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.

Kinetics and products of Thermotoga maritima ß-glucosidase with lactose and cellobiose.

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother's milk. They are synthesized by ß-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many ß-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable ß-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the ß-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly ß(1 → 3) and ß(1 → 6) elongating activity, but also some ß(1 → 4) elongation was observed. The ß-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose. KEY POINTS: • ß-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield. • Thermotoga maritima ß-glucosidase has high activity and high thermostability. • Thermotoga maritima ß-glucosidase GOS contains mainly (ß1-3) and (ß1-6) linkages.

A new ß-galactosidase from Paenibacillus wynnii with potential for industrial applications.

Commercial ß-galactosidases exhibit undesirable kinetic properties regarding substrate affinity (Michaelis-Menten constant [KM] for lactose) and product inhibition (inhibitor constant [Ki] for galactose). An in silico screening of gene sequences was done and identified a putative ß-galactosidase (Paenibacillus wynnii ß-galactosidase, BgaPw) from the psychrophilic bacterium Paenibacillus wynnii. The cultivation of the wild-type P. wynnii strain resulted in very low ß-galactosidase activities of a maximum of 150 nkat per liter of medium with o-nitrophenyl-ß-d-galactopyranoside (oNPGal) as substrate. The recombinant production of BgaPw in Escherichia coli BL21(DE3) increased the yield ∼9,000-fold. Here, a volumetric activity of 1,350.18 ± 11.82 µkatoNPGal/Lculture was achieved in a bioreactor cultivation. The partly purified BgaPw showed a pH optimum at 7.0, a temperature maximum at 40°C, and an excellent stability at 8°C with a half-life of 77 d. Kinetic studies with BgaPw were done in milk or in milk-imitating synthetic buffer (Novo buffer), respectively. Remarkably, the KM value of BgaPw with lactose was as low as 0.63 ± 0.045 mM in milk. It was found that the resulting products of lactose hydrolysis, namely galactose and glucose, did not inhibit the ß-galactosidase activity of BgaPw, but instead showed a striking activating effect in both cases (up to 144%). In a comparison study in milk, lactose was completely hydrolyzed by BgaPw in 72 h at 8°C, whereas 2 other known ß-galactosidases were less powerful and converted only about 90% of lactose in the same time. Finally, the formation of galactooligosaccharides (GOS) was demonstrated with the new BgaPw, starting with pharma-lactose (400 g/L). A GOS production of about 144 g/L was achieved after 24 h (36.0% yield).

Biochemical characterization of a novel C-terminally truncated ß-galactosidase from Paenibacillus antarcticus with high transglycosylation activity.

The transgalactosylase activity of ß-galactosidases offers a convenient and promising strategy for conversion of lactose into high-value oligosaccharides, such as galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMOs). In this study, we cloned and biochemically characterized a novel C-terminally truncated ß-galactosidase (PaBgal2A-D) from Paenibacillus antarcticus with high transglycosylation activity. PaBgal2A-D is a member of glycoside hydrolase (GH) family 2. The optimal pH and temperature of PaBgal2A-D were determined to be pH 6.5 and 50°C, respectively. It was relatively stable within pH 5.0-8.0 and up to 50°C. PaBgal2A-D showed high transglycosylation activity for GOS synthesis, and the maximum yield of 50.8% (wt/wt) was obtained in 2 h. Moreover, PaBgal2A-D could synthesize lacto-N-neotetraose (LNnT) using lactose and lacto-N-triose II (LNT2), with a conversion rate of 16.4%. This study demonstrated that PaBgal2A-D could be a promising tool to prepare GOS and LNnT.

Transcriptomic and metabolomic analysis of prebiotics utilization by Bifidobacterium animalis.

In this study, the utilization mechanism of oligosaccharides by Bifidobacterium was investigated through the transcriptome sequencing and non-targeted metabolomics technology of Bifidobacterium animalis cultured with fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS). The results showed that FOS affected the synthesis of adenosine triphosphate binding transporters (ABC transporters) by increasing the expression levels of msmE, msmG, and gluA. Similarly, GOS improved aminoacyl-tRNA synthases by upregulating the expression of tRNA-Ala, tRNA-Pro, and tRNA-Met. Bifidobacterium animalis cultured with FOS and GOS produced different metabolites, such as histamine, tartaric acid, and norepinephrine, with the functions of inhibiting inflammation, alleviating depression and diseases related to brain and nervous system and maintaining body health. Furthermore, the transcriptome and metabolome analysis results revealed that FOS and GOS promoted the growth and metabolism of Bifidobacterium animalis by regulating the related pathways of carbohydrate, energy, and amino acid metabolism. Overall, the experimental results provided significant insights into the prebiotic effects of FOS and GOS.

Prebiotic Consumption Alters Microbiota but Not Biological Markers of Stress and Inflammation or Mental Health Symptoms in Healthy Adults: A Randomized, Controlled, Crossover Trial.

BACKGROUND: Chronic stress contributes to systemic inflammation and diminished mental health. Although animal work suggests strong links with the microbiota-gut-brain axis, clinical trials investigating the effectiveness of prebiotics in improving mental health and reducing inflammation are lacking. OBJECTIVES: We aimed to determine fructooligosaccharide (FOS) and galactooligosaccharide (GOS) effects on biological markers of stress and inflammation and mental health symptoms in adults. Secondary outcomes included fecal microbiota and metabolites, digestive function, emotion, and sleep. METHODS: Twenty-four healthy adults (25-45 y; 14 females, 10 males; BMI, 29.3 ± 1.8 kg/m2) from central Illinois participated in a 2-period, randomized, controlled, single-blinded crossover trial. Interventions included the prebiotic (PRE) treatment (237 mL/d Lactaid low-fat 1% milk, 5 g/d FOS, 5 g/d GOS) and control (CON) (237 mL/d Lactaid), which were consumed in counterbalanced order for 4 wk each, separated by ≥4-wk washout. Inflammatory markers were measured in blood plasma (>10-h fast) and cortisol in urine. The Depression Anxiety Stress Scales-42 assessed mental health symptoms. Fecal samples were collected for 16S rRNA gene (V4 region) sequencing and analysis. Emotion was measured by rating images from a computer task. Sleep was assessed using 7-d records and accelerometers. Change scores were analyzed using linear mixed models with treatment and baseline covariate as fixed effects and participant ID as the random effect. RESULTS: There were no differences in change scores between PRE and CON treatments on biological markers of stress and inflammation or mental health. PRE increased change in percent sequences (q = 0.01) of Actinobacteriota (CON: 0.46 ± 0.70%; PRE: 5.40 ± 1.67%) and Bifidobacterium (CON: -1.72 ± 0.43%; PRE: 4.92 ± 1.53%). There were also no differences in change scores between treatments for microbial metabolites, digestive function, emotion, or sleep quality. CONCLUSIONS: FOS+GOS did not affect biological markers of stress and inflammation or mental health symptoms in healthy adults; however, it increased Bifidobacterium. CLINICAL TRIAL REGISTRY: NCT04551937, www. CLINICALTRIALS: gov.

Application of oligosaccharides in meat processing and preservation.

In recent decades, consumer preference and attention to foodstuff presented as healthy and with desirable nutritional information, has increased significantly. In this field, the meat industry has a challenging task since meat and meat products have been related to various chronic diseases. Functional ingredients have emerged in response to the increasing demand for healthier and more nutritious foods. On this matter, oligosaccharides such as fructooligosaccharides (FOS), xylooligosaccharides (XOS), galactooligosaccharides (GOS