Artificial intelligence applied to enzymatic hydrolysis of lactose: improving the control of industrial processes / Inteligência artificial aplicada a hidrólise enzimática da lactose: melhorando o controle dos processos industriais

Lactose is the main carbohydrate in milk, and its absorption occurs via enzymatic hydrolysis, generating glucose and galactose. Lactose intolerance is the reduction of intestinal hydrolysis capacity due to hypolactasia, which results in the need to consume dairy foods with low levels of this carbohydrate. ß-galactosidase enzymes are used in dairy industries to hydrolyze lactose, thereby allowing intolerant consumers access to dairy products without the negative health implications. Alternative and official analytical methods are used to quantify the carbohydrate content resulting from enzymatic hydrolysis. The objective of this study was to evaluate the enzymatic hydrolysis of two distinct industrial enzymes produced by the microorganisms Bacillus licheniformis and Kluyveromyces lactis using three analytical methods: enzymatic method, cryoscopy, and high performance liquid chromatography (HPLC) using artificial intelligence to improve the control of the industrial processes. After adding the enzymes to skim milk, time kinetics was performed by collecting samples at time 0, every 10 min for 1 h, and every 30 min until the end of 5 h of hydrolysis. In 97% of the cases, a decrease in lactose concentration was observed by HPLC, followed by the deepening of the cryoscopic point. Glucose measurements by absorbance and HPLC quantification were correlated (r = 0.79; p < 0.01) but not concordant (p < 0.01). It was concluded that by means of artificial intelligence, it was possible to indirectly estimate lactose concentration using an algorithm that associates cryoscopy and glucose concentration.(AU)

O principal carboidrato do leite é a lactose e a sua absorção ocorre devido à hidrólise enzimática, gerando glicose e galactose. A intolerância à lactose é a redução da capacidade de hidrólise intestinal devido à hipolactasia, gerando a necessidade do consumo de alimentos lácteos com baixo teor deste carboidrato. As enzimas ß-galactosidase são utilizadas nas indústrias de laticínios para hidrolisar a lactose, proporcionando ao consumidor intolerante a possibilidade de ingerir os produtos lácteos sem prejuízos à saúde. Para quantificar o conteúdo de carboidratos resultante da hidrólise enzimática, são utilizados métodos analíticos alternativos e oficiais. O objetivo deste estudo foi avaliar a hidrólise enzimática de duas enzimas industriais distintas produzidas pelos microrganismos Bacillus licheniformis e Kluyveromyces lactis, por meio de três métodos analíticos: método enzimático, crioscopia e HPLC. A inteligência artificial foi utilizada para melhorar o controle dos processos industriais. Após a adição das enzimas ao leite desnatado, foi realizada a cinética de tempo coletando as amostras no tempo 0, a cada 10 minutos, até completar 1 hora de reação, e a cada 30 minutos até serem atingidas 5 horas de reação de hidrólise. Em 97% dos casos, a diminuição da concentração de lactose por HPLC acompanhou o aprofundamento do ponto crioscópico. As medições de glicose por absorbância e HPLC foram correlacionadas (r = 0,79; p < 0,01), mas não concordantes (p < 0,01). Concluiu-se que, por meio da inteligência artificial, é possível estimar indiretamente a concentração de lactose a partir de um algoritmo que associa a crioscopia e a concentração de glicose.(AU)

Cell permeabilization of Kluyveromyces and Saccharomyces species to obtain potential biocatalysts for lactose hydrolysis

Yeast's beta-galactosidase is an intracellular enzyme, through which it is possible to determine in vivo its activity as a biocatalyst in the lactose hydrolysis. Permeabilization process was used for transforming the microorganisms cells into biocatalysts with an enhanced enzyme activity. The potential application of this enzyme technology in industrial process depends mainly on the enzyme activity. Beta-galactosidase enzyme that hydrolyzes lactose, for instance, is largely dependent on the reaction time and its stability under different physical conditions, such as pH, temperature and enzyme concentration. The objective of this study was to optimize the cellular permeabilization process of Kluyveromyces marxianusCCT 3172 and Saccharomyces fragilisCCT 7586 cultured in cheese whey for lactose hydrolysis. Box-Behnken design was carried out for cell permeabilization with three independent variables, ethanol concentration, permeabilization time and temperature. The best permeability conditions for K. marxianusCCT 3172 were 27% (v v-1) ethanol, 3 min at 20ºC, with specific enzymatic activity of 0.98 U mg-1.For S. fragilisCCT 7586, a specific enzymatic activity of 1.31 U mg-1was achieved using 45% (v v-1) of ethanol, 17 min. of reaction under 17ºC. Thus, it was concluded that cellular permeabilization with ethanolis an efficient process to determine beta-galactosidase activity.(AU)

Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.(AU)

Agave syrup as a substrate for inulinase production by Kluyveromyces marxianus NRRL Y-7571 / Xarope de agave como substrato para produção de inulinase por Kluyveromyces marxianus NRRL Y-7571

The factorial planning was used to plan and optimize inulinase production by the yeast Kluyveromyces marxianus NRRL Y-7571. The experiments were conducted using a Central Composite Design (CCD) 22 , at different concentrations of agave syrup (3.6 to 6.4%) and yeast extract (2.2 to 3.0%). After 96 hours of fermentation, the best condition for the inulinase production was 5% agave syrup and 2.5% yeast extract, which yielded an average of 129.21 U mL-1 of inulinase. Partial characterization of the crude enzyme showed that the optimal pH and temperature were 4.0 and 60C, respectively. The enzyme showed thermal stability at 55C for 4 hours.(AU)

O planejamento fatorial foi utilizado para planejar e otimizar a produção de inulinase pela levedura Kluyveromyces marxianus NRRL Y-7571. Os experimentos foram conduzidos por meio de Delineamento Composto Central Rotacional (DCCR) 22 em diferentes concentrações de xarope de agave (3,6 a 6,4%) e extrato de levedura (2,2 a 3,0%). Depois de 96 horas de fermentação, a melhor condição para produção de inulinase foi xarope de agave 5% e extrato de levedura 2,5%, com uma produção média de 129,21 U mL-1. A caracterização parcial do extrato enzimático bruto mostrou que a enzima apresenta pH e temperatura ótimos de 4,0 e 60o C, respectivamente. A enzima mostrou estabilidade térmica a 55o C durante 4 horas.(AU)

Effects of Kluyveromyces marxianus Isolated from Tibetan Mushrooms on the Plasma Lipids, Egg Cholesterol Level, Egg Quality and Intestinal Health of Laying Hens

The effects of the Kluyveromyces marxianus M3 strain, isolated from Tibetan mushrooms, on plasma lipids, egg cholesterol level, egg quality, and intestinal health of laying hens were evaluated. In total, 160 Beijing fatty laying hens (43 weeks old) were divided into four groups and fed a basal diet supplemented with 0%, 0.1%, 0.3%, or 0.5% freeze-dried K. marxianus M3 powder for four weeks. The results showed that yeast supplementation reduced serum total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C), and very low density lipoprotein-cholesterol (VLDL-C) levels (p 0.01), and increased serum high density lipoprotein-cholesterol (HDL-C) level (p 0.05). Moreover, regardless of K. marxianus M3 dietary addition level, the cholesterol content of the eggs decreased by more than 26%. When0.3% yeast was supplemented, significant differences were found in the egg weights, shell strength, albumen height, Haugh unit and nutrient content of the eggs (p 0.01). Finally, 0.3% yeast supplementation improved the intestinal flora conditions of the hens by decreasing the Salmonella and Staphylococcus aureus counts (p 0.01) and increasing the Bifidobacterium count (p 0.01). The results in this work demonstrated that yeast culture supplementation to the diets deceased the serum and egg yolk cholesterol, and increased egg quality.

