, an aldehyde precursor of the betalains, a class of natural pigments that replaces anthocyanins in the pigmentation of these species. Some basidiomycete fungi also produce betalains, such as the fly agaric (Amanita muscaria). In this organism, DODA is able to catalyze an additional 2,3-cleavage of L-DOPA, yielding muscaflavine, an isomer of betalamic acid that gives rise to another class of natural pigments: the hygroaurins. Since the characterization of the dodA gene, which encodes the A. muscaria DODA (AMAMU), there are no reports in the literature that explore the catalytic promiscuity of this enzyme, its relation to other DODAs and the chemoenzymatic synthesis of betalains from this enzyme. Thus, we seek to contextualize the phylogenetic and functional relationships between AMAMU and different DODA lineages, as well as to establish a method that enable the cloning, heterologous expression and functional characterization of this enzyme. Phylogenetic analysis revealed that AMAMU has a convergent evolutionwith plant and bacterial DODAs and that although AMAMU is functionally homologous to the DODA of the Escherichia coli bacteria, this latter is homologous to the plant DODAs. Therefore, there is no direct relationship between the primary sequence of DODAs and their function. We have also shown that there is no relationship between the expression of BvDODA1 transcripts, and its BvDODA2 paralogue, and the pigment difference between yellow and red beet varieties. Cloning of the published coding sequence (CDS) for the dodA gene of A. muscaria resulted in the retention of the first intron, which prevented its expression. Then, a new CDS of 558 nucleotides was proposed for this gene, which includes a translation start codon that remains in the open reading frame and encodes for a protein 185 residues long, 43 less than AMAMU. Expression of this CDS resulted in the recombinant AmDODA protein, able to catalyze the synthesis of betalamic acid and muscaflavine from L-DOPA and D-DOPA. AmDODA has an approximate size of 22 kDa, with an optimum activity pH of 8.5 and a Michaelis constant (KM) of 3.7 ± 0.9 mmol L-1 and a maximum velocity (Vmax) of 3.3 ± 0.4 µmol min-1 mg-1. Its use was demonstrated in the chemoenzymatic synthesis of betalains-model with potential application as probes for confocal microscopy of two-photon fluorescence. In this context, this thesis explores the molecular, biochemical and biological aspects of the DODA of the fungus A. muscaria and brings important contributions about the pigmentation by betalains in nature

Study of acid-base indicator property of flowers of Ipomoea biloba.

Indicators help to determine the equivalence point in acid – base titrations (neutralization titrations). They show sharp color change with respect to change in pH. Commonly used indicators for neutralization titrations are synthetic in nature. They are found to posses hazardous effects in human body. The highly colored pigments obtained from plants are found to exhibit color changes with variation of pH. A study has been done to investigate the indicator activity of aqueous extract of flower pigments and compared with that of already existing synthetic indicators. Pigments were extracted using hot water and a definite volume was added which gave accurate and reliable results for all the four different types of neutralization titrations - strong acid against strong base, strong acid against a weak base, weak acid against strong base and weak acid against weak base. The work proved to be acceptable in introduc-ing flower pigments as a substitute to the synthetic acid-base indicators.

Study of acid-base indicator property of flowers of Ipomoea biloba.

Indicators help to determine the equivalence point in acid – base titrations (neutralization titrations). They show sharp color change with respect to change in pH. Commonly used indicators for neutralization titrations are synthetic in nature. They are found to posses hazardous effects in human body. The highly colored pigments obtained from plants are found to exhibit color changes with variation of pH. A study has been done to investigate the indicator activity of aqueous extract of flower pigments and compared with that of already existing synthetic indicators. Pigments were extracted using hot water and a definite volume was added which gave accurate and reliable results for all the four different types of neutralization titrations - strong acid against strong base, strong acid against a weak base, weak acid against strong base and weak acid against weak base. The work proved to be acceptable in introduc-ing flower pigments as a substitute to the synthetic acid-base indicators.