In vitro fermentation characteristics of whole grain wheat flakes and the effect of toasting on prebiotic potential

Population studies have shown a positive correlation between diets rich in whole grains and a reduced risk of developing metabolic diseases, like diabetes, cardiovascular disease, and certain cancers. However, little is known about the mechanisms of action, particularly the impact different fermentable components of whole grains have on the human intestinal microbiota. The modulation of microbial populations by whole grain wheat flakes and the effects of toasting on digestion and subsequent fermentation profile were evaluated. Raw, partially toasted, and toasted wheat flakes were digested using simulated gastric and small intestinal conditions and then fermented using 24-hour, pH-controlled, anaerobic batch cultures inoculated with human feces. Major bacterial groups and production of short-chain fatty acids were compared with those for the prebiotic oligofructose and weakly fermented cellulose. Within treatments, a significant increase (P<.05) in bifidobacteria numbers was observed upon fermentation of all test carbohydrates, with the exception of cellulose. Toasting appeared to have an effect on growth of lactobacilli as only fermentation of raw wheat flakes resulted in a significant increase in levels of this group.

In vitro evaluation of Cu and Fe bioavailability in cashew nuts by off-line coupled SEC-UV and SIMAAS

In this work Cu and Fe bioavailability in cashew nuts was evaluated using in vitro method. Extractions with simulated gastric and intestinal fluids and dialysis procedures were applied for this purpose. The proteins separation and quantification were performed by size exclusion chromatography (SEC) coupled on-line to ultra-violet (UV) and off-line to simultaneous multielement atomic absorption spectrometry (SIMAAS). The SEC-UV and SIMAAS profiles of the protein fractions obtained by alkaline extraction (NaOH) and precipitation with HCl indicated the presence of high and low molecular weight species in the range between >75 kDa and 9.3 kDa. Almost 83% of Cu and 78% of Fe were extracted during cashew nut digestion and 90% of both elements were dialyzed. With these results it is possible to assume that 75% of Cu and 70% of Fe present in cashew nut could be bioavailable. The SEC-UV and SIMAAS chromatographic profiles obtained after in vitro gastrointestinal digestion reveal that Cu and Fe not dialyzed can be associated to a compound of 9.2 kDa. (C) 2010 Elsevier B.V. All rights reserved.

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)). Piper solmsianum is a shrub from Southeast Brazil in which many biologically active compounds were identified. The aim of this work was to establish a cell suspension culture system for this species. With this in mind, petiole and leaf explants obtained from in vitro plantlets were cultured in the presence of different plant growth regulator combinations (IAA, NAA, 2,4-D and BA). Root and indirect shoot adventitious formation, detected by histological analysis, was observed. Besides the different combinations of plant growth regulators, light regime and the supplement of activated charcoal (1.5 mg.l(-1)) were tested for callus induction and growth. Cultures maintained in light, on a 0.2 mg.l(-1) 2,4-D and 2 mg.l(-1) BA supplemented medium, and in the absence of activated charcoal, showed the highest calli fresh matter increment. From a callus culture, cell suspension cultures were established and their growth and metabolite accumulation studied. The achieved results may be useful for further characterization of the activated secondary metabolites pathways in in vitro systems of P. solmsianum.

Water-binding capacity and viscosity of Australian sweet lupin kernel fibre under in vitro conditions simulating the human upper gastrointestinal tract

There is currently little understanding of the physicochemical properties in the human gastrointestinal tract of Australian sweet lupin (Lupinus angustifolius) kernel fibre (LKF), a novel food ingredient with potential for the fibre enrichment of foods such as baked goods. Since physicochemical properties of dietary fibres have been related to beneficial physiological effects in vitro, this study compared water-binding capacity and viscosity of LKF with that of other fibres currently used for fibre-enrichment of baked goods, under in vitro conditions simulating the human upper gastrointestinal tract. At between 8.47 and 11.07g water/g dry solids, LKF exhibited water-binding capacities that were significantly higher (P<0.05) than soy fibre, pea hull fibre, cellulose and wheat fibre at all of the simulated gastrointestinal stages examined. Similarly, viscosity of LKF was significantly higher (P<0.05) than that of the other fibres at all simulated gastrointestinal stages. The relatively high water-binding capacity and viscosity of LKF identified in this study suggests that this novel fibre ingredient may elicit different and possibly more beneficial physiological effects in the upper human gastrointestinal tract than the conventional fibre ingredients currently used in fibre-enriched baked goods manufacture. We are now performing human studies to investigate the effect of LKF in the diet on health-related gastrointestinal events.

