Production of transgalactosylated oligosaccharides (TOS) by galactosyltransferase activity from Penicillium simplicissimum

Ingestion of transgalactosylated oligosaccharides (TOS) and other non-digestible oligosaccharides (NDOs) induces a significant increase in Bifidobacterium, Lactobacillus and some desirable species of Streptococcus populations in the gut of human and other animals (prebiotic effect). This change in the intestinal flora is responsible for several beneficial physiological effects such as a decrease of putrefactive products in the feces, lower blood cholesterol content, higher Ca2+ absorption, a smaller loss of bone tissue in ovariotomized female rats and a lower incidence of colon cancer. beta-Galactosidase from Penicillium simplicissimum, a strain isolated from soil, showed high galactosyltransferase activity when incubated with a highly concentrated lactose solution. Optimum pH and temperature ranges for hydrolytic activity were 4.0-4.6 and 55-60 degrees C, respectively, for a lactose concentration of 5.0% (w/v). Maximal galactosyltransferase activity was obtained at pH 6.5 and 50 degrees C and TOS synthesis was positively associated with lactose concentration in the reaction medium. Thus, when 50 ml of a 60% (w/v) lactose solution was incubated with 26.6 U of beta-galactosidase under the best pH and temperature conditions for transferase activity, a final product with 30.5% TOS (183 mg ml(-1)), 27.5% residual lactose and 42.0% monosaccharides was obtained. (C) 1999 Elsevier B.V. Ltd. All rights reserved.

Enzymatic production of functional oligosaccharides - A review

Interest in oligosaccharide production and its general characteristics is growing. The physiological effects resulting from its ingestion make them more attractive than its sweetness, and the versatility of these carbohydrates allows their use for human and animal nutrition, pharmacology and the cosmetics industry, among others. Several microorganisms are involved in enzyme production to create oligomers with biological activity, including fructooligosaccharides, galactooligosaccharides and aminoglucanoligosaccharides. Some oligomers are currently on the market, but the search for new microorganisms producing enzymes, high-yield processes for obtaining oligosaccharides, different physiological effects, and a correlation between chemical structure and function continues.

Metabolismo di oligosaccaridi prebiotici in Bifidobacterium per il potenziale sviluppo di nuovi prodotti alimentari funzionali

The growth and the metabolism of Bifidobacterium adolescentis MB 239 fermenting GOS, lactose, galactose, and glucose were investigated. An unstructerd unsegregated model for growth of B. adolescentis MB 239 in batch cultures was developed and kinetic parameters were calculated with a Matlab algorithm. Galactose was the best carbon source; lactose and GOS led to lower growth rate and cellular yield, but glucose was the poorest carbon source. Lactate, acetate and ethanol yields allowed calculation of the carbon fluxes toward fermentation products. Similar distribution between 3- and 2-carbon products was observed on all the carbohydrates (45 and 55%, respectively), but ethanol production was higher on glucose than on GOS, lactose and galactose, in decreasing order. Based on the stoichiometry of the fructose 6-phosphate shunt and on the carbon distribution among the products, ATP yield was calculated on the different carbohydrates. ATP yield was the highest on galactose, while it was 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondance among ethanol production, low ATP yields, and low biomass production was established demonstrating that carbohydrate preferences may result from different sorting of carbon fluxes through the fermentative pathway. During GOS fermentation, stringent selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were first to be consumed, and a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β-(1-4) galactosides can be hydrolysed before they are taken up. The physiology of Bifidobacterium adolescentis MB 239 toward xylooligosaccharides (XOS) was also studied and our attention was focused on an extracellular glycosyl-hydrolase (β-Xylosidase) expressed by a culture of B. adolescentis grown on XOS as sole carbon source. The extracellular enzyme was purified from the the supernatant, which was dialyzed and concentrated by ultrafiltration. A two steps purification protocol was developed: the sample was loaded on a Mono-Q anion exchange chromatography and then, the active fractions were pooled and β-Xylosidase was purified by gel filtration chromatography on a Superdex-75. The enzyme was characterized in many aspects. β- Xylosidase was an homo-tetramer of 160 kDa as native molecular mass; it was a termostable enzyme with an optimum of temperature at 53 °C and an optimum of pH of 6.0. The kinetics parameter were calculated: km = 4.36 mM, Vmax = 0.93 mM/min. The substrate specificity with different di-, oligo- and polysaccharides was tested. The reactions were carried out overnight at pH 7 and at the optimum of temperature and the carbohydrates hydrolysis were analyzed by thin layer chromatography (TLC). Only glycosyl-hydrolase activities on XOS and on xylan were detected, whereas sucrose, lactose, cellobiose, maltose and raffinose were not hydrolyzed. It’s clearly shown that β-Xylosidase activity was higher than the Xylanase one. These studies on the carbohydrate preference of a strain of Bifidobacterium underlined the importance of the affinity between probiotics and prebiotics. On the basis of this concept, together with Barilla G&R f.lli SpA, we studied the possibility to develop a functional food containing a synbiotic. Three probiotic strains Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 were studied to assess their suitability for utilization in synbiotic products on the basis of antioxidative activity, glutathione production, acid and bile tolerance, carbohydrates fermentation and viability in food matrices. Bile and human gastric juice resistance was tested in vitro to estimate the transit tolerance in the upper gastrointestinal tract. B. lactis and L. plantarum were more acid tolerant than S. thermophilus. All the strains resisted to bile. The growth kinetics on 13 prebiotic carbohydrates were determined. Galactooligosaccharides and fructo-oligosaccharides were successfully utilized by all the strains and could be considered the most appropriate prebiotics to be used in effective synbiotic formulations. The vitality of the three strains inoculated in different food matrices and maintained at room temperature was studied. The best survival of Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 was found in food chocolate matrices. Then an in vivo clinical trial was carried out for 20 healthy volunteers. The increase in faecal bifidobacteria and lactobacilli populations and the efficacy of the pre-prototype was promising for the future develop of potential commercial products.

