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.

Amination of naringinase to improve citrus juice debittering using a catalyst immobilized on glyoxyl-agarose.

A naringinase complex was chemically aminated prior to its immobilization on glyoxyl-agarose to develop a robust biocatalyst for juice debittering. The effects of amination on the optimal pH and temperature, thermal stability, and debittering performance were analyzed. Concentration of amino groups on catalysts surface increased in 36 %. Amination reduced the ß-glucosidase activity of naringinase complex; however, did not affect optimal pH and temperature of the enzyme and it favored immobilization, obtaining α-l-rhamnosidase and ß-d-glucosidase activities of 1.7 and 4.2 times the values obtained when the unmodified enzymes were immobilized. Amination favored the stability of the immobilized biocatalyst, retaining 100 % of both activities after 190 h at 30 °C and pH 3, while its non-aminated counterpart retained 80 and 52 % of α-rhamnosidase and ß-glucosidase activities, respectively. The immobilized catalyst showed a better performance in grapefruit juice debittering, obtaining a naringin conversion of 7 times the value obtained with the non-aminated catalyst.

Acidophilic heterotrophs: basic aspects and technological applications.

Acidophiles comprise a group of microorganisms adapted to live in acidic environments. Despite acidophiles are usually associated with an autotrophic metabolism, more than 80 microorganisms capable of utilizing organic matter have been isolated from natural and man-made environments. The ability to reduce soluble and insoluble iron compounds has been described for many of these species and may be harnessed to develop new or improved mining processes when oxidative bioleaching is ineffective. Similarly, as these microorganisms grow in highly acidic media and the chances of contamination are reduced by the low pH, they may be employed to implement robust fermentation processes. By conducting an extensive literature review, this work presents an updated view of basic aspects and technological applications in biomining, bioremediation, fermentation processes aimed at biopolymers production, microbial electrochemical systems, and the potential use of extremozymes.

Use of whey permeate containing in situ synthesised galacto-oligosaccharides for the growth and preservation of Lactobacillus plantarum.

Galacto-oligosaccharides (GOS) are prebiotics that have a beneficial effect on human health by promoting the growth of probiotic bacteria in the gut. GOS are commonly produced from lactose in an enzymatic reaction catalysed by ß-galactosidase, named transglycosylation. Lactose is the main constituent of whey permeate (WP), normally wasted output from the cheese industry. Therefore, the main goal of this work was to optimise the synthesis of GOS in WP using ß-galatosidase from Aspergillus oryzaea. WP and whey permeate enzymatically treated (WP-GOS) were used as culture media of Lactobacillus plantarum 299v. Lb. plantarum 299v attained the stationary phase in approximately 16 h, reaching 3·6 and 4·1×108 CFU/ml in WP and WP-GOS, respectively. The in situ synthesised GOS were not consumed during growth. No significant differences were observed in the growth kinetics of microorganisms in both media. After fermentation, microorganisms were dehydrated by freeze-drying and spray-drying and stored. The recovery of microorganisms after fermentation, dehydration and storage at 4 °C for at least 120 d was above 108 CFU/g. These studies demonstrated that WP is an appropriate substrate for the synthesis of GOS and the obtained product is also adequate as culture medium of Lb. plantarum 299v. The coexistence of GOS and dehydrated viable probiotic microorganisms, prepared using an effluent as raw material, represents the main achievement of this work, with potential impact in the development of functional foods.

[Genetic variants associated to male infertility in Mexican patients]. / Variantes genéticas asociadas con infertilidad masculina en pacientes mexicanos.

Recently Mexican Federation of Obstetrics and Gynecology Colleges (Federación Mexicana de Colegios de Obstetricia y Ginecologia, FEMECOG) published the Mexican guideline forthe management of male infertility, which suggests performing genetic laboratory tests as part of diagnosis and management of infertile patients and states that these should receive genetic counseling. This paper reviews the genetic approach proposed by Mexican guideline. A systematic review of medical literature was performed in Pubmed and Web of Knowledge from 1980 to 2012 in order to find reports of genetic variants associated to male infertility in Mexican patients. Also it is discussed the current knowledge of these variants, their clinical implications and finally the guidelines and recommendations for their molecular diagnosis. Most genetic variants in Mexican infertile patients are chromosome abnormalities. In relation to other variants there is only a report of Y chromosome microdeletions, repeated CAG in androgen receptor and more common mutations in CFTR, and other article reporting mutations in CFTR in patients with congenital absence of vas deferens. Little is known about the genetics of Mexican infertile patients apart from chromosome abnormalities. However, the contribution of genetics as etiology of male infertility is taking more relevance and currently the consensual management of infertile male should include the screening of genetic background. This review pretends to be a quick guide for clinicians who want to know about reports of genetic variants related to male infertility in Mexican population and how to approach their diagnosis.

