Os alimentos desempenham papel fundamental na nutrição e também na imunidade, pois podem conter substâncias que interagem direta ou indiretamente com o sistema imune, principalmente o de mucosas. Alguns polissacarídeos não-amido (PNAs) originados de plantas, algas e fungos comestíveis podem modular a função imune, contribuindo para a manutenção da saúde. A banana apresenta composição monossacarídica da parede celular similar à de plantas com efeitos imunomoduladores, permitindo formular a hipótese de que os PNAs dessa fruta também tenham essas propriedades. Assim, o objetivo foi extrair, purificar e caracterizar PNAs hidrossolúveis da polpa de banana das cultivares Nanicão e Thap Maeo e testá-los em cultivos de macrófagos RAW 267.4, células THP-1, macrófagos diferenciados por PMA THP-1 e células HL-60 in vitro avaliando a ação imunomoduladora através da dosagem de citocinas, NO e ensaios de fagocitose. As frações hidrossolúveis foram caracterizadas quanto ao seu conteúdo monossacarídico e ligações por hidrólise enzimática, identificando homogalacturonanos, mananos, arabinogalactanos, xilogalacturonanos e galactoglucomananos. As frações foram testadas para ausência de endotoxinas e viabilidade celular por MTT. Foram estabelecidas as doses não-tóxicas e ensaiadas as concentrações de 10, 50 e 200 µg.ml-1 das frações polissacarídicas para os testes in vitro. Não houve toxicidade para macrófagos e monócitos, enquanto que para a linhagem de células pro-mielóciticas de leucemia humana HL-60, as frações se mostraram citotóxicas. Houve aumento de atividade fagocítica nas frações WSP, UFP e HTP quando comparadas ao controle negativo de células, assim como a produção de citocinas inflamatórias como IL-1β, TNF-α, IL-6, além da quimiocina IL-8 nas células oriundas de THP-1 e diminuição nos marcadores TLR-2 com aumento de CD14 nas células THP-1, além do aumento do tamanho celular promovido pelas frações polissacarídicas mostrando diferenciação para série granulocítica. Portanto, PNAs de ambas as cultivares de banana possuem potencial imunomodulador seja inflamatório, anti-inflamatório ou de diferenciação de células THP-1 imaturas in vitro. / Food plays a major role in nutrition as in immunity, as they may contain substances that interact directly or indirectly with the immune system, especially those located on mucous membranes. Some non-starch polysaccharides (NSPs) originate from plants, algae, and edible fungi can modulate immune function, contributing to the maintenance of health. The banana presents cell wall monosaccharide composition like the other plant polysaccharides with immunomodulatory effects allowing to formulate the hypothesis that this fruit NSPs also have these properties. The objective was to extract, purify and characterize water-soluble banana pulp NSPs of Nanicão and Thap Maeo cultivars and test them in vitro on cell lineages of RAW 267.4 macrophages, PMA differentiated THP-1 macrophages, THP-1 monocytes, and HL-60, evaluating their immunomodulatory action by the dosage of cytokines, NO and phagocytosis assays. Water-soluble fractions were characterized as to their content and monosaccharide linkages by enzymatic hydrolysis, identifying homogalacturonans, mannans, arabinogalactans, xylogalacturonan, and galactoglucomannans in its composition. Fractions were tested for the absence of endotoxins and cell viability by MTT. Non-toxic concentrations doses of 10, 50 and 200 µg.ml-1 of the polysaccharide fractions were established for in vitro testing. The NSPs fractions WSP, UFP, and HTP tested promoted an increase in phagocytic activity in THP-1 PMA derived macrophages compared to negative control cells, as well as the production of inflammatory cytokines such as IL-1β, TNF-α, IL-6. An increase in the THP-1 CD14 cell receptor and decrease in CD33, CD123, CD11c and TLR-2 receptors were promoted by the banana polysaccharides. Besides, there was an increase in cell size showing cell differentiation towards granulocyte line and demonstrating that NSPs from both cultivars were immunomodulatory with a capacity of immature cell differentiation and proving that in vitro screening test is an efficient method to test other foodborne substances with immunomodulatory potential.

