RESUMO
ß-D-galactosidase is a hydrolase enzyme capable of hydrolyzing lactose in milk-based foods. Its free form can be inactivated in solution during the production of low-dosage lactose foods. Then, it is important to study strategies for avoiding the free enzyme inactivation with the aim of circumventing this problem. The stabilization of ß-D-galactosidase in aqueous solution after interactions with chitosan/eucalyptus sawdust composite membrane proved to be a potential strategy when optimized by central composite rotatable (CCR) design. In this case, the best experimental conditions for ß-D-galactosidase partitioning and stability in an aqueous medium containing the chitosan-based composite membrane reinforced with eucalyptus sawdust were i) enzyme/buffer solution ratio of 0.0057, ii) pH 5.6, iii) membrane mass of 50 mg, and iv) temperature lower than 37 °C. Significance was found for the linear enzyme/buffer solution ratio, linear temperature, and quadratic pH (p < 0.05) in the interval between 0 and 60 min of study. In the interval between 60 and 120 min, there was significance (p < 0.12) for linear temperature, the temperature-enzyme/buffer solution ratio interaction and the interaction between linear pH and linear enzyme/buffer solution ratio. The Pareto charts and response surfaces clearly showed all the effects of the experimental variables on the stabilization of ß-D-galactosidase in solution after interactions with the chitosan composite membrane. In this case, industrial food reactors covered with chitosan/eucalyptus sawdust composite membrane could be a strategy for the hydrolysis of lactose during milk-producing processes.
Assuntos
Quitosana , Estabilidade Enzimática , beta-Galactosidase , Quitosana/química , beta-Galactosidase/química , beta-Galactosidase/metabolismo , Concentração de Íons de Hidrogênio , Membranas Artificiais , Soluções , Temperatura , Lactose/químicaRESUMO
BACKGROUND: The general assumption that prebiotics reach the colon without any alterations has been challenged. Some in vitro and in vivo studies have demonstrated that 'non-digestible' oligosaccharides are digested to different degrees depending on their structural composition. In the present study, we compared different methods aiming to assess the digestibility of oligosaccharides synthesized by ß-galactosidase (ß-gal) of Lactobacillus delbruecki subsp. bulgaricus CRL450 (CRL450-ß-gal) from lactose, lactulose and lactitol. RESULTS: In the simulated gastrointestinal fluid method, no changes were observed. However, the oligosaccharides synthesized by CRL450-ß-gal were partially hydrolyzed in vitro, depending on their structure and composition, with rat small intestinal extract (RSIE) and small intestinal brush-border membrane vesicles (BBMV) from pig. Digestion of some oligosaccharides increased when mixtures were fed to C57BL/6 mice used as in vivo model; however, lactulose-oligosaccharides were the most resistant to the physiological conditions of mice. In general ß (1â6) linked products showed higher resistance compared to ß (1â3) oligosaccharides. CONCLUSION: In vitro digestion methods, without disaccharidases, may underestimate the importance of carbohydrates hydrolysis in the small intestine. Although BVMM and RSIE digestion assays are appropriate in vitro methods for these studies, in vivo studies remain the most reliable for understanding what actually happens in the digestion of oligosaccharides. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Assuntos
Digestão , Camundongos Endogâmicos C57BL , Oligossacarídeos , Prebióticos , beta-Galactosidase , Prebióticos/análise , Animais , beta-Galactosidase/metabolismo , beta-Galactosidase/química , Oligossacarídeos/metabolismo , Oligossacarídeos/química , Camundongos , Ratos , Suínos , Masculino , Lactulose/metabolismo , Lactulose/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Intestino Delgado/metabolismo , Intestino Delgado/enzimologia , Lactobacillus/metabolismo , Lactobacillus/enzimologia , Hidrólise , Lactose/metabolismo , Lactose/químicaRESUMO
The objective of this study was to develop a bioprocess for lactose hydrolysis in diverse dairy matrices, specifically skim milk and cheese whey, utilizing column reactors employing a core-shell enzymatic system featuring ß-galactosidase fused to a Cellulose Binding Domain (CBD) tag (ß-galactosidase-CBD). The effectiveness of reactor configurations, including ball columns and toothed columns operating in packed and fluidized-bed modes, was evaluated for catalyzing lactose hydrolysis in both skim milk and cheese whey. In a closed system, these reactors achieved lactose hydrolysis rates of approximately 50% within 5 h under all evaluated conditions. Considering the scale of the bioprocess, the developed enzymatic system was capable of continuously hydrolyzing 9.6 L of skim milk while maintaining relative hydrolysis levels of approximately 50%. The biocatalyst, created by immobilizing ß-galactosidase-CBD on magnetic core-shell capsules, exhibited exceptional operational stability, and the proposed bioprocess employing these column reactors showcases the potential for scalability.