Ultrasound-assisted enzymatic synthesis of galacto-oligosaccharides using native whey with two commercial ß-galactosidases: Aspergillus oryzae and Kluyveromyces var lactis.

Native whey obtained during casein micelle microfiltration was used as a novel source to produce galacto-oligosaccharides (GOS). Since the presence of macromolecules and other interferers reduces biocatalyst performance, this work evaluated the effect of different ultrasound processing conditions on GOS synthesis using concentrated native whey. Ultrasonic intensities (UI) below 11 W/cm2 tended to increase the activity in the enzyme from Aspergillus oryzae for several minutes but accelerated the inactivation in that from Kluyveromyces lactis. At 40 °C, 40 % w/w native whey, 70 % wave amplitude, and 0.6 s/s duty-cycle, a UI of 30 W/cm2 was achieved, and the increased specific enzyme productivity was similar to the values obtained with pure lactose (∼0.136 g GOS/h/mgE). This strategy allows for obtaining a product containing prebiotics with the healthy and functional properties of whey proteins, avoiding the required purification steps used in the production of food-grade lactose.

Trends in lactose-derived bioactives: synthesis and purification.

Lactose obtained from cheese whey is a low value commodity despite its great potential as raw material for the production of bioactive compounds. Among them, prebiotics stand out as valuable ingredients to be added to food matrices to build up functional foods, which currently represent the most active sector within the food industry. Functional foods market has been growing steadily in the recent decades along with the increasing awareness of the World population about healthy nutrition, and this is having a strong impact on lactose-derived bioactives. Most of them are produced by enzyme biocatalysis because of molecular precision and environmental sustainability considerations. The current status and outlook of the production of lactose-derived bioactive compounds is presented with special emphasis on downstream operations which are critical because of the rather modest lactose conversion and product yields that are attainable. Even though some of these products have already an established market, there are still several challenges referring to the need of developing better catalysts and more cost-effective downstream operations for delivering high quality products at affordable prices. This technological push is expected to broaden the spectrum of lactose-derived bioactive compounds to be produced at industrial scale in the near future.

A pseudo steady-state model for galacto-oligosaccharides synthesis with ß-galactosidase from Aspergillus oryzae.

A pseudo steady-state model for the kinetically controlled synthesis of galacto-oligosaccharides (GOS) with Aspergillus oryzae ß-galactosidase is presented. The model accounts for the dynamics of lactose consumption and production of galactose, glucose, di, tri, tetra, and penta-oligosaccharides during the synthesis, being able to describe the total GOS content in the reaction medium at the experimental conditions evaluated. Experimental results show that the formation of GOS containing only galactose residues is significant at high conversions of substrate, which was taken into account in the model. The formation of enzyme transition complexes was considered and reasonable assumptions were made to reduce the number of parameters to be determined. The model developed has 8 parameters; 2 of them were experimentally determined and the other 6 were estimated by fitting to the experimental data using multiresponse regression. Temperature effect on kinetic and affinity constants was determined in the range from 40 to 55°C, and the data were fitted to Arrhenius type equation. Parameters of the proposed model are independent from the enzyme load in the reaction medium and, differently from previously reported models, they have a clear biochemical meaning. The magnitude of the kinetic and affinity constants of the enzyme suggests that the liberation of galactose from the galactosyl-enzyme complex is a very slow reaction and such complex is driven into GOS formation. It also suggests that the affinity for sugars of the galactosyl-enzyme complex is higher than that of the free enzyme.

Immobilization of Aspergillus oryzae ß-galactosidase in cation functionalized agarose matrix and its application in the synthesis of lactulose.

Aspergillus oryzae ß-galactosidase was immobilized in in-house quaternary ammonium agarose (QAA) and used for the first time in the synthesis of lactulose. A biocatalyst was obtained with a specific activity of 24,690 IUH∙g-1; protein immobilization yield of 97% and enzyme immobilization yield of 76% were obtained at 30 °C in 10 mM phosphate buffer pH 7 for standard size agarose at 100 mgprotein∙gsupport-1 which the maximum protein load of QAA. Highest yield and specific productivity of lactulose were 0.24 g∙g-1 and 9.78 g∙g-1 h-1 respectively, obtained at pH 6, 100 IUH∙g lactose-1 enzyme/lactose ratio and 12 lactose/fructose molar ratio. In repeated-batch operation with the immobilized enzyme, the cumulative mass of lactulose per unit mass of contacted protein and cumulative specific productivity were higher than obtained with the soluble enzyme since the first batch. After enzyme activity exhaustion, the enzyme was desorbed and QAA support was reused without alteration in its maximum enzyme load capacity and without detriment in yield, productivity and selectivity in the batch synthesis of lactulose with the resulting biocatalyst. This significantly decreases the economic impact of the support, presenting itself as a distinctive advantage of immobilization by ionic interaction.