In vitro carbohydrate digestibility of whole-chickpea and chickpea bread products

Pulses such as the chickpea are generally considered to be valuable dietary sources of slowly digestible starch, a form of starch that is considered beneficial to health since it results in relatively low post-meal blood glucose levels compared with more rapidly digested starch. The development of novel chickpea-based foods is necessary to help expand the worldwide consumption of the chickpea. However, the effect of different processing methods on the starch digestibility of chickpea-based foods has not been widely investigated. This study used an in vitro method simulating human carbohydrate digestion to determine levels of slowly digestible starch, rapidly digestible starch (RDS), resistant starch, total starch and rapidly available glucose (RAG) of: (i) whole-chickpea products (domestically boiled, commercially canned and commercially precooked/vacuum-packaged); and (ii) standard white bread, chickpea flour bread (25% replacement of wheat flour by chickpea flour) and extruded chickpea flour bread (25% replacement of wheat flour by extruded chickpea flour). The RAG levels were then used to predict the relative in vivo glycaemic indices of the products. The commercially precooked/vacuum-packaged whole chickpeas demonstrated higher levels of RDS than the commercially canned and domestically boiled products (P<0.05). In addition, the domestically boiled product had lower levels of RAG (g/100 g available carbohydrate) compared with the canned and precooked/vacuum-packaged products (P<0.05). There were no significant differences between any of the carbohydrate digestibility measures of the white bread, chickpea flour bread and extruded chickpea flour bread (P>0.05) and all bread products demonstrated far higher RAG (g/100 g available carbohydrate) values than the whole-chickpea products. The findings suggest that the commercially precooked/vacuum-packaged whole chickpeas and the canned product may have higher and less beneficial glycaemic indices than the domestically boiled chickpeas. It appears unlikely that the use of chickpea flour or extruded chickpea flour, at the incorporation rate investigated in this study, would modify the glycaemic index of bread. It is probable, however, that the chickpea bread products investigated would demonstrate higher and potentially less beneficial glycaemic indices than the whole-chickpea products.

Amylase and glucosidase enzyme inhibitory activity of ginger (Zingiber officinale Roscoe) an in vitro study

The prevalence of type 2 diabetes has reached to an epidemic proportion in Sri Lanka. The need for achieving better control of blood glucose level has been evident in diabetes management. However it is not easy to achieve this goal in a large proportion of patients. This is partly due to limitations of currently available pharmacological agents which stimulate research on novel anti-diabetic agents with different mechanisms. Digestive enzymes have been targeted as potential avenues for modulation of blood glucose concentration through inhibition of the enzymatic breakdown of complex carbohydrates to meal derived glucose absorption. Acarbose is a widely used oral anti-diabetic drug which inhibits the α-glucosidase, enzyme responsible for breaking down of disaccharides and polysaccharides into glucose. Many herbal extracts have been found to posses similar inhibitory effects. Ginger (Zingiber officinale Roscoe) has developed a reputation in treatment of several diseases. In vitro enzymic inhibitory effect of ginger was investigated in this study. Enzymes α -amylase and α -glucosidase treated with either Acarbose or ginger extract were allowed to react with cooked rice and percentages of glucose content were measured. The glucosidase and amylase activities on the rice were inhibited by addition of ginger cause significant reduction in glucose percentages (36.86± 1.05 to 26.87± 2.17, P<0.05 and 49.04±0.65 to 35.35±2.22, P<0.05) which showed comparable results with Acarbose on glucosidase activity (36.86± 1.05 to, 27.8±1.32 P<0.05). Results of the study indicates ginger as a potential plant based amylase and glucosidase inhibitor in carbohydrate digestion but usage in glycaemic control in human has to be investigated further.

Ingrowth of human mesenchymal stem cells into porous silk particle reinforced silk composite scaffolds : an in vitro study

Silk fibroin protein is biodegradable and biocompatible, exhibiting excellent mechanical properties for various biomedical applications. However, porous three-dimensional (3-D) silk fibroin scaffolds, or silk sponges, usually fall short in matching the initial mechanical requirements for bone tissue engineering. In the present study, silk sponge matrices were reinforced with silk microparticles to generate protein-protein composite scaffolds with desirable mechanical properties for in vitro osteogenic tissue formation. It was found that increasing the silk microparticle loading led to a substantial increase in the scaffold compressive modulus from 0.3 MPa (non-reinforced) to 1.9 MPa for 1:2 (matrix:particle) reinforcement loading by dry mass. Biochemical, gene expression, and histological assays were employed to study the possible effects of increasing composite scaffold stiffness, due to microparticle reinforcement, on in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs). Increasing silk microparticle loading increased the osteogenic capability of hMSCs in the presence of bone morphogenic protein-2 (BMP-2) and other osteogenic factors in static culture for up to 6 weeks. The calcium adsorption increased dramatically with increasing loading, as observed from biochemical assays, histological staining, and microcomputer tomography (μCT) analysis. Specifically, calcium content in the scaffolds increased by 0.57, 0.71, and 1.27 mg (per μg of DNA) from 3 to 6 weeks for matrix to particle dry mass loading ratios of 1:0, 1:1, and 1:2, respectively. In addition, μCT imaging revealed that at 6 weeks, bone volume fraction increased from 0.78% for non-reinforced to 7.1% and 6.7% for 1:1 and 1:2 loading, respectively. Our results support the hypothesis that scaffold stiffness may strongly influence the 3-D in vitro differentiation capabilities of hMSCs, providing a means to improve osteogenic outcomes.