The role of bifidobacteria in newborn health and the intestinal microbial balance

Gut microbial acquisition during the early stage of life is an extremely important event since it affects the health status of the host. In this contest the healthy properties of the genus Bifidobacterium have a central function in newborns. The aim of this thesis was to explore the dynamics of the gut microbial colonization in newborns and to suggest possible strategies to maintain or restore a correct balance of gut bacterial population in infants. The first step of this work was to review the most recent studies on the use of probiotics and prebiotics in infants. Secondly, in order to prevent or treat intestinal disorders that may affect newborns, the capability of selected Bifidobacterium strains to reduce the amount of Enterobacteriaceae and against the infant pathogen Streptococcus agalactiae was evaluated in vitro. Furthermore, the ability of several commercial fibers to stimulate selectively the growth of bifidobacterial strains was checked. Finally, the gut microbial composition in the early stage of life in response to the intrapartum antibiotic prophylaxis (IAP) against group B Streptococcus was studied using q-PCR, DGGE and next generation sequencing. The results globally showed that Bifidobacterium breve B632 strain is the best candidate for the use in a synbiotic product coupled to a mixture of two selected prebiotic fibers (galactooligosaccharides and fructooligosaccharides) for gastrointestinal disorders in infants. Moreover, the early gut microbial composition was affected by IAP treatment with infants showing lower counts of Bifidobacterium spp. and Bacteroides spp. coupled to a decrement of biodiversity of bacteria, compared to control infants. These studies have shown that IAP could affect the early intestinal balance in infants and they have paved the way to the definition of new strategies alternative to antibiotic treatment to control GBS infection in pregnant women.

Bioreaction and separation in preparative batch chromatographic columns : the hydrology of lactose to yield glucose,galactose and oligosaccharides