Effect of product partition on the synthesis of butyl-ß-D-galactoside from Aspergillus oryzae.

The effect of donor substrate and products partitioning on the performance of butyl-ß-galactoside synthesis with Aspergillus oryzae ß-galactosidase was studied. Firstly, the partition coefficient of the donor substrate (lactose) and the reaction products (glucose, galactose and butyl-ß-galactoside) were determined in the aqueous and organic phases of the reaction medium. In the temperature range studied (30 to 50 °C), butyl ß-galactoside was roughly 130 and 30-fold more soluble in the organic phase than lactose and the monosaccharides, respectively. Afterward, the effect of the 1-butanol/ aqueous phase ratio (α) on the reaction was evaluated in the range from 0.25 to 4. Results show that higher values of α reduce the incidence of secondary hydrolysis by favoring the extraction of butyl-ß-galactoside into the organic phase where it is not hydrolyzed, leading to higher yields. Also, major interfacial properties for butyl-ß-galactoside were determined at 25 °C.

Enzymatic production of lactulose by fed-batch and repeated fed-batch reactor.

The effects of the most significant operational variables on reactor performance of fed-batch and repeated fed-batch were evaluated in the lactulose production by enzymatic transgalactosylation. Feed flowrate in the fed stage (F) and fructose to lactose molar ratio (Fr/L) were the variables that mostly affected the values ​​of lactulose yield (YLu), lactulose productivity (πLu) and selectivity of transgalactosylation (SLu/TOS). Maximum YLu of 0.21 g lactulose per g lactose was obtained at 50% w/w inlet carbohydrates concentration (IC) of, 50 °C, Fr/L 8, F 1 mL⋅min-1, 200 IU∙gLactose-1 reactor enzyme load and pH 4.5. At these conditions the selectivity was 7.4, productivity was 0.71 gLu∙g-1∙h-1and lactose conversion was 0.66. The operation by repeated fed batch increases the efficiency of use of the biocatalysts (EB) and the accumulated productivity compared to batch and fed batch operation with the same biocatalyst. EB obtained was 4.13 gLu∙mgbiocatalyst protein-1, 10.6 times higher than in fed-batch.

Conventional and non-conventional applications of ß-galactosidases.

ß-Galactosidase is one of the most important industrial enzymes, that has been used for many decades in the dairy industry. The main application of ß-galactosidase is related to the production of low-lactose and lactose-free milk and dairy products, which are now common consumer goods in supermarket shelves. This is a well-established market that is expected to keep on growing as these products become more accessible to mid-income people worldwide. However, a fresh air has come into the ß-galactosidase business as non-conventional applications arose in recent decades based on its transgalactosylation activity. This capacity is certainly a major asset for a commodity enzyme that can be used now as a catalyst for the upgrading of readily available and cheap lactose into high added-value glycosides in processes of organic synthesis in tune with green chemistry principles within the framework of sustainability. This is a reflection of a paradigm shift, where enzymes are now being considered as apt catalysts for the synthesis of valuable organic compounds. This article reviews the main applications of ß-galactosidase, going from its conventional use related to its hydrolytic activity to the ongoing non-conventional applications in the synthesis of high added-value oligosaccharides based on its transgalactosylation activity.

Improvements in the production of Aspergillus oryzae ß-galactosidase crosslinked aggregates and their use in repeated-batch synthesis of lactulose.

Aspergillus oryzae ß-galactosidase was immobilized by aggregation and crosslinking, obtaining catalysts (CLAGs) well-endowed for lactulose synthesis. Type and concentration of the precipitating agent were determinants of immobilization yield, specific activity and thermal stability. CLAGs with specific activities of 64,007, 48,374 and 44,560 IUH g-1 were obtained using 50% v/v methanol, ethanol and propanol as precipitating agents respectively, with immobilization yields over 90%. Lactulose synthesis was conducted at 50 °C, pH 4.5, 50% w/w total sugars, 200 IUH g-1 of enzyme and fructose/lactose molar ratio of 8 in batch and repeated-batch operation. Lactulose yields were 0.19 g g-1 and 0.24 g g-1 for fructose to lactose molar ratios of 4 mol mol-1 and 8 mol mol-1 while selectivities were 3.3 mol mol-1 and 6.6 mol mol-1 respectively for CLAGs obtained by ethanol and propanol precipitation. Based on these results, both CLAGs were selected for the synthesis in repeated-batch mode. The cumulative mass of lactulose in repeated-batch was higher with CLAGs produced by ethanol and propanol precipitation than with the free enzyme. 86 and 93 repeated-batches could have been respectively performed with those CLAGs considering a catalyst replacement criterion of 50% of residual activity, as determined by simulation.