Effects of Kluyveromyces marxianus Isolated from Tibetan Mushrooms on the Plasma Lipids, Egg Cholesterol Level, Egg Quality and Intestinal Health of Laying Hens

The effects of the Kluyveromyces marxianus M3 strain, isolated from Tibetan mushrooms, on plasma lipids, egg cholesterol level, egg quality, and intestinal health of laying hens were evaluated. In total, 160 Beijing fatty laying hens (43 weeks old) were divided into four groups and fed a basal diet supplemented with 0%, 0.1%, 0.3%, or 0.5% freeze-dried K. marxianus M3 powder for four weeks. The results showed that yeast supplementation reduced serum total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C), and very low density lipoprotein-cholesterol (VLDL-C) levels (p 0.01), and increased serum high density lipoprotein-cholesterol (HDL-C) level (p 0.05). Moreover, regardless of K. marxianus M3 dietary addition level, the cholesterol content of the eggs decreased by more than 26%. When0.3% yeast was supplemented, significant differences were found in the egg weights, shell strength, albumen height, Haugh unit and nutrient content of the eggs (p 0.01). Finally, 0.3% yeast supplementation improved the intestinal flora conditions of the hens by decreasing the Salmonella and Staphylococcus aureus counts (p 0.01) and increasing the Bifidobacterium count (p 0.01). The results in this work demonstrated that yeast culture supplementation to the diets deceased the serum and egg yolk cholesterol, and increased egg quality. (AU)

Kluyveromyces lactis beta-galactosidase immobilization in calcium alginate spheres and gelatin for hydrolysis of cheese whey lactose / Imobilização da beta-galactosidase de Kluyveromyces lactis em esferas de alginato de cálcio e gelatina para hidrólise da lactose presente no soro de queijo

One of the greatest challenges for dairy industries is the correct destination of all the whey generated during cheese making, considering its high impact, the large volume created, and its technological potential. Enzymatic hydrolysis of cheese whey lactose is a biotechnological alternative. However, one of the limiting factors of its use is the relatively high cost of the enzymes, which could be lowered with the immobilization of these biocatalysts. Considering this context, the objective of this research was to evaluate the commercial Kluyveromyces lactis -galactosidase enzyme immobilized in calcium alginate spheres and gelatin, using glutaraldehyde and concanavalin A (ConA) as modifying agents in the hydrolysis of cheese whey lactose process. Results have shown that the enzyme encapsulation complexed with ConA in alginate-gelatin spheres, without glutaraldehyde in the immobilization support, has significantly increased the hydrolysis of lactose rate, achieving a maximum conversion of 72%.

Um dos grandes desafios das indústrias de laticínios é destinar de forma correta todo o soro gerado durante a produção de queijo, devido ao seu impacto ambiental, grande volume gerado e potencial tecnológico. A hidrólise enzimática da lactose presente no soro de queijo é uma alternativa biotecnológica. Contudo, um dos fatores limitantes de sua utilização é o custo relativamente alto das enzimas, o que poderia ser minimizado com a imobilização destes biocatalisadores. Baseado nesse contexto, o objetivo do presente trabalho foi avaliar a enzima comercial -galactosidase de Kluyveromyces lactis , imobilizada em esferas de alginato de cálcio e gelatina, empregando o glutaraldeído e a concanavalina A (ConA) como agentes modificadores, no processo de hidrólise da lactose presente no soro de queijo. Os resultados obtidos demonstraram que o encapsulamento da enzima complexada com ConA em esferas de alginato-gelatina, sem a presença de glutaraldeído no meio de imobilização, aumentou de modo significativo o teor de hidrólise da lactose, obtendo conversão máxima de 72%.

Kluyveromyces lactis beta-galactosidase immobilization in calcium alginate spheres and gelatin for hydrolysis of cheese whey lactose / Imobilização da beta-galactosidase de Kluyveromyces lactis em esferas de alginato de cálcio e gelatina para hidrólise da lactose presente no soro de queijo

One of the greatest challenges for dairy industries is the correct destination of all the whey generated during cheese making, considering its high impact, the large volume created, and its technological potential. Enzymatic hydrolysis of cheese whey lactose is a biotechnological alternative. However, one of the limiting factors of its use is the relatively high cost of the enzymes, which could be lowered with the immobilization of these biocatalysts. Considering this context, the objective of this research was to evaluate the commercial Kluyveromyces lactis -galactosidase enzyme immobilized in calcium alginate spheres and gelatin, using glutaraldehyde and concanavalin A (ConA) as modifying agents in the hydrolysis of cheese whey lactose process. Results have shown that the enzyme encapsulation complexed with ConA in alginate-gelatin spheres, without glutaraldehyde in the immobilization support, has significantly increased the hydrolysis of lactose rate, achieving a maximum conversion of 72%.(AU)