In vitro and in situ screening systems for morphological and phytochemical analysis of Withania somnifera germplasms

We report, for the first time for Withania somnifera, the use of a modified in vitro system for morphological and phytochemical screening of true to type plants as compared with those grown in a conventional in situ system. Eleven germplasms of cultivated W. somnifera from different regions of India were collected to examine chemotypic variation in withaferin A (WA). Methods were developed to optimize WA extraction. The maximum concentration of WA was extracted from manually ground leaf and root material to which 60 % methanol was added followed by sonication in a water bath sonicator. Variation in WA concentration in whole plants was observed amongst the different germplasms. In the in vitro system, the concentration of WA ranged between 0.27 and 7.64 mg/g dry weight (DW) and in the in situ system, the range in concentration was between 8.06 and 36.31 mg/g DW. The highest amount of WA found in leaves was 7.37 and 41.42 mg/g DW in the in vitro and the in situ systems respectively. In roots, the highest WA concentration was 0.27 mg/g DW in the in vitro and 0.60 mg/g DW in the in situ system. There are distinct advantages in using the in vitro grown plants rather than those grown in the in situ system including the simplicity of design, efficient use of space and nutrition and a system which is soil and contaminant free. The proposed in vitro system is therefore ideal for utilization in molecular, enzymatic and biochemical studies.

Creatine transporter (SLC6A8) knockout mice display an increased capacity for in vitro creatine biosynthesis in skeletal muscle.

The present study aimed to investigate whether skeletal muscle from whole body creatine transporter (CrT; SLC6A8) knockout mice (CrT(-/y)) actually contained creatine (Cr) and if so, whether this Cr could result from an up regulation of muscle Cr biosynthesis. Gastrocnemius muscle from CrT(-/y) and wild type (CrT(+/y)) mice were analyzed for ATP, Cr, Cr phosphate (CrP), and total Cr (TCr) content. Muscle protein and gene expression of the enzymes responsible for Cr biosynthesis L-arginine:glycine amidotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) were also determined as were the rates of in vitro Cr biosynthesis. CrT(-/y) mice muscle contained measurable (22.3 ± 4.3 mmol.kg(-1) dry mass), but markedly reduced (P < 0.05) TCr levels compared with CrT(+/y) mice (125.0 ± 3.3 mmol.kg(-1) dry mass). AGAT gene and protein expression were higher (~3 fold; P < 0.05) in CrT(-/y) mice muscle, however GAMT gene and protein expression remained unchanged. The in vitro rate of Cr biosynthesis was elevated 1.5 fold (P < 0.05) in CrT(-/y) mice muscle. These data clearly demonstrate that in the absence of CrT protein, skeletal muscle has reduced, but not absent, levels of Cr. This presence of Cr may be at least partly due to an up regulation of muscle Cr biosynthesis as evidenced by an increased AGAT protein expression and in vitro Cr biosynthesis rates in CrT(-/y) mice. Of note, the up regulation of Cr biosynthesis in CrT(-/y) mice muscle was unable to fully restore Cr levels to that found in wild type muscle.

Comparison of micronized whole soybeans to common protein sources in dry dog and cat diets

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Desenvolvimento inicial e crescimento in vitro de Cattleya violacea (Kunth) Rolfe em diferentes concentrações de sacarose

O presente estudo teve por objetivo analisar os aspectos da germinação e avaliar o efeito de concentrações de sacarose no crescimento in vitro de Cattleya violacea. Sementes provenientes de cápsulas fechadas foram semeadas em meio de cultura Murashige e Skoog (MS) e a morfologia externa da semente à plântula foi fotodocumentada em estereomicroscópio e microscópio eletrônico de varredura. Plântulas com 90 dias após a semeadura foram repicadas em meio de cultura ½ MS (com metade da concentração de macronutrientes) com diferentes concentrações de sacarose (0, 10, 20, 30 e 40 g L-1), incubadas nas mesmas condições in vitro por mais 150 dias e em seguida as plântulas foram avaliadas quanto ao número de raízes, comprimento da maior raiz, número de folhas, comprimento da parte aérea, massa fresca e seca total. Os dados biométricos foram submetidos à análise estatística e a eles ajustadas curvas de regressão. As sementes apresentaram testa reticulada com uma extremidade micropilar (aberta) e calazal (fechada); o embrião originou uma estrutura tuberiforme clorofilada denominada protocormo que pode apresentar rizóides, folíolos e quando provido de raiz é considerado plântula. A ausência de açúcar ou a maior concentração avaliada de sacarose foram prejudiciais ao crescimento da planta. A concentração de 27 g L-1 proporcionou maior crescimento in vitro possibilitando maior eficiência para a propagação massal dessa espécie de elevado potencial ornamental.