Metabolic Costs of a 58-minute Multi-Intensity Exercise Session with and Without Music and Cueing.

It is unclear if the presence or absence of music and cueing influence total energy expenditure (TEE) during a multi-intensity exercise program. The purpose of this study was to determine the difference between TEE with or without music and cueing during a 58-minute exercise session using heart rate estimation (HRe) and indirect calorimetry (IC). Using a randomized crossover design, 22 participants (6 males; 16 females; 27.64 ± 10.33 yrs.) were randomized into two groups (Group A = 11; Group B = 11). All participants performed the same 58-minute exercise session under two conditions: with music (WM) and without music and cueing (WOM). TEE was obtained through the Activio heart rate system for all 22 participants. TEE and excess post-exercise oxygen consumption (EPOC) were also obtained in a subset of eight participants (4 males; 4 females; 28.25 ± 5.9 yrs.) via IC through a ParvoMedics metabolic cart. Paired samples t-tests were performed to compare TEE between conditions using HRe and IC. Statistical analysis was performed using IBM Analytics, SPSS v24 with significance set at p < 0.05. A significant difference (p = 0.008) was found between TEE WM and WOM using IC (475.74 ± 98.50 vs. 429.37 ± 121.42), but not between TEE WM and WOM using HRe (p = 2.04; 482.67 ± 151.79 vs. 452.90 ± 164.59). The presence of music and cueing increased TEE when monitored via IC, but not when measured via wearable heart rate technology. Music and cueing does aid in additional caloric expenditure.

Synthesis of Lactulose in Continuous Stirred Tank Reactor With ß-Galactosidase of Apergillus oryzae Immobilized in Monofunctional Glyoxyl Agarose Support.

Lactulose synthesis from fructose and lactose in continuous stirred tank (CSTR) reactor operation with glyoxyl-agarose immobilized Aspergillus oryzae ß-galactosidase is reported for the first time. The effect of operational variables: inlet concentrations of sugar substrates, temperature, feed substrate molar ratio, enzyme loading and feed flow rate was studied on reactor performance. Even though the variation of each one affected to a certain extent lactulose yield (Y Lactulose ), specific productivity (π Lactulose ) and selectivity of the reaction (lactulose/transgalactosylated oligosaccharides molar ratio) (S Lu/TOS ), the most significant effects were obtained by varying the inlet concentrations of sugar substrates and the feed substrate molar ratio. Maximum Y Lactulose of 0.54 g⋅g-1 was obtained at 50°C, pH 4.5, 50% w/w inlet concentrations of sugar substrates, feed flowrate of 12 mL⋅min-1, fructose/lactose molar ratio of 8 and reactor enzyme load of 29.06 IU H ⋅mL-1. At such conditions S Lu/TOS was 3.7, lactose conversion (X Lactose ) was 0.39 and total transgalactosylation yield was 0.762 g⋅g-1, meaning that 76% of the reacted lactose corresponded to transgalactosylation and 24% to hydrolysis, which is a definite advantage of this mode of operation. Even though X Lactose in CSTR was lower than in other reported modes of operation for lactulose synthesis, transgalactosylation was more favored over hydrolysis which reduced the inhibitory effect of galactose on ß-galactosidase.

Synthesis of Butyl-ß-D-Galactoside in the Ternary System: Acetone/1-Butanol/Aqueous Solution.