The initial aim of this project was to improve the performance of a chromatographic bioreactor-separator (CBRS). In such a system, a dilute enzyme solution is pumped continuously through a preparative chromatographic column, while pulses of substrate are periodically injected on to the column. Enzymic reaction and separation are therefore performed in a single unit operation. The chromatographic columns used were jacketed glass columns ranging from 1 to 2 metres long with an internal diameter of 1.5 cm. Linking these columns allowed 1, 2, 3 and 4 metre long CBRS systems to be constructed. The hydrolysis of lactose in the presence of β~galactosidase was the reaction of study. From previous work at Aston University, there appeared to be no difficulties in achieving complete lactose hydrolysis in a CBRS. There did, however, appear to be scope for improving the separative performance, so this was adopted as an initial goal. Reducing the particle size of the stationary phase was identified as a way of achieving this improvement. A cation exchange resin was selected which had an average particle size of around half that previously used when studying this reaction. A CBRS system was developed which overcame the operational problems (such as high pressure drop development) associated with use of such a particle size. A significant improvement in separative power was achieved. This was shown by an increase in the number of theoretical plates (N) from about 500 to about 3000 for a 2 metre long CBRS, coupled with higher resolution. A simple experiment with the 1 metre column showed that combined bioreaction and separation was achievable in this system. Having improved the separative performance of the system, the factors affecting enzymic reaction in a CBRS were investigated; including pulse volume and the degree of mixing between enzyme and substrate. The progress of reaction in a CBRS was then studied. This information was related to the interaction of reaction and separation over the reaction zone. The effect of injecting a pulse over a length of time as in CBRS operation was simulated by fed batch experiments. These experiments were performed in parallel with normal batch experiments where the substrate is mixed almost instantly with the enzyme. The batch experiments enabled samples to be taken every minute and revealed that reaction is very rapid. The hydrodynamic characteristics of the two injector configurations used in CBRS construction were studied using Magnetic Resonance Imaging, combined with hydrodynamic calculations. During the optimisation studies, galactooligosaccharides (GOS) were detected as intermediates in the hydrolysis process. GOS are valuable products with potential and existing applications in food manufacture (as nutraceuticals), medicine and drug targeting. The focus of the research was therefore turned to GOS production. A means of controlling reaction to arrest break down of GOS was required. Raising temperature was identified as a possible means of achieving this within a CBRS. Studies were undertaken to optimise the yield of oligosaccharides, culminating in the design, construction and evaluation of a Dithermal Chromatographic Bioreactor-separator.

Effects of early nutrition on the wnt and bone morphogenic protein pathways in the developing intestine

Infant formula is consumed by the majority of infants in the United States for at least part of the first year of life. Infant formula lacks many of the bioactive compounds that are naturally occurring in breast milk. Because of this, there has been an increased interest by the companies that manufacture infant formula to include additives that would potentially allow formula to more closely mimic breast milk activity. One such ingredient currently being added to infant formula is prebiotics. Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth of specific healthful bacteria in the colon. It is speculated that prebiotics replicate the activity of breast milk oligosaccharides, which through the production of butyrate by intestinal microbiota, may interact with the Wnt/BMP pathways. The Wnt/BMP pathways regulate intestinal stem cells, which determine the growth, development and maintenance of the intestine. Therefore, the objective of this study was to explore the effects that the addition of prebiotics to formula have on the regulation of the Wnt/BMP pathways when fed to neonatal piglets, a model commonly used in the study of infant nutrition. Piglets (n=5) were randomized into sow-reared (SR), fed control formula (F), or fed formula with added prebiotics (F+P). Fructooligosaccharides (FOS) (2 g/L) and polydextrose (PDX) (2 g/L) were chosen as the prebiotics for this study, because this combination had been less studied than other combinations. Ileum and ascending colon were collected at 7 and 14 days-of-age. Dry matter content, pH, and short chain fatty acid (SCFA) content was measured. The mRNA expression of β-catenin, sFRP3, sFRP4, frizzled 6, DKK1 (Wnt pathway), gremlin (BMP pathway), TNF-a, HNF-4α and osteopontin (OPN) was measured by RT-qPCR. Piglets fed the F+P diet had greater acetate concentration and lower pH in the ileum at day 14 and in the colon at day 7 and day 14 than F piglets. Butyrate concentrations were highest in SR with F+P not differing from F in ileum at day 14 and colon at day 7 and day 14. Effects of age were seen in all genes, with the exception of OPN, sFRP-3 and sFRP-4. On day 7, no effect of diet was observed in the ileum, however, mRNA expression of DKK1 and frizzled 6 were greater in F+P than SR (p≤0.05). On day 14, gremlin expression was lower and OPN was greater in the ileum of SR piglets compared to F and F+P. Also on day 14, HNF-4α mRNA expression was greater in both ileum and colon of F+P piglets and sFRP3 mRNA expression was greater in the colon than F or SR . In summary, differences were observed between gene expression of F+P and SR piglet intestines, but the supplementation of 2 g/L scFOS and 2 g/L PDX to formula did not shift expression of genes in the Wnt/BMP pathways to be more similar to SR than F. As the Wnt/BMP pathway is known to exist in a gradient along the crypt-villus axis, with Wnt expression dominating in the crypt region and BMP expression dominating in the villi, it was possible that pooling whole tissue reduced our ability to detect treatment effects that would be concentrated in either region. A method was therefore developed to remove intestinal epithelial cells along the villus-to-crypt axis. Twenty-five-day-old F and SR piglets were euthanized and ileal tissue was collected and placed in a dissociation buffer in a shaking water bath. Exfoliated cells were removed at increasing time points from 5 to 100 minutes in order to remove cells along the villus-to-crypt axis. After the final incubation, remaining mucosal tissue was removed using a sterile glass microscope slide and pooled with the final exfoliated cell isolation. After each cell collection, a section of tissue was fixed in formalin for histomorphological examination. Expression of genes in the Wnt/BMP pathways, along with crypt marker genes (CDK5 and v-myb), were measured in both whole ileal tissue, pooled epithelial cells, and separate epithelial cell isolations from the same piglet. The expression of β-catenin, HNF-4α, TNF-α, TGF-β and the crypt marker v-myb matched the expected villus-to-crypt pattern in cells collected after 10 (incubation 1), 30 (incubation 2) and 60 (incubation 3) minutes. However, expression of expression in cells collected after 100 minutes (incubation 4) was variable, which may be due to the fact that crypt cells were not efficiently removed and the presence of unwanted non-epithelial tissue. Gremlin, OPN, DKK1, sFRP3 and sFRP4 expression was not statistically different along the villus-to-crypt axis. Frizzled 6 and CDK5 did not express as we had predicted, with expression highest towards the villi. In summary, the epithelial cell collection method used was not entirely successful. While much of the gene data suggests that cells were removed along the villus-to-crypt axis through the first three incubations, the last incubation, which involved scraping the tissue, removed non-epithelial components of the mucosa, while leaving the crypts intact. In conclusion, the addition of 2 g/L PDX and 2 g/L scFOS did not cause gene expression of the Wnt/BMP pathways to mirror either F or SR expression. New isolation methods to extract cells along the crypt-villus axis should be considered, including the use of a laser capture microdissection. While this combination of prebiotics did not yield the intended effects, future research should be done on other combinations, such as the inclusion of galactooligosaccharides (GOS), which is commonly added to food products including infant formula.

