Enzymes for production of whey protein hydrolysates and other value-added products.

Whey is a byproduct of dairy industries, the aqueous portion which separates from cheese during the coagulation of milk. It represents approximately 85-95% of milk's volume and retains much of its nutrients, including functional proteins and peptides, lipids, lactose, minerals, and vitamins. Due to its composition, mainly proteins and lactose, it can be considered a raw material for value-added products. Whey-derived products are often used to supplement food, as they have shown several physiological effects on the body. Whey protein hydrolysates are reported to have different activities, including antihypertensive, antioxidant, antithrombotic, opioid, antimicrobial, cytomodulatory, and immuno-modulatory. On the other hand, galactooligosaccharides obtained from lactose can be used as prebiotic for beneficial microorganisms for the human gastrointestinal tract. All these compounds can be obtained through physicochemical, microbial, or enzymatic treatments. Particularly, enzymatic processes have the advantage of being highly selective, more stable than chemical transformations, and less polluting, making that the global enzyme market grow at accelerated rates. The sources and different products associated with the most used enzymes are particularly highlighted in this review. Moreover, we discuss metagenomics as a tool to identify novel proteolytic enzymes, from both cultivable and uncultivable microorganisms, which are expected to have new interesting activities. Finally enzymes for the transformation of whey sugar are reviewed. In this sense, carbozymes with ß-galactosidase activity are capable of lactose hydrolysis, to obtain free monomers, and transgalactosylation for prebiotics production. KEY POINTS: • Whey can be used to obtain value-added products efficiently through enzymatic treatments • Proteases transform whey proteins into biopeptides with physiological activities • Lactose can be transformed into prebiotic compounds using ß-galactosidases.

Galactooligosaccharides: Physiological benefits, production strategies, and industrial application.

The concern for better life quality has been encouraging the bioprocess industries to develop technological strategies to obtain new biomolecules. Galactooligosaccharides (GOS) are an important class of food-grade oligosaccharides, being classified as non-digestible, and which present prebiotic potential, promoting better conditions of health and well-being. The main benefits include the selective stimulation of beneficial microorganisms, the decrease in the formation of toxic compounds, the increase in the absorption of minerals, improvement of an immune response, and a reduction in the severity of obesity and diabetes. This review approaches the recent methodologies and strategies to obtain GOS, their health benefits, purification, and technological properties for industrial application. Improvements in the process are continuously being investigated, with the technique of enzyme immobilization representing a potentially promising strategy. Sustainable GOS productions have been reported by the use of agro-industrial residues, such as cheese whey. Despite these advances, the main concern of the process consists in the low yield, which implies high investments in the purification of the bioproducts. Technological and nutritional approaches to the GOS application in different industrial sectors are also reported.

Absorción mineral y retención ósea en ratas normales en crecimiento por el consumo de un yogur experimental reducido en lactosa que contiene galactooligosacáridos (GOS) / Mineral absorption and bone retention in normal growing rats by consumption of an experimental lactose-reduced yogurt containing galactooligosaccharides (GOS)

Introducción: Los GOS son prebióticos naturales presentes en la leche materna que pue-den obtenerse enzimáticamente a partir de la lactosa de leche de vaca durante la fabricación de yogur. El producto lácteo resultante será reducido en lactosa y contendrá prebióticos y bacterias potencialmente probióticas. Sin embargo, mantendrá la baja relación Ca/Pi que aporta la leche de vaca, lo que podría alterar el remodelamiento óseo y la mineralización. Objetivo: comparar si un yogur reducido en lactosa que contiene GOS (YE) ofrece ventajas adicionales respecto de un yogur regular sin GOS (YR) sobre las absorciones (Abs) de Ca y Pi, retención y calidad ósea durante el crecimiento normal. Al destete, ratas machos fueron divididas en 3 grupos alimentados con AIN ́93-G (C), YE o YR durante 28 días. Resultados: YE mostró el mayor aumento de lactobacilos fecales; producción de ácidos grasos de cadena corta especialmente p, profundidad de las criptas colónicas y menor pH cecal. El %AbsCa y %AbsPi aumentó en el siguiente órden: YE> YR> C (p < 0,05). El contenido de Ca y Pi en fémur, la densidad y contenido mineral óseos y los parámetros biomecánicos fueron similares en YE y C, mientras que YR mostró valores significativa-mente menores (p < 0,05). Conclusiones: YE aumentó las Abs y biodisponibilidad de minerales, alcanzando la retención y calidad ósea de C. El aumento en las Abs observado en YR no logró obtener la retención y calidad ósea de C. Conclusión: YE habría contrarrestado el efecto negativo del mayor aporte de Pi de la leche de vaca y sería una buena estrategia para lograr el pico de masa ósea y calidad del hueso adecuados, especialmente en individuos intolerantes a la lactosa. (AU)