A major challenge for the pig industry is to formulate starter diets that primarily fit the digestive capacity, maintain GIT health and promote growth without recourse to in-feed antimicrobials. Experiments were conducted to evaluate the efficacy of carbohydrase enzymes (CE) targeting non-starch polysaccharides (NSP) in enhancing gut health and function in piglets. First, an experiment was conducted to evaluate the effects of adding CE in piglet diets on growth performance, GIT bacterial activity and nutrient digestibility. Pigs fed diets containing CE had a higher ileal lactobacilli count, total organic acids concentrations, NSP digestibility and low ammonia compared with control. The effectiveness of CE targeting NSP was further evaluated using enterotoxigenic E. coli (ETEC) in a challenge model to evaluate the impact on gut health and function. Two approaches for the ETEC challenge were adopted; an in situ small intestine segments perfusion model and an in vivo model. Initially, a pilot study was conducted to establish and validate the in situ model. In the pilot study, conventional anti-diarrhea agents; fumaric acid, ZnO, egg yolk antibodies against ETEC K88 fimbriae and carbadox, attenuated fluid losses in ETEC-infected jejunal segments. Following the establishment of the in situ model, four experiments were conducted to study the effects NSP hydrolysis products (HP) from various feedstuffs (i.e. wheat, soybean meal, canola meal and flaxseed) on ETEC-induced secretory diarrhea. The results demonstrated that HP protected against ETEC-induced fluid and electrolyte losses. A further study was conducted to investigate the response of piglets fed diets containing HP and EYA singly or in combination upon oral challenge with ETEC. Feeding HP and EYA alone or in combination attenuated ETEC-enteritis symptoms such that piglets fed additives showed less pronounced acute phase responses and superior performance. Piglets fed diets containing additives had lower gastric pH, fewer ETEC adhered to ileal mucosa and lower incidence of diarrhea. Overall, reduction of intestinal pathogens or toxic bacterial metabolites contributes to enhanced GIT health and function. These novel results expand the scope of enzyme technology in animal nutrition within the new paradigm of dietary approaches to gut health and function.

A major challenge for the pig industry is to formulate starter diets that primarily fit the digestive capacity, maintain GIT health and promote growth without recourse to in-feed antimicrobials. Experiments were conducted to evaluate the efficacy of carbohydrase enzymes (CE) targeting non-starch polysaccharides (NSP) in enhancing gut health and function in piglets. First, an experiment was conducted to evaluate the effects of adding CE in piglet diets on growth performance, GIT bacterial activity and nutrient digestibility. Pigs fed diets containing CE had a higher ileal lactobacilli count, total organic acids concentrations, NSP digestibility and low ammonia compared with control. The effectiveness of CE targeting NSP was further evaluated using enterotoxigenic E. coli (ETEC) in a challenge model to evaluate the impact on gut health and function. Two approaches for the ETEC challenge were adopted; an in situ small intestine segments perfusion model and an in vivo model. Initially, a pilot study was conducted to establish and validate the in situ model. In the pilot study, conventional anti-diarrhea agents; fumaric acid, ZnO, egg yolk antibodies against ETEC K88 fimbriae and carbadox, attenuated fluid losses in ETEC-infected jejunal segments. Following the establishment of the in situ model, four experiments were conducted to study the effects NSP hydrolysis products (HP) from various feedstuffs (i.e. wheat, soybean meal, canola meal and flaxseed) on ETEC-induced secretory diarrhea. The results demonstrated that HP protected against ETEC-induced fluid and electrolyte losses. A further study was conducted to investigate the response of piglets fed diets containing HP and EYA singly or in combination upon oral challenge with ETEC. Feeding HP and EYA alone or in combination attenuated ETEC-enteritis symptoms such that piglets fed additives showed less pronounced acute phase responses and superior performance. Piglets fed diets containing additives had lower gastric pH, fewer ETEC adhered to ileal mucosa and lower incidence of diarrhea. Overall, reduction of intestinal pathogens or toxic bacterial metabolites contributes to enhanced GIT health and function. These novel results expand the scope of enzyme technology in animal nutrition within the new paradigm of dietary approaches to gut health and function.