Assuntos
Lactose , Leite , Animais , Lactose/química , Hidrólise , Leite/química , Leite/metabolismo , beta-Galactosidase/química , Fenômenos Magnéticos , Enzimas Imobilizadas/metabolismoRESUMO
OBJECTIVE: The investigation of benznidazole (BZN), excipients, and tablets aims to evaluate their thermal energy and tableting effects. They aim to understand better the molecular and pharmaceutical processing techniques of the formulation. SIGNIFICANCE: The Product Quality Review, part of Good Manufacturing Practices, is essential to highlight trends and identify product and process improvements. METHODS: A set of technique approaches, infrared spectroscopy, X-ray diffraction, and thermal analysis with isoconversional kinetic study, were applied in the protocol. RESULTS: X-ray experiments suggest talc and α-lactose monohydrate dehydration and conversion of ß-lactose to stable α-lactose upon tableting. The signal crystallization at 167 °C in the DSC curve confirmed this observation. A calorimetric study showed a decrease in the thermal stability of BZN tablets. Therefore, the temperature is a critical process parameter. The specific heat capacity (Cp) of BZN, measured by DSC, was 10.04 J/g at 25 °C and 9.06 J/g at 160 °C. Thermal decomposition required 78 kJ mol-1. Compared with the tablet (about 200 kJ mol-1), the necessary energy is two-fold lower, as observed in the kinetic study by non-isothermal TG experiment at 5; 7.5; 10; and 15 °C min-1. CONCLUSIONS: These results indicate the necessity of considering the thermal energy and tableting effects of BZN manufacturing, which contributes significantly to the molecular mechanistic understanding of this drug delivery system.
Assuntos
Química Farmacêutica , Temperatura Alta , Lactose/química , Comprimidos/químicaRESUMO
The present study described the chemical composition and somatic cell score (SCS) of samples of refrigerated raw milk collected from commercial farms in the state of Rio Grande do Sul in order to better understand the behavior of constituents present in non-fatty solids (NFS) in milk according to the season of the year. Means were used to describe statistical data. To estimate the probability of NFS levels meeting IN 76 (BRAZIL, 2018), binary logistic regression was used. It was reported that 18.2% (233.817) of analytical results showed NFS below 8.4%, representing the minimum required by IN 76. The highest average NFS level observed in the five-year period was registered in the micro-region of Passo Fundo (8.70%) in winter. The microregion with the lowest results was Porto Alegre (8.53%); however, it still demonstrated levels within the limits established by IN 76. The study indicates that milk constituents show differences between seasons. In autumn and winter, the constituents remained equal to or higher than those required by current legislation, while spring and summer were the periods with the lowest NFS values. The SCS was also influenced by the seasons, with the highest rates in spring, summer, and autumn.
O presente estudo teve como objetivo descrever os resultados de composição química e escore de células somáticas (ECS) de amostras de leite cru refrigerado coletado em fazendas comerciais no estado do Rio Grande do Sul, para melhor entendimento do comportamento dos constituintes presentes nos sólidos não gordurosos (SNG) no leite de acordo com as estações do ano. As médias foram estudadas para descrever as estatísticas dos dados. Para estimar a probabilidade de os teores de SNG atenderem à IN 76 de 2018, foi utilizada a regressão logística binária. Foi constatado que 18,2% (233.817) dos resultados analíticos apresentaram SNG abaixo de 8,4%, que representa o mínimo exigido pela IN 76 (BRASIL, 2018). A maior média de SNG observada no período de cinco anos foi registrada na microrregião de Passo Fundo (8,70%), no inverno. A microrregião com menores resultados foi a de Porto Alegre (8,53%), no entanto com teores dentro do estabelecido pela IN 76/2018. O estudo demonstrou que os constituintes do leite apresentaram diferenças entre as estações do ano. O outono e inverno foram os períodos em que os constituintes se mantiveram iguais ou superiores aos exigidos pela legislação vigente, enquanto que a primavera e o verão foram os períodos com os menores valores de SNG. O ECS também foi influenciado pelas estações do ano. Na primavera, verão e outono ocorreram os maiores índices.
Assuntos
Estações do Ano , Leite/química , Fazendas , Lactose/químicaRESUMO
In this work, we studied the development of a biocomposite formulated with alginate and gelatin, crosslinked with genipin for application as support for ß-galactosidase immobilization. Also, the biocomposites with the immobilized enzyme were characterized by thermal analyses and SAXS (size, density, and interconnectivity of alginate rods) for a detailed analysis of the microstructure, as well as the thermal and operational stabilities of the enzyme. The structural modifications of the biocomposite determined by SAXS demonstrate that the addition of both genipin and enzyme produced a significant reduction in size and density of the Ca(II)-alginate rods. Immobilized ß-galactosidase could be stored for 175 days under refrigeration maintaining 80% of its initial activity. Moreover, 90% of its relative activity was kept after 11 reuses in a batch process of lactose hydrolysis. Thus, the biocomposite proved to be effective as support for enzyme immobilization.