Breast milk contains an optimal calcium/phosphate (Ca/Pi) ratio and GOS. These natural prebiotics can be enzymatically produced via cow's milk lactose inyogurt manufacture. This milk product is low in lactose and contains prebiotics and potentially probiotic bacteria but maintains a low Ca/Pi ratio that could alter bone remodeling and mineralization. We evaluated if a lactose-reduced yogurt containing GOS (YE) offers additional advantages over regular yogurt without GOS (YR) on Ca and Pi absorption (Abs), bone retention and quality during normal growth. Weaning male rats were divided into 3 groups fed AIN'93-G (C), YE or YR for 28 days. Results: YE showed the highest increase in fecal lactobacilli; short-chain fatty acids production, especially propionate and butyrate; intestine crypt depth, and the lowest cecal pH. AbsCa% and AbsPi% increased in this order: YE> YR> C (p <0.05). Ca and Pi content in femur, bone density and mineral content, and biomechanical parameters were similar in YE and C, while YR showed the significantly lowest value (p < 0.05). Conclusions: YE increased mineral Abs reaching the retention and bone quality of C. Although YR increased Abs, bone retention and quality did not achieve C values. Seemingly, YE compensated for the negative effect of the higher Pi supply and would be a good strategy to achieve adequate peak bone mass and bone quality, especially in lactose intolerant individuals. (AU)

Quality indicators in lactose hydrolyzed milks and soy beverages from Colombia.

Worldwide there is great interest in producing low lactose milk and drinks, such as soy beverages, suitable for consumption by lactose-intolerant people. These products have different carbohydrate compositions, which affect quality indicators derived from Maillard reaction (furosine and 5-hydroxyl-methyl furfural, HMF). In this study, quality parameters have been determined in 11 commercial samples (8 lactose-hydrolyzed milks and 3 soy beverages). Presence of ß-galactooligosaccharides in milks (22.2-458.1 mg/100 mL) correlating roughly with the remaining lactose content (1.9-357.7 mg/100 mL). Soy beverages contained α-galactosides in concentrations of 30-75 mg/100 mL. HMF and furosine were detected in all milk samples. In addition, powdered milks subjected to controlled storage (40 °C, aw 0.44) showed a furosine increase up to 88%. In conclusion, a great composition diversity was observed in the different products, probably favored by the lack of regulation and underline the importance of controlling processing and storage conditions to preserve product quality.

Microwave Assisted Extraction of Bioactive Carbohydrates from Different Morphological Parts of Alfalfa (Medicago sativa L.).