Intensive and semi-intensive aquaculture systems are dependent on nutrient input either in the form of supplemental or complete feeds. Most complete diets still include high fish meal (FM) levels (≥10%). However, as the industry attempts to reduce its reliance on FM, feeds must now be formulated with much lower levels especially for omnivorous species such as tilapia. By 2015, mean FM inclusion in tilapia diets was projected to fall below 3% and be further reduced to 1% by 2020. In the global context of competition for crops, finding suitable plant-based replacers for FM and meeting the increasing demand for seafood, lower-cost and under-utilised plant feedstuffs are now receiving greater attention. The study was divided into three distinct components – field survey, growth experiments, and life cycle assessment. Field surveys were used to contextualise the growth experiments and assess commercialisation opportunities for multi-enzyme inclusion in tilapia feeds. Two sets of digestibility and growth experiments were designed to evaluate the feasibility of using high inclusions of plant-based ingredients sourced from locally available feedstuffs in Thailand to substitute FM at low inclusion levels (0 – 5%). The research evaluated the hypothesis regarding the potential of exogenous enzymes (protease, xylanase and phytase) to minimize anti-nutritional effects on nutrient digestibility of proteins, polysaccharides and phosphorus in tilapia. The research also assessed the secondary effects of enzyme supplementation on economic efficiency and life cycle environmental impacts. Tilapia is the second most cultured finfish globally and Thailand is the sixth largest producer. Based on the findings of the field survey, feeding practices of Thai tilapia farmers were confirmed to be diverse. Feed inputs included, but were not limited to, agro-industrial by-products (e.g. rice bran, corn bran etc.) and commercial diets. Commercial diets contained 15 – 30% crude protein and lower protein livestock diets (i.e. pig ration) were often used for supplemental feeding or “fattening”. The experimental low FM diets were therefore formulated as grow-out or “fattening” diets for semi-intensive green-water systems, a prominent feature (>60%) of Thai tilapia farming. In Phase 1, the digestibility experiment assessed the digestibility and growth in tilapia fed 0%, 3% and 5% FM diets with and without xylanase (0.385 g kg-1) and phytase (0.075 g kg-1). Performance decreased significantly with declining FM levels. No differences in feed intake, feed conversion ratio (FCR), specific growth rate (SGR) and weight gain were observed between the enzyme and control diets. Nevertheless, tilapia fed the enzyme supplemented 3% FM and control 5% FM performed similarly (P < 0.05). No enzyme-related effects were noticed for protein digestibility but phosphorus (P) digestibility improved by 9%, except at 0% FM level (P > 0.05). The enzymes had no apparent influence on nitrogen (N) retention contrary to previous studies, however, higher retention for P was observed. Villus length decreased with declining FM levels yet no improvements were seen in tilapia fed enzyme diets. In a simultaneous grow-out experiment, the six experimental diets were compared to an industry 10% FM standard. Conversion ratio was the lowest (1.66) in adult tilapia fed 10% FM diet however the enzyme supplemented 0% FM fed fish had a comparatively low FCR of 1.67. There were no significant enzyme-related effects on weight gain¸ SGR and protein efficiency. Proximal villi results were inconsistent. The cost of feed decreased with declining FM levels but increased with enzyme inclusion. Nevertheless, the economic returns per kg of whole fish produced were better using enzyme supplemented diets compared to the controls. Though the size of the effects on growth and nutrient utilisation were modest, the findings suggested that xylanase and phytase had some level of synergistic action on the targeted anti-nutrients. However, further research was required. In Phase 2, two control diets (2% FM, negative control (NC) and 10% FM, positive