Degradação in vitro de tecidos da lâmina foliar e do colmo de gramíneas forrageiras tropicais, em função do estádio de desenvolvimento

O experimento foi realizado com o objetivo de avaliar o efeito da idade sobre o potencial de degradação dos diferentes tecidos da lâmina foliar e do colmo de capim-braquiária (Brachiaria decumbens), capim-gordura (Melinis minutiflora) e capim-tifton 85 (Cynodon sp). Foram amostradas a 7ª (capim-braquiária e capim-gordura) e a 11ª (capim-tifton 85) lâminas foliares, no dia da exposição da lígula e 20 dias após. Por meio de observações ao microscópio foram estimadas a extensão da digestão in vitro dos tecidos da lâmina e do colmo e a redução na espessura da parede de células do esclerênquima do colmo. Lâminas foliares e segmentos de colmos jovens apresentaram maiores áreas digeridas. Permaneceram intactos os tecidos com células de parede espessada e lignificada, a bainha parenquimática dos feixes, o esclerênquima, o xilema e a epiderme do colmo. Tecidos com células de parede delgada, normalmente não-lignificada, o mesofilo, o floema e o parênquima, desapareceram completamente. O avanço na idade reduziu a digestão do mesofilo, em lâminas de capim-braquiária e capim-gordura, e do parênquima em colmos, principalmente de capim-gordura. A epiderme na lâmina foliar foi parcialmente digerida, independentemente da idade e da espécie. Embora aparentemente intactas, células esclerenquimáticas do colmo sofreram redução da espessura da parede com a incubação em líquido ruminal. A porcentagem de redução variou de 7 a 37% e a taxa de redução da espessura de 0,007 a 0,018 µm/h.

Resposta do híbrido de sorgo-sudão à adubação nitrogenada e potássica: composição química e digestibilidade in vitro da matéria orgânica

A pesquisa foi instalada no Setor de Forragicultura da FCAV/UNESP-Jaboticabal, objetivando avaliar a composição química e a digestibilidade in vitro da matéria orgânica (DIVMO) do híbrido de Sorgo-sudão cv. AG 2501C, no outono e inverno. O manejo da pastagem foi conduzido simulando o sistema de lotação intermitente. O experimento foi desenvolvido de março a setembro de 2002. A forrageira foi submetida a nove tratamentos: três níveis de nitrogênio (100, 200 e 300 kg de N/ha) e três níveis de potássio (0, 80 e 160 kg de K2O/ha), em delineamento experimental em blocos casualizados e parcelas subdivididas. A adubação nitrogenada e potássica não foram significativas para a DIVMO. O nitrogênio influenciou a proteína bruta (PB) com valores de 15,1; 16,4 e 15,7 %, a fibra em detergente neutro (FDN) com valores de 65,3; 65,8 e 64,5% e fibra em detergente ácido (FDA) com 35,5; 37,8 e 39,6% para 100; 200 e 300 kg N/ha. O potássio aumentou significativamente a lignina das plantas. O melhor nível obtido foi 100 kg/ha de nitrogênio, sem potássio.

Produção de amônia na fermentação in vitro de rações com levedura, uréia ou farelo de algodão

O objetivo deste experimento foi avaliar a liberação de nitrogênio amoniacal (N-NH3) in vitro proveniente de rações contendo levedura, uréia e farelo de algodão, usadas na alimentação de ruminantes. Os dados relativos ao N-NH3 encontrado nas amostras obtidas durante a primeira hora de fermentação apresentaram diferenças entre as rações preparadas com farelo de algodão e levedura, quando comparadas à ração com uréia, com valores de 7,52; 8,66; e 29,84 mg de N-NH3/100 mL de fluido ruminal, respectivamente. A mesma tendência foi observada até as 12 horas de fermentação, com valores de 1,05; 1,57; e 8,57 mg N-NH3/100 mL de fluido, para as rações com farelo de algodão, levedura e uréia, respectivamente. A análise dos dados relativos às amostras com 12 horas de fermentação mostrou tendência de diminuição da concentração de N-NH3, atingindo os menores valores neste tempo de incubação.