Despite the nutritional properties of alfalfa, its production is mainly for animal feed and it is undervalued as a food source. In this study, the valorization of alfalfa as a potential source of bioactive carbohydrates [inositols, α-galactooligosaccharides (α-GOS)] is presented. A Box-Behnken experimental design was used to optimize the extraction of these carbohydrates from leaves, stems, and seeds of alfalfa by solid-liquid extraction (SLE) and microwave-assisted extraction (MAE). Optimal extraction temperatures were similar for both treatments (40 °C leaves, 80 °C seeds); however, SLE required longer times (32.5 and 60 min vs. 5 min). In general, under similar extraction conditions, MAE provided higher yields of inositols (up to twice) and α-GOS (up to 7 times); hence, MAE was selected for their extraction from 13 alfalfa samples. Pinitol was the most abundant inositol of leaves and stems (24.2-31.0 mg·g-1 and 15.5-22.5 mg·g-1, respectively) while seed extracts were rich in α-GOS, mainly in stachyose (48.8-84.7 mg·g-1). In addition, inositols and α-GOS concentrations of lyophilized MAE extracts were stable for up to 26 days at 50 °C. These findings demonstrate that alfalfa is a valuable source of bioactive carbohydrates and MAE a promising alternative technique to obtain functional extracts.

Synthesis of b(1 ? 3) and b(1 ? 6) galactooligosaccharides from lactose and whey using a recombinant b-galactosidase from Pantoea anthophila

BACKGROUND: Milk whey, a byproduct of the dairy industry has a negative environmental impact, can be used as a raw material for added-value compounds such as galactooligosaccharides (GOS) synthesis by bgalactosidases. RESULTS: B-gal42 from Pantoea anthophila strain isolated from tejuino belonging to the glycosyl hydrolase family GH42, was overexpressed in Escherichia coli and used for GOS synthesis from lactose or milk whey. Crude cell-free enzyme extracts exhibited high stability; they were employed for GOS synthesis reactions. In reactions with 400 g/L lactose, the maximum GOS yield was 40% (w/w) measured by HPAEC-PAD, corresponding to 86% of conversion. This enzyme had a strong predilection to form GOS with b(1 ? 6) and b (1 ? 3) galactosyl linkages. Comparing GOS synthesis between milk whey and pure lactose, both of them at 300 g/L, these two substrates gave rise to a yield of 38% (60% of lactose conversion) with the same product profile determined by HPAEC-PAD. CONCLUSIONS: B-gal42 can be used on whey (a cheap lactose source) to produce added value products such as galactooligosaccharides.

Production of ß-galactosidase by Trichoderma sp. through solid-state fermentation targeting the recovery of galactooligosaccharides from whey cheese.

AIMS: Optimization of ß-galactosidase production by Trichoderma sp. under solid-state fermentation using wheat bran as solid substrate through an experimental design and its application targeting the recovery of galactooligosaccharides (GOS) from whey cheese. METHODS AND RESULTS: The ß-galactosidase production by Trichoderma sp. increased 2·3-fold (2·67 U g-1 of substrate) culturing the fungus at 30°C for 187 h, at an inoculum of 105 spores per ml, and a 1 : 1·65 (w/v) ratio of wheat bran to tap water. The best enzyme activity was obtained at 55°C and pH 4·5. The catalytic activity was maintained for up to 180 min incubating at 35-45°C, and above 50% at acidic or alkaline pH for up to 24 h. It also presented resistance to chemical compounds. ß-galactosidase catalysed the hydrolysis of the lactose and the transgalactosylation reaction leading to the production of GOS. CONCLUSION: Trichoderma sp. produced ß-galactosidase with transgalactosylation activity that may be used to recover GOS, products with high added value, from whey cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: ß-galactosidases are used in different industrial sectors. Therefore, the Trichoderma ß-galactosidase is a promising alternative for the production of GOS as prebiotic from the dairy effluents, contributing to the reduction in the environmental impact.

Effect of the lactose source on the ultrasound-assisted enzymatic production of galactooligosaccharides and gluconic acid.

