Haroldo Yukio Kawaguti

Potential Applications of Carbohydrases Immobilization in the Food Industry

Carbohydrases find a wide application in industrial processes and products, mainly in the food industry. With these enzymes, it is possible to obtain different types of sugar syrups (viz. glucose, fructose and inverted sugar syrups), prebiotics (viz. galactooligossacharides and fructooligossacharides) and isomaltulose, which is an interesting sweetener substitute for sucrose to improve the sensory properties of juices and wines and to reduce lactose in milk. The most important carbohydrases to accomplish these goals are of microbial origin and include amylases (α-amylases and glucoamylases), invertases, inulinases, galactosidases, glucosidases, fructosyltransferases, pectinases and glucosyltransferases. Yet, for all these processes to be cost-effective for industrial application, a very efficient, simple and cheap immobilization technique is required. Immobilization techniques can involve adsorption, entrapment or covalent bonding of the enzyme into an insoluble support, or carrier-free methods, usually based on the formation of cross-linked enzyme aggregates (CLEAs). They include a broad variety of supports, such as magnetic materials, gums, gels, synthetic polymers and ionic resins. All these techniques present advantages and disadvantages and several parameters must be considered. In this work, the most recent and important studies on the immobilization of carbohydrases with potential application in the food industry are reviewed.

Production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans 191.

This study concerned the production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans strain 191. In shaken flasks the maximum yield of chitinase was 6.9 U/mL after 72 h of cultivation at 25oC and 200 rpm. In a 5 L fermenter with 1.5 vvm aeration, the highest yield obtained was 4.19 U/mL after 168 h of fermentation at 25oC and 200 rpm, and using 3 vvm, it was 4.38 U/mL after 144 h of fermentation. The chitinase (61 KDa) was purified about 6.65 times by Sepharose CL 4B 200 gel filtration with a yield of 46.61%. The purified enzyme was able to lyse the cell walls of some fungi and to form protoplasts.

Application of response surface methodology for glucosyltransferase production and conversion of sucrose into isomaltulose using free Erwinia sp. cells

Isomaltulose is a structural isomer of sucrose commercially used in food industries. Glucosyltransferase produced by Erwinia sp. D12 catalyses an intramolecular transglucosylation of sucrose giving isomaltulose. The Experimental design and response surface methodology were applied for the optimization of the nutrient concentration in the culture medium for the enzyme production in shaken flasks at 200 rpm and 30oC. The three variables involved in this study were sugar cane molasses, bacteriological peptone and yeast extract Prodex Lac SD ® . The statistical analysis of the results showed that, in the range studied, all the factors had a significant effect (p ® (15 g/L). Maximum glucosyltransferase activity of 29.88 U/mL was achieved in a 6.6-L fermenter using the optimized medium. Free Erwinia sp. D12 cells were used for isomaltulose production from sucrose during fifteen successive batches. The final isomaltulose concentration of 75.6% obtained in the first batch increased to 77.21% (mean value) in the other fourteen batches and the productivity of 1.1 g/L x hr was obtained in batch process.

Production of glucosyltransferase by Erwinia sp. using experimental design and response surface methodology

Glucosyltransferase produced by strain Erwinia sp. is an intracellular enzyme that catalyzes the formation of isomaltulose from sucrose. Isomaltulose is a non-cariogenic reducing dissacharide commercially used in foods. Response surface methodology and 23-factorial central composite design were employed to optimize a fermentation medium for the production of glucosyltransferase by Erwinia sp. in shaken flasks at 200 rpm and 30oC. The three variables involved in this study were sugar cane molasses (SCM), corn steep liquor (CSL) and yeast extract Prodex Lac SD (YEP). The statistical analysis of the results showed that, in the range studied, all the factors had a significant effect on glucosyltransferase production and the optimum medium composition for enzyme production was (in g l-1) SCM-100, CSL-60 and YEP-8, which lead to a glucosyltransferase activity of 6.65 U mL-1.

Immobilization of Erwinia sp. D12 Cells in Alginate-Gelatin Matrix and Conversion of Sucrose into Isomaltulose Using Response Surface Methodology

Isomaltulose is a noncariogenic reducing disaccharide and also a structural isomer of sucrose and is used by the food industry as a sucrose replacement. It is obtained through enzymatic conversion of microbial sucrose isomerase. An Erwinia sp. D12 strain is capable of converting sucrose into isomaltulose. The experimental design technique was used to study the influence of immobilization parameters on converting sucrose into isomaltulose in a batch process using shaken Erlenmeyer flasks. We assessed the effect of gelatin and transglutaminase addition on increasing the reticulation of granules of Erwinia sp. D12 cells immobilized in alginate. Independent parameters, sodium alginate concentration, cell mass concentration, CaCl2 concentration, gelatin concentration, and transglutaminase concentration had all a significant effect (P < 0.05) on isomaltulose production. Erwinia sp. D12 cells immobilized in 3.0% (w/v) sodium alginate, 47.0% (w/v) cell mass, 0.3 molL−1 CaCl2, 1.7% (w/v) gelatin and 0.15% (w/v) transglutaminase presented sucrose conversion into isomaltulose, of around 50–60% in seven consecutive batches.