Assuntos
Alginatos , Aspergillus oryzae , Aspergillus oryzae/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/química , Gelatina , Hidrólise , Iridoides , Lactose/química , Espalhamento a Baixo Ângulo , Difração de Raios X , beta-Galactosidase/metabolismoRESUMO
This study aimed to develop single-step purification and immobilization processes on cellulosic supports of ß-galactosidase from Kluyveromyces sp. combined with the Cellulose-Binding Domain (CBD) tag. After 15 min of immobilization, with an enzymatic load of 150 U/gsupport, expressed activity values reached 106.88 (microcrystalline cellulose), 115.03 (alkaline nanocellulose), and 108.47 IU/g (acid nanocellulose). The derivatives produced were less sensitive to the presence of galactose in comparison with the soluble purified enzyme. Among the cations assessed (Na+, K+, Mg2+, and Ca2+), magnesium provided the highest increase in the enzymatic activity of ß-galactosidases immobilized on cellulosic supports. Supports and derivatives showed no cytotoxic effect on the investigated cell cultures (HepG2 and Vero). Derivatives showed high operational stability in the hydrolysis of milk lactose and retained from 53 to 64% of their hydrolysis capacity after 40 reuse cycles. This study obtained biocatalyzers with promising characteristics for application in the food industry. Biocatalyzers were obtained through a low-cost one-step sustainable bioprocess of purification and immobilization of a ß-galactosidase on cellulose via CBD.
Assuntos
Enzimas Imobilizadas , Lactose , Celulose , Estabilidade Enzimática , Enzimas Imobilizadas/química , Hidrólise , Lactose/química , beta-Galactosidase/químicaRESUMO
The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using ß-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant ß-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized ß-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant ß-galactosidase, showing its potential application in other high-molecular-weight enzymes.
Assuntos
Histidina/química , Lactose/química , Níquel/química , beta-Galactosidase/metabolismo , Queijo/análise , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Nanopartículas de Magnetita , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura , Soro do Leite/química , beta-Galactosidase/químicaRESUMO
The objective of this research was to evaluate the immobilization of the enzyme ß-galactosidase in a genipin-activated chitosan support. The influence of the number of spheres and substrate concentration on immobilization yield (IY) and enzyme activity (EA) was analyzed using experimental design. Thermal, operational and storage stabilities were assessed, and the enzymatic derivatives were characterized by thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The TGA showed that the enzymatic derivatives kept their thermal behavior, and the SEM images revealed smooth surfaces in all the spheres. The optimized conditions for the immobilization process were 4.57 mg·mL-1 of spheres and a substrate concentration of 10 mM (IY = 84.13%; EA = 24.97 U·g-1). Thermal stability was enhanced at 10 and 37 °C, enabling four successive cycles of lactose hydrolysis in diluted UHT milk. Therefore, the immobilized enzyme in genipin-activated chitosan has potential for lactose hydrolysis and applications in the food industry.
Assuntos
Quitosana/química , Enzimas Imobilizadas/química , Iridoides/química , Kluyveromyces/enzimologia , Leite/química , beta-Galactosidase/química , Animais , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Hidrólise , Lactose/química , beta-Galactosidase/metabolismoRESUMO
ß-Galactosidase production, partial purification and characterization by a new fungal were investigated. Partial purification was performed by aqueous two-phase system (ATPS) using polyethylene glycol (PEG) molar mass, PEG concentration, citrate concentration and pH as the independent variables. Purification factor (PF), partition coefficient (K) and yield (Y) were the responses. After identification by rDNA sequencing and classification as Cladosporium tenuissimum URM 7803, this isolate achieved a maximum cell concentration and ß-galactosidase activity of 0.48 g/L and 462.1 U/mL, respectively. ß-Galactosidase partitioned preferentially for bottom salt-rich phase likely due to hydrophobicity and volume exclusion effect caused in the top phase by the high PEG concentration and molar mass. The highest value of PF (12.94) was obtained using 24% (w/w) PEG 8000 g/mol and 15% (w/w) citrate, while that of Y (79.76%) using 20% (w/w) PEG 400 g/mol and 25% (w/w) citrate, both at pH 6. The enzyme exhibited optimum temperature in crude and ATPS extracts in the ranges 35-50 °C and 40-55 °C, respectively, and optimum pH in the range 3.0-4.5, with a fall of enzyme activity under alkaline conditions. Some metal ions and detergents inhibited, while others stimulated enzyme activity. Finally, C. tenuissimum URM 7803 ß-galactosidase showed a profile suitable for prebiotics production.