It is well known that one of the main problems in galactooligosaccharide production (GOS) via tranglycosylation of lactose is the presence of monosaccharides that contribute to increasing the glycaemic index, as is the case of glucose. In this work, as well as studying the effect of ultrasound (US) on glucose oxidase (Gox) activation during gluconic acid (GA) production, we have carried out an investigation into the selective oxidation of glucose to gluconic acid in multienzymatic reactions (ß-galactosidase (ß-gal) and Gox) assisted by power US using different sources of lactose as substrate (lactose solution, whey permeate, cheese whey). In terms of the influence of matrix on GOS and GA production, lactose solution gave the best results, followed by cheese whey and whey permeate, salt composition being the most influential factor. The highest yields of GOS production with the lowest glucose concentration and highest GA production were obtained with lactose solution in multienzymatic systems in the presence of ultrasound (30% amplitude) when Gox was added after 1 h of treatment with ß-gal. This work demonstrates the ability of US to enhance efficiently the obtainment of prebiotic mixtures of low glycaemic index.

Proteomic study of Enterococcus durans LAB18S growing on prebiotic oligosaccharides.

This study evaluates the influence of prebiotic carbohydrates, namely fructooligosaccharides (FOS) and galactooligosaccharides (GOS), on the protein expression of Enterococcus durans LAB18S. The strain was cultivated in 10 g L-1 FOS, GOS or glucose (control) and cellular proteins were extracted for mass spectrometry analysis. A total of 771 proteins were identified and 135 E. durans proteins were validated by the Scaffold algorithm. The proteins were functionally categorized according to Gene Ontology terms. Both FOS and GOS were used as carbon source by E. durans LAB18S, upregulating the production of proteins that may be associated with intestinal mucosa adhesion, carbohydrate and nitrogen metabolism, and stress response. Cells grown with GOS showed an increased expression of the cell division protein divIVA, EF-Tu and glyceraldehyde 3-phosphate dehydrogenase that have been associated with epithelial cell adhesion. The use of FOS stimulated the production of proteins related to amino acid metabolism and energy conversion, and ClpX protein, which plays an important role in protein turnover. The results of this study indicate that FOS and GOS can be metabolized by E. durans and stimulate the microorganism to produce proteins related to some desirable characteristics for a probiotic strain.

Determining 1-kestose, nystose and raffinose oligosaccharides in grape juices and wines using HPLC: method validation and characterization of products from Northeast Brazil.

The objective of this work was to validate a method for direct determination in grape juice and wine of 1-kestose, nystose and raffinose oligosaccharides by reversed-phase high-performance liquid chromatography with refractive index detection using a new type of RP-C18 column (150 × 4.6 mm, 4 µm) with polar end-capping. The validated methodology was also used to characterize grape juice and fine wine products from Northeastern Brazil; and presented suitable linearity, precision, recovery, limits of detection and quantification. The method presented good specificity, revealing that sugars, organic acids, and ethanol (the main interferences in refraction detection) did not influence the quantification of the studied oligosaccharides. The main oligosaccharide found was 1-kestose (approximately 50% of the samples), followed by raffinose (20% of the samples). The results obtained in this are an indication that grape juices and wines have the potential to be functional beverages in relation to the presence of prebiotics.

Co-immobilized ß-galactosidase and Saccharomyces cerevisiae cells for the simultaneous synthesis and purification of galacto-oligosaccharides.

Simultaneous synthesis and purification (SSP) of galacto-oligosaccharides (GOS) from lactose was conducted using a combi-biocatalyst formed by crosslinked enzyme aggregates of Aspergillus oryzae ß-galactosidase and Saccharomyces cerevisiae cells co-immobilized by entrapment in calcium alginate gel particles. Product yield obtained with the combi-biocatalyst was similar than obtained with the soluble enzyme (23.3%), having a final purity of 25.7%. During the simultaneous process, ethyl-ß-galactoside was produced from the ethanol generated as a metabolic product of yeast cells, but ethyl-ß-galactoside was considerably decreased at high aeration (4 vvm). The combi-biocatalyst can be recovered and reused but its performance is limited by the reduction of the metabolic capacity of the cells. In this way, a process was developed for the SSP of GOS from lactose, obtaining a comparable product yield and higher specific productivity than in a conventional synthesis.

Response surface methodology as a tool for modelling galacto-oligosaccharide production.