Um dos grandes desafios das indústrias de laticínios é destinar de forma correta todo o soro gerado durante a produção de queijo, devido ao seu impacto ambiental, grande volume gerado e potencial tecnológico. A hidrólise enzimática da lactose presente no soro de queijo é uma alternativa biotecnológica. Contudo, um dos fatores limitantes de sua utilização é o custo relativamente alto das enzimas, o que poderia ser minimizado com a imobilização destes biocatalisadores. Baseado nesse contexto, o objetivo do presente trabalho foi avaliar a enzima comercial -galactosidase de Kluyveromyces lactis , imobilizada em esferas de alginato de cálcio e gelatina, empregando o glutaraldeído e a concanavalina A (ConA) como agentes modificadores, no processo de hidrólise da lactose presente no soro de queijo. Os resultados obtidos demonstraram que o encapsulamento da enzima complexada com ConA em esferas de alginato-gelatina, sem a presença de glutaraldeído no meio de imobilização, aumentou de modo significativo o teor de hidrólise da lactose, obtendo conversão máxima de 72%.(AU)

Volatile metabolites produced from agro-industrial wastes by Na-alginate entrapped Kluyveromyces marxianus

The aim of this study was to evaluate the effects of alginate entrapment on fermentation metabolites of Kluyveromyces marxianus grown in agrowastes that served as the liquid culture media. K. marxianus cells entrapped in Na-alginate were prepared using the traditional liquid-droplet-forming method. Whey and pomaces from processed tomatoes, peppers, and grapes were used as the culture media. The changes in the concentrations of sugar, alcohol, organic acids, and flavor compounds were analyzed using gas chromatography-mass spectrometry (GC-MS) and high pressure liquid chromatography (HPLC). Both free and entrapped, K. marxianus were used individually to metabolize sugars, organic acids, alcohols, and flavor compounds in the tomato, pepper, grape, and acid whey based media. Marked changes in the fermentation behaviors of entrapped and free K. marxianus were observed in each culture. A 1.45-log increase was observed in the cell numbers of free K. marxianus during fermentation. On the contrary, the cell numbers of entrapped K. marxianus remained the same. Both free and entrapped K. marxianus brought about the fermentation of sugars such as glucose, fructose, and lactose in the agrowaste cultures. The highest volume of ethanol was produced by K. marxianus in the whey based media. The concentrations of flavor compounds such as ethyl acetate, isoamyl alcohol, isoamyl acetate, 2-phenylethyl isobutyrate, phenylethyl acetate, and phenylethyl alcohol were higher in fermented agrowaste based media compared to the control.(AU)

Hypocholesterolemic effects of Kluyveromyces marxianus M3 isolated from Tibetan mushrooms on diet-induced hypercholesterolemia in rat

To investigate the effects of Kluyveromyces marxianus M3 isolated from Tibetan mushrooms on diet-induced hypercholesterolemia in rats, female Wistar rats were fed a high-cholesterol diet (HCD) for 28 d to generate hyperlipidemic models. Hyperlipidemic rats were assigned to four groups, which were individually treated with three different dosages of K. marxianus M3+HCD or physiological saline+HCD via oral gavage for 28 d. The total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels in the serum and liver of the rats were measured using commercially available enzyme kits. In addition, the liver morphology was also examined using hematoxylin and eosin staining and optical microscopy. According to our results, the serum and liver TC, TG, LDL-C levels and atherogenic index (AI) were significantly decreased in rats orally administered K. marxianus M3 (p <0.01), and the HDL-C levels and anti atherogenic index (AAI) were significantly increased (p <0.01) compared to the control group. Moreover, K. marxianus M3 treatment also reduced the build-up of lipid droplets in the liver and exhibited normal hepatocytes, suggesting a protective effect of K. marxianus M3 in hyperlipidemic rats.(AU)

Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors..(AU)

Whole cells in enantioselective reduction of benzyl acetoacetate

The β-ketoester benzyl acetoacetate was enantioselectively reduced to benzyl (S)-3-hydroxybutanoate by seven microorganism species. The best result using free cells was obtained with the yeast Hansenula sp., which furnished 97% ee and 85% of conversion within 24 h. After immobilization in calcium alginate spheres, K.marxianus showed to be more stable after 2 cycles of reaction.