control (PC)) were compared with three enzyme supplemented 2% FM diets (NO-PRO, 0.385 g kg-1 xylanase and 0.075 g kg-1 phytase only; LO-PRO, xylanase + phytase + 0.2 g kg-1 protease and HI-PRO, xylanase + phytase + 0.4 g kg-1 PRO). Growth performances improved with enzyme supplementation compared to the NC (P < 0.05). Of the enzyme supplemented diets, the LO-PRO diet showed the highest improvements in weight gain (26%) and feed intake (19%), the latter comparing statistically to the 10% FM PC diet. The HI-PRO diet had the best FCR (1.88), again comparable to the PC (1.73). The NO-PRO diet had the highest protein, P, lipid and energy digestibility, suggesting no additive effect of protease on these coefficients. In terms of gut histomorphology, the LO-PRO and PC diets had the highest measurements and were statistically similar which may have explained similarities in feed intake. Compared to the NC, the HI-PRO diet produced the highest level of change in net profit due to gains in feeding efficiency however, the LO-PRO showed better improvements in terms of growth. Based on these findings, the ternary combination of protease with xylanase and phytase (LOPRO) has potential in limiting FM use for tilapia grow-out feeds, however, the economic efficiencies were still below that of a 10% FM diet. Future considerations for research should target the indigestible dietary components in order to optimise enzyme dosages and maximise the benefits of each enzymes. In conclusion, a comparative life cycle assessment (LCA) was used to evaluate the environmental impacts of low FM diets and commercial feeds associated with tilapia production in Thailand. The study showed that the low FM enzyme supplemented diets had lower impact potentials and were environmental superior to the average (10% FM) commercial standard. LCA modules are recommended for least-cost formulation programmes as an option going forward. Additionally, LCA can be used as a predictive tool to guide farmers, especially small-scale producers, on the potential impacts of feed input choices and feeding practices. This will ensure higher product quality but also demonstrate environmental responsibility on the part of aquafeed and fish producers to final seafood consumers.

Neste trabalho, polissacarídeos não amiláceos (PNAs) da banana madura com potencial de propriedades imunomoduladoras foram obtidos por meio da remoção dos açúcares solúveis (glicose, frutose e sacarose) do purê de banana submetido a duas técnicas de extração: sólido-líquido (ESL) com agitação mecânica e assistida por ultrassom (EAU) usando etanol a 99,5 g/100 g como solvente. Para o estudo em batelada, diferentes razões da matéria-prima/solvente (1:5, 1:7 e 1:10) g/mL, tempos de extração (30, 60 e 90) min e duas temperaturas de (25 e 65) °C foram estudados. No estudo cinético, o impacto da redução da razão matéria-prima/solvente de 1:5 para 1:3 g/mL foi estudado até 90 min, nas mesmas condições estudadas na extração em batelada. O teor de açúcares solúveis (AS) medido nos extratos foi superior na temperatura de 65°C, porém às razões de 1:7 e 1:10, não resultou em maior quantidade de AS nos extratos, portanto, menores quantidades de etanol podem ser usadas diminuindo custos. Com o emprego da técnica EAU à 25 °C e tempo de extração de 30 min, foi observado que uma redução da razão matéria-prima/solvente até 1:3 g/mL produz maior rendimento de processo. No entanto, foi observado que maiores tempos de extração promoveram degradação da parede celular da matéria-prima. Entre os modelos cinéticos testados, o ajuste do modelo de Patricelli aos dados experimentais indicou que a ESL é regida pela fase de lavagem em que ocorreu 85 % da extração dos AS. Frações molares de glicose, ácidos galacturônicos, manose, arabinose, xilose, galactose de conteúdo monossacarídeo foram observadas nos rafinados, indicando possivelmente serem parte de polissacarídeos do tipo ?