Glucosyltransferase production by Klebsiella sp. K18 and conversion of sucrose to palatinose using immobilized cells

The strain Klebsiella sp. K18 produces the enzyme glucosyltransferase and catalyses the conversion of sucrose to palatinose, an alternative sugar that presents low cariogenicity. Response Surface Methodology was successfully employed to determine the optimal concentration of culture medium components. Maximum glucosyltransferase production (21.78 U mL-1) was achieved using the optimized medium composed by sugar cane molasses (80 g L-1), bacteriological peptone (7 g L-1) and yeast extract (20 g L-1), after 8 hours of fermentation at 28°C. The conversion of sucrose to palatinose was studied utilizing immobilized cells in calcium alginate. The effects of the alginate concentration (2-4%), cell mass concentration (20-40%) and substrate concentration (25-45%) were evaluated and the yield of palatinose was approximately 62.5%.

Produção de isomaltulose, um substituto da sacarose, utilizando glicosiltransferase microbiana

Isomaltulose, a functional isomer of sucrose, is a non-cariogenic reducing disaccharide; has a low glycemic index; selectively promotes growth of beneficial bifidobacteria in the human intestinal microflora; and has greater stability than sucrose in some foods and beverages. Isomaltulose is a nutritional sugar that is digested more slowly than sucrose, and has health advantages for diabetics and nondiabetics. Immobilization techniques, especially entrapment of the cells, are widely used for conversion of sucrose into isomaltulose. Immobilization offers advantages such as minimum downstream processing, continuous operation and reusability of cells. Isomaltulose is currently considered to be a promising sugar substitute.

Exploration of Microorganisms Producing Bioactive Molecules of Industrial Interest by Solid State Fermentation

The prospect of biomolecules using microorganisms in fermentation processes is widely used, in this context to solid state fermentation (SSF) has advantages such as the possibility of using agricultural and industrial waste and reduction of water waste. Studies show that different microorganisms can be used in SSF; actinomyces and fungi are the most used due to growth in media with low water activity. Among the highlight biomolecules produced are antibiotics, anticarcinogenic agents, anticoccidians, antiviral, neuroactive, antioxidants, and enzymes. The enzymes are produced in greater scale among the different classes; hydrolases have gained importance because of cellulases, hemicellulases, proteases, chitinases, lipases, and phytases. Cellulases are a complex capable of acting on cellulosic materials, promoting its hydrolysis to release sugars, of which glucose is the one with largest industrial interest. Xylanolytic enzymes act on xylan, hemicellulose components, which may be attached to the cellulose and lignin in the plant cell wall. The study of chitinase has been stimulated by their possible involvement as agents of defense against pathogenic organisms that contain chitin, such as insects, nematodes, and fungi. Proteases catalyze the hydrolysis of peptide bonds of proteins and may have activity on ester and amide bonds. Lipases allow catalysis of the hydrolysis and synthesis, often in chemo, regal, or enantioselective reactions. Furthermore, phytase catalyzes the hydrolysis of phytate to phosphate and inorganic phosphorus, increasing the bioavailability of phosphorus for monogastric animals.

Production of isomaltulose obtained by Erwinia sp. cells submitted to different treatments and immobilized in calcium alginate.

In recent decades, there has been an increase in the studies of isomaltulose obtainment, due to its physicochemical properties and physiological health benefits. These properties, which include low cariogenicity, low glycemic index and greater stability, allow the use of this sweetener as a substitute for sucrose in foods; besides the fact that it can be converted to isomalt, a dietary non-cariogenic sugar alcohol used in pharmaceuticals as well as in the food industry. Isomaltulose (6-O-α-D-glucopyronosyl-1-6-D-fructofuranose) is a disaccharide reducer obtained by the enzymatic conversion of sucrose - the α-glucosyltransferase enzyme. Different treatments were performed for the preparation of whole cells; lysed cells; and crude enzyme extract of Erwinia sp. D12 strain immobilized in calcium alginate. The packed bed column of granules, containing Erwinia sp. cells sonicated and immobilized in calcium alginate (CSI), reached a maximum conversion of 53-59% sucrose into isomaltulose and it presented activity for 480 hours. The converted syrup was purified and the isomaltulose crystallization was performed through the lowering of temperature. The isomaltulose crystals presented purity of 96.5%.

PERFIL SENSORIAL E ACEITAÇÃO DE NÉCTARES DE GOIABA INDUSTRIALIZADOS

Provadores selecionados e treinados avaliaram aparencia, aroma, sabor de nectares de goiaba industrializados e comercializados na regiao de Campinas - SP, utilizando a Analise Descritiva Quantitativa (ADQ). O teste de aceitacao e intencao de compra foi conduzido em laboratorio com 40 provadores nao treinados, em cabines individuais, utilizando a escala hedonica nao estruturada de 9 cm. As amostras foram apresentadas segundo delineamento experimental de blocos completos balanceados e os resultados foram analisados aplicando a Analise de Variância (ANOVA). A ADQ mostrou que houve diferenca significativa em relacao a cor vermelho-rosado, viscosidade aparente, aroma e sabor de goiaba fresca e arenosidade. O teste de consumidor indicou que apesar da aceitacao das amostras 1 e 3 nao serem significativamente diferentes (p<0,05), a amostra 1 apresentou a maior parte das respostas (77%) distribuidas na regiao central da escala, enquanto que a amostra 3 obteve 35% de respostas acima de 8, ou seja, na regiao proxima ao extremo da escala, “gostei muitissimo”. A atitude de compra revelou que 44% dos provadores certamente compraria a amostra 3, confirmando a correlacao entre a aceitacao e a atitude de compra. A Analise Descritiva Quantitativa proporcionou uma completa descricao e quantificacao do perfil sensorial das amostras. Os atributos sensoriais que mais caracterizaram a amostra 3 (cor vermelho-rosado, viscosidade visual, aroma e sabor de goiaba fresca e textura arenosa) podem ter influenciado na maior aceitacao e intencao de compra deste produto.