The experiments reported in this research paper describe the effects of ß-galactosidase enzyme dose and cheese whey amount, on the maximum concentration and yield of galacto-oligosaccahride (GOS) and reaction time. The experimental plan was based on central composite rotational design (CCRD) and modelled by response surface methodology (RSM). The results indicate that the proposed mathematical models could adequately describe the concentration and yield of GOS and the reaction time within the limits of the factors that are being investigated. The variance analysis shows high values of coefficients of determination (>0·97) while no significant lack of fit was evident. Hence, the models could be employed to select reaction conditions applied in the manufacture of products enriched in bioactive compounds with high value-added.

Improvement of covalent immobilization procedure of ß-galactosidase from Kluyveromyces lactis for galactooligosaccharides production: Modeling and kinetic study.

Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by ß-galactosidase (EC 3.2.1.23) from Kluyveromyces lactis covalently immobilized to a polysiloxane-polyvinyl alcohol (POS-PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL-1 initial lactose concentration and 6 U mL-1 enzyme concentration, obtaining 25.46 ± 0.01 gL-1 yield of trisaccharides. Although below the HPLC-IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL-1 of trisaccharides and 13.79 ± 0.21 gL-1 of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady-state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1568-1578, 2017.

Synthesis and purification of galacto-oligosaccharides: state of the art.

Lactose-derived non-digestible oligosaccharides are prominent components of functional foods. Among them, galacto-oligosaccharides (GOS) outstand for being prebiotics whose health-promoting effects are supported on strong scientific evidences, having unique properties as substitutes of human milk oligosaccharides in formulas for newborns and infants. GOS are currently produced enzymatically in a kinetically-controlled reaction of lactose transgalactosylation catalyzed by ß-galactosidases from different microbial strains. The enzymatic synthesis of GOS, although being an established technology, still offers many technological challenges and opportunities for further development that has to be considered within the framework of functional foods which is the most rapidly expanding market within the food sector. This paper presents the current technological status of GOS production, its main achievements and challenges. Most of the problems yet to be solved refer to the rather low GOS yields attainable that rarely exceed 40 %, corresponding to lactose conversions around 60 %. This means that the product or reaction (raw GOS) contains significant amounts of residual lactose and monosaccharides (glucose and galactose). Efforts to increase such yields have been for the most part unsuccessful, even though improvements by genetic and protein engineering strategies are to be expected in the near future. Low yields impose a burden on downstream processing to obtain a GOS product of the required purity. Different strategies for raw GOS purification are reviewed and their technological significance is appraised.

Influence of lactose hydrolysis on galacto-oligosaccharides, lactose, volatile profile and physicochemical parameters of different yogurt varieties.

BACKGROUND: Different types of reduced-lactose yogurt, obtained by lactose hydrolysis using ß-galactosidase enzyme, are commercially available. The breakdown of lactose modifies the carbohydrate profile, including the production of prebiotic galacto-oligosaccharides (GOS), which could affect the survival and activity of starter and probiotic cultures and the parameters of yogurt quality. The extension of these changes is dependent on the yogurt matrix composition. This study aimed to evaluate the influence of lactose hydrolysis on GOS, lactose, volatile profile and physicochemical parameters of different yogurt varieties during storage. RESULTS: The presence of ß-galactosidase enzyme did not affect either the global composition or the survival of cultures. Overall, the hydrolyzed products had lower acidity than traditional ones. GOS were found at similar levels in fresh hydrolyzed yogurts, whereas in traditional yogurts they were not detected. The proportion of ketones, acids and aldehydes seems to be more dependent on yogurt variety than on addition of the enzyme. Likewise, the storage period affected the volatile fraction to different degree; the increase in acid compounds was more pronounced in hydrolyzed than in traditional yogurts. CONCLUSION: This work shows that it is possible to obtain different varieties of reduced-lactose yogurt, some of them with additional benefits to health such as reduced fat, reduced calories, added with probiotic/inulin and enriched in GOS, with similar characteristics to traditional products. © 2016 Society of Chemical Industry.

Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus plantarum after Spray-Drying.

In this work maltodextrins were added to commercial galacto-oligosaccharides (GOS) in a 1:1 ratio and their thermophysical characteristics were analyzed. GOS:MD solutions were then used as matrices during spray-drying of Lactobacillus plantarum CIDCA 83114. The obtained powders were equilibrated at different relative humidities (RH) and stored at 5 and 20°C for 12 weeks, or at 30°C for 6 weeks. The Tgs of GOS:MD matrices were about 20-30°C higher than those of GOS at RH within 11 and 52%. A linear relation between the spin-spin relaxation time (T2) and T-Tg parameter was observed for GOS:MD matrices equilibrated at 11, 22, 33, and 44% RH at 5, 20, and 30°C. Spray-drying of L. plantarum CIDCA 83114 in GOS:MD matrices allowed the recovery of 93% microorganisms. In contrast, only 64% microorganisms were recovered when no GOS were included in the dehydration medium. Survival of L. plantarum CIDCA 83114 during storage showed the best performance for bacteria stored at 5°C. In a further step, the slopes of the linear regressions provided information about the rate of microbial inactivation for each storage condition (k values). This information can be useful to calculate the shelf-life of spray-dried starters stored at different temperatures and RH. Using GOS:MD matrices as a dehydration medium enhanced the recovery of L. plantarum CIDCA 83114 after spray-drying. This strategy allowed for the first time the spray-drying stabilization of a potentially probiotic strain in the presence of GOS.

Effect of a mixture of GOS/FOS® on calcium absorption and retention during recovery from protein malnutrition: experimental model in growing rats.

INTRODUCTION: During growth, protein deprivation impairs epiphyseal growth plate (EGP) height, bone volume (BV) and endochondral ossification. During catch-up growth, Ca availability becomes essential to ensure the extra amount needed to achieve optimal peak bone mass and strength. GOS and FOS improve mineral absorption in the colon. PURPOSE: The effect of a mixture of GOS/FOS® 9:1 added to a 0.5 %Ca (NCa) and a 0.3 %Ca (LCa) diets on Ca, P and Mg absorptions and bone mineralization, density and structure using an experimental model of growing rats recovering from early protein malnutrition was investigated. METHODS: To induce protein malnutrition, rats were fed a low protein diet: 4 % (LPD) during 1 week and then were randomly assigned to recovery groups (R) until day 50 (T = 50) as follows: R0.5 %: NCa; RP0.5 %: NCa + 5.3 % GOS/FOS®; R0.3 %: LCa and RP0.3 %: LCa + 5.3 % GOS/FOS®. Control groups received the 0.5 %Ca or 0.3 %Ca diet from weaning until day 40 or 50. RESULTS: Body weight and length increased in C groups throughout the study; both were arrested in all R during LPD consumption and increased immediately after re-feeding. Independently of dietary Ca content, LS counts, ß-glucosidase and Ca, P and Mg absorption increased, whereas cecum pH, ß-glucuronidase, urease and tryptophanase decreased in RP0.5 %: and RP0.3 %: as compared to the other studied groups (p < 0.01). Prebiotic consumption decreased CTX levels and increased femur Ca, Mg and P contents, total skeleton bone mineral content, proximal tibia and spine BMD, BV, EGP height and hypertrophic zone thickness, stiffness and elastic modulus as compared to recovery groups fed the prebiotic-free diets. CONCLUSION: Under the present experimental conditions, GOS/FOS® mixture induced colonic positive effects, which increased Ca, P and Mg absorption. Thus, consuming the prebiotic-containing diet resulted in an extra amount of minerals that improved bone development in growing rats recovering from protein malnutrition.

In vitro fermentation of prebiotic carbohydrates by intestinal microbiota in the presence of Lactobacillus amylovorus DSM 16998.