-glicanos, xilomananos glucomananos, arabinogalactanos e arabinoxilanos. / In this work, non-starch polysaccharides (PNAs) of ripe bananas with potential for immunomodulatory properties were obtained by means removing the soluble sugars (glucose, fructose and sucrose) from banana puree submitted to two extraction techniques: solid-liquid (SLE) with mechanical stirring and ultrasonic assisted (UAE) using 99.5 g/ 100 g ethanol as solvent. For the batch study, different ratios of the raw material/ solvent (1:5, 1:7 and 1:10) g/ mL, extraction times of (30, 60 and 90) min and two temperatures of (25 and 65 ) °C were studied. In the kinetic study, the impact of reducing the raw material/ solvent ratio from 1:5 to 1:3 g/ mL was studied up to 90 min, under the same conditions studied in batch extraction. The soluble sugar content (AS) measured in the extracts was higher at temperature of 65 °C, however, at the ratios of 1:7 and 1:10, did not result in higher amount of AS in the extracts, therefore, smaller amounts of ethanol can be costs. With the use of the UAE technique at 25 °C and extraction time of 30 min, it was observed that a reduction of the raw material / solvent ratio up to 1:3 g / mL produces a higher process yield. However, it was observed that longer extraction times promoted degradation of the cell wall of the raw material. Among the kinetic models tested, the adjustment of Patricelli model to the experimental data indicated that the SLE the predominance is by the washing phase in which 85% of the AS extractions occurred. Glucose molar fractions, galacturonic acids, mannose, arabinose, xylose, galactose of monosaccharide content were observed in the raffinates, indicating possibly being part of polysaccharides as ?-glycan, xylomannans, glucomannans, arabinogalactans and arabinoxylans.

Tyrimas atliktas 2012-2014 metų laikotarpiu Lietuvos sveikatos mokslų universitete, Veterinarijos akademijoje, GTF Paukščių lesalų ir paukštininkystės laboratorijoje, prie Gyvulininkystės katedros, AB „Vilniaus paukštynas“ ir KTU maisto instituto juslinių tyrimų laboratorijoje. Darbas turi 46 puslapius, 18 lentelių, 6 paveikslus. Darbo tikslas yra ištirti lesalų, papildytų skirtingų koncentracijų fermentinio priedo, įtaką viščiukų broilerių linijos Ross-308 produktyvumui bei mėsos kokybei. Darbo uždaviniai yra ištirti fermentinio priedo įtaka viščiukų broilerių produktyvumui, išgyvenimui, virškinamojo trakto turinio sausųjų medžiagų kiekiui, krūtinės ir kojų raumenų pH, vidinių organų, žarnyno svoriui ir ilgiui, viščiukų skerdenos rodikliams ir viščiukų mėsos juslinėms savybėms. Gauti rezultatai: Skirtingi fermentinio preparato kiekiai viščiukų broilerių augimo intensyvumui esminės įtakos neturėjo, tačiau juos naudojant lesalų konversija pagerėjo 1-2 proc. Tiriamosiose grupėse buvo nustatytas didesnis broilerių išsaugojimas 1,5-2 proc., palyginus su kontroline grupe. Analizuojant sausųjų medžiagų ir vandenilio jonų koncentracijas viščiukų broilerių virškinamajame trakte statistiškai patikimų skirtumų tarp kontrolinės ir tiriamųjų paukščių grupių nenustatyta. Tiriamųjų viščiukų broilerių žarnyno ir aklosios žarnos bendras ilgis padidėjo 4-5 proc., liaukinio skrandžio išsivystymas 5-43 proc., vidaus organų, kaip širdies ir kepenų, svoriai didėjo 8-27 proc., tačiau kasos ir... [toliau žr. visą tekstą] / Name of work: “The influence of different concentrations of xylanase, beta-glucanase and cellulase on productivity and meat quality of broilers chickens cross line Ross-308”. The work was performed in year 2012-2014 at Lithuanian University of Health Sciences, Veterinary Academy, in Poultry Feed and Poultry Products Laboratory, JSC „Vilniaus paukšynas“ and Food Institute of Kaunas University of Technology, in Sensory Analysis Laboratory. The work has 46 pages, 18 tables and 6 images. The aim of the work was to investigate the effect of different concentrations of NSP enzymes on Productivity and meat quality of broiler chickens. The tasks of the work were to investigate the effect of different concentrations of non-starch polysaccharides (NSP) enzymes for productivity of broiler chickens; effect on broiler chickens mortality, intestinal dry mass, intestinal pH value, breast muscle and leg muscle pH, internal organ weight, intestinal length, broiler carcass results and meat sensory and textual properties. Results: Different enzyme concentrations did not influence broiler chicken growth performance in general. Though feed conversion was better by 1-2 percent than in the control group. In enzyme treatment groups Broiler chicken mortality was lower by 1,5-2 percent compared to the control group. During the analysis of dry mass and hydrogen ion concentrations in the gastrointestinal tract of Broiler chickens, results showed that statistically significant differences could not... [to full text]