Conversão da lactose e síntese de galacto-oligossacarídeos: uma abordagem experimental e teórica

O presente trabalho avaliou, na etapa experimental, um processo simultâneo de catálise e fermentação láctica visando obter um iogurte com potenciais características nutracêuticas e, na sua etapa teórica, estabeleceu uma interlocução entre a vivência experimentalista e a teoria da cinética enzimática, no que se refere à conversão da lactose e à síntese de galactooligossacarídeos (GOS). Na abordagem experimental, para um substrato específico, avaliouse biocatálise conduzida simultaneamente à fermentação, defasando a adição da enzima em relação ao início do processo fermentativo. A fermentação foi realizada a partir de cultura láctica liofilizada comercial contendo dois micro-organismos probióticos, Bifidobacterium animalis e Lactobacillus acidophilus, associados aos micro-organismos característicos do iogurte, Lactobacillus bulgaricus e Streptococcus thermophilus. Foi utilizado um preparado enzimático contendo -galactosidases obtidas de duas origens distintas: Kluyveromyces lactis e Aspergillus niger. Foram avaliados os efeitos da concentração da enzima e do tempo de adição da enzima em um planejamento experimental 2 2 . As respostas foram às concentrações, ao final do processo, de lactose, de GOS, de glicose e de galactose e a hidrólise dos galactooligossacarídeos ao longo do tempo. No que se refere à abordagem teórica, o presente trabalho considerou modelos matemáticos de hidrólise de dissacarídeos e conversão da lactose, em que a inibição foi representada a partir do incremento da concentração dos produtos da reação. No que se refere à conversão da lactose e síntese de GOS, o presente trabalho buscou estabelecer um modelo matemático em que a inibição ocorreu por efeito do incremento das concentrações de glicose e de galactose, comparando-o com os modelos conhecidos na literatura. Verificou-se que o desempenho do modelo obtido no presente trabalho foi robusto em relação às premissas estabelecidas. Na comparação com resultados experimentais de conversão enzimática, o modelo mostrou-se capaz de minimizar o erro e de ajustar-se aos dados experimentais.