The aim of this study was to evaluate the assimilation of the prebiotics fructooligosaccharides (FOS), galactooligosaccharides (GOS), and Konjac glucomannan oligosaccharides (KGMO) by three human (H1, H2 and H3) and pig (P1, P2 and P3) faecal microbiotas in the presence of the potentially probiotic strain Lactobacillus amylovorus DSM 16698, using an in vitro batch fermentation model. Total bacteria and L. amylovorus populations were quantified using qPCR and biochemical features (pH, production of short chain fatty acids (SCFA), lactate, ammonia, and carbohydrate assimilation) were determined. L. amylovorus did not have a competitive advantage under in vitro conditions, reflected by its reduced relative abundance during fermentation despite the carbohydrate sources added. Pig microbiota sustained more stable probiotic counts. Intermittently produced lactate was possibly assimilated by the microbiota and converted to other SCFA as the carbohydrates were assimilated, with H3 probably having a methanogenic metabolism with high lactate and acetate consumption except in the presence of FOS, which assimilation resulted in the highest total SCFA for this volunteer. Addition of FOS also resulted in lower pH and ammonia, which might have been used as nitrogen source by pig microbiota. KGMO needed longer fermentation periods to be completely assimilated by both human and porcine faecal microbiotas. Overall, our results reinforce the notion that care must be taken when generalising the effects claimed for a given probiotic or potentially probiotic strain, including the combination with different prebiotic substrates, since they may vary considerably among individuals, which is important when studying potentially pro- and prebiotic combinations for application as functional foods and feed ingredients.

Scientific evidence for health effects attributed to the consumption of probiotics and prebiotics: an update for current perspectives and future challenges.

Probiotics and prebiotics, mainly commercialised as food ingredients and also as supplements, are considered highly profitable niche markets. However, in recent years, the food industry has suffered from a series of health claim restrictions on probiotics and prebiotics in many parts of the world, including those made by the European Food Safety Authority. Therefore, we reviewed the core benefits of probiotic and prebiotic consumption on health. A number of studies have examined the prevention and/or management of intestinal infections, respiratory tract infections, CVD, osteoporosis, urogenital infections, cavities, periodontal disease and halitosis, allergic reactions, inflammatory bowel disease and irritable bowel syndrome and Helicobacter pylori gastric infections. In fact, a deeper understanding of the mechanisms involved in human microbiota and immune system modulation by probiotics and prebiotics relies on continuous efforts to establish suitable biomarkers of health and diseases risk factors for the design of clinical trials required for health claim approval. In spite of the promising results, the performance of large, long-term, well-planned, well-aligned clinical studies is crucial to provide more reliability and a more solid basis for the outcomes achieved and to support the potential use of probiotics and prebiotics in clinical practice.

Simultaneous synthesis of mixtures of lactulose and galacto-oligosaccharides and their selective fermentation.

Lactulose and galacto-oligosaccharides (GOS) are well recognized prebiotics derived from lactose. In the synthesis of lactulose with ß-galactosidases GOS are also produced, but the ratio of lactulose and GOS in the product can be tuned at will, depending on the operation conditions, so to obtain an optimal product distribution in terms of prebiotic potential. The selectivity of fermentation of each carbohydrate alone as well as mixtures of both was determined using pH-controlled anaerobic batch cultures with faecal inoculum. Within the experimental range considered, lactulose/GOS molar ratio of 4 resulted in the highest selectivity for Bifidobacterium and Lactobacillus/Enterococcus, so this ratio was selected as the target for the synthesis of lactulose from fructose and lactose with Aspergillus oryzae ß-galactosidase. Synthesis was optimized using response surface methodology, considering temperature, initial concentrations of acceptor sugars and fructose/lactose molar ratio as key variables, with the aim of maximizing lactulose yield at the optimal product distribution in terms of prebiotic potential (lactulose/GOS molar ratio of 4). Under optimal conditions (50°C, 50%w/w total initial concentrations of sugars and fructose/lactose molar ratio of 6.44), lactulose yield of 0.26g of lactulose produced per g of initial lactose was obtained at the optimal product distribution.