Neste trabalho, polissacarídeos não amiláceos (PNAs) da banana madura com potencial de propriedades imunomoduladoras foram obtidos por meio da remoção dos açúcares solúveis (glicose, frutose e sacarose) do purê de banana submetido a duas técnicas de extração: sólido-líquido (ESL) com agitação mecânica e assistida por ultrassom (EAU) usando etanol a 99,5 g/100 g como solvente. Para o estudo em batelada, diferentes razões da matéria-prima/solvente (1:5, 1:7 e 1:10) g/mL, tempos de extração (30, 60 e 90) min e duas temperaturas de (25 e 65) °C foram estudados. No estudo cinético, o impacto da redução da razão matéria-prima/solvente de 1:5 para 1:3 g/mL foi estudado até 90 min, nas mesmas condições estudadas na extração em batelada. O teor de açúcares solúveis (AS) medido nos extratos foi superior na temperatura de 65°C, porém às razões de 1:7 e 1:10, não resultou em maior quantidade de AS nos extratos, portanto, menores quantidades de etanol podem ser usadas diminuindo custos. Com o emprego da técnica EAU à 25 °C e tempo de extração de 30 min, foi observado que uma redução da razão matéria-prima/solvente até 1:3 g/mL produz maior rendimento de processo. No entanto, foi observado que maiores tempos de extração promoveram degradação da parede celular da matéria-prima. Entre os modelos cinéticos testados, o ajuste do modelo de Patricelli aos dados experimentais indicou que a ESL é regida pela fase de lavagem em que ocorreu 85 % da extração dos AS. Frações molares de glicose, ácidos galacturônicos, manose, arabinose, xilose, galactose de conteúdo monossacarídeo foram observadas nos rafinados, indicando possivelmente serem parte de polissacarídeos do tipo ?-glicanos, xilomananos glucomananos, arabinogalactanos e arabinoxilanos. / In this work, non-starch polysaccharides (PNAs) of ripe bananas with potential for immunomodulatory properties were obtained by means removing the soluble sugars (glucose, fructose and sucrose) from banana puree submitted to two extraction techniques: solid-liquid (SLE) with mechanical stirring and ultrasonic assisted (UAE) using 99.5 g/ 100 g ethanol as solvent. For the batch study, different ratios of the raw material/ solvent (1:5, 1:7 and 1:10) g/ mL, extraction times of (30, 60 and 90) min and two temperatures of (25 and 65 ) °C were studied. In the kinetic study, the impact of reducing the raw material/ solvent ratio from 1:5 to 1:3 g/ mL was studied up to 90 min, under the same conditions studied in batch extraction. The soluble sugar content (AS) measured in the extracts was higher at temperature of 65 °C, however, at the ratios of 1:7 and 1:10, did not result in higher amount of AS in the extracts, therefore, smaller amounts of ethanol can be costs. With the use of the UAE technique at 25 °C and extraction time of 30 min, it was observed that a reduction of the raw material / solvent ratio up to 1:3 g / mL produces a higher process yield. However, it was observed that longer extraction times promoted degradation of the cell wall of the raw material. Among the kinetic models tested, the adjustment of Patricelli model to the experimental data indicated that the SLE the predominance is by the washing phase in which 85% of the AS extractions occurred. Glucose molar fractions, galacturonic acids, mannose, arabinose, xylose, galactose of monosaccharide content were observed in the raffinates, indicating possibly being part of polysaccharides as ?-glycan, xylomannans, glucomannans, arabinogalactans and arabinoxylans.

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