The enzymatic synthesis of short-tailed alkyl glucosides is generally carried out in an aqueous-organic biphasic reaction medium with a rather low fatty alcohol concentration in the aqueous phase (where the synthesis occurs). Thus, hydrolytic reactions have a significant impact on the synthesis performance. Given this background, the use of acetone as cosolvent was studied for the synthesis of butyl-ß-galactoside with Aspergillus oryzae ß-galactosidase. The liquid-liquid equilibrium of the reaction mixture components (acetone/1-butanol/aqueous solution) was determined and the single- and two-phase regions were defined at 30, 40, and 50°C. It was observed that the liquid-liquid equilibrium of the ternary system acetone/1-butanol/water differs significantly from the one obtained using an aqueous solution (50 mM McIlvaine buffer pH 4.5; 5 g L-1) instead of water. This is mainly because of the salting-out effect of the buffer; nevertheless, the presence of lactose also altered the equilibrium. Having this in mind, the effects of temperature (30 and 50°C) and reaction mixture composition were assessed. Three general conditions were evaluated: single-phase ternary system (30% acetone), two-phase ternary system (10% acetone) and two-phase binary system (0% acetone). Acetone had a deleterious effect on enzyme stability at 50°C, leading to low reaction yields. However, no enzyme deactivation was detected at 30°C. Moreover, a reaction yield of 0.98 mol mol-1 was attained in the 30/50/20% (w/w) mixture of acetone/1-butanol/aqueous solution. This very high yield can be explained by the huge increase in the concentration of 1-butanol and the reduction of water activity. The synthesis was carried out using also the ß-galactosidase immobilized in glyoxal-agarose and amino-glyoxal-agarose, and by aggregation and crosslinking. In the case of agarose-derived catalysts, two average particle diameters were assessed to evaluate the presence of internal mass transfer limitations. Best yield (0.88 mol mol-1) was obtained with glyoxal-agarose derivatives and the particle size had non-effect on yield. The chemical structure of butyl-ß-galactoside was determined by NMR and FT-IR.

Continuous enzymatic synthesis of lactulose in packed-bed reactor with immobilized Aspergillus oryzae ß-galactosidase.

Lactulose synthesis from fructose and lactose in continuous packed-bed reactor operation with glyoxyl-agarose immobilized Aspergillus oryzae ß-galactosidase is reported for the first time. Alternative strategies to conventional batch synthesis have been scarcely explored for lactulose synthesis. The effect of flow rate, substrates ratio and biocatalyst-inert packing material mass ratio (MB/MIM) were studied on reactor performance. Increase in any of these variables produced an increase in lactulose yield (YLu) being higher than obtained in batch synthesis at comparable conditions. Maximum YLu of 0.6 g·g-1 was obtained at 50 °C, pH 4.5, 50% w/w total sugars, 15 mL·min-1, fructose/lactose molar ratio of 12 and MB/MIM of 1/8 g·g-1; at such conditions yield of transgalactosylated oligosaccharides (YTOS) was 0.16 g·g-1, selectivity (lactulose/TOS molar ratio) was 5.4 and lactose conversion (XLactose) was 28%. Reactor operation with recycle had no significant effect on yield, producing only some decrease in productivity.

ß-Galactosidase from Exiguobacterium acetylicum: Cloning, expression, purification and characterization.

The main goal of this work was to evaluate the performance of ß-galactosidase from Exiguobacterium acetylicum MF03 in both hydrolysis and transgalactosylation reactions from different substrates. The enzyme gene was expressed in Escherichia coli BL21 (DE3), sequenced, and subjected to bioinformatic and kinetic assessment. Results showed that the enzyme was able to hydrolyze lactulose and o-nitrophenyl-ß-d-galactopyranoside, but unable to hydrolyze lactose, o-nitrophenyl-ß-d-glucopyranoside, butyl- and pentyl-ß-d-galactosides. This unique and novel substrate specificity converts the E. acetylicum MF03 ß-galactosidase into an ideal catalyst for the formulation of an enzymatic kit for lactulose quantification in thermally processed milk. This is because costly steps to eliminate glucose (resulting from hydrolysis of lactose when a customary ß-galactosidase is used) can be avoided.

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.

Fed-batch operation for the synthesis of lactulose with ß-galactosidase of Aspergillus oryzae.

Fed-batch synthesis of lactulose from lactose and fructose with Aspergillus oryzae ß-galactosidase was evaluated, obtaining a concentration of 40.4g·L-1, which is 20% higher than obtained in batch, while the concentration of transgalactosylated oligosaccharides (TOS) was reduced by 98%. Therefore, selectivity of lactulose synthesis can be significantly higher by operating in fed-batch mode. The enzyme-limiting substrate mass ratio (E/S) is a critical variable in fed-batch operation. Higher values favor lactose hydrolysis over transgalactosylation, being 400IU/g the limit for proper lactulose synthesis in fed-batch operation. Selectivity of lactulose synthesis increased with E/S being quite high at 800IUH·g-1 or higher. However, this increase was obtained at the expense of lactulose yield. Lactulose synthesis in fed-batch operation was a better option than conventional batch synthesis, since higher product concentration and selectivity of lactulose over TOS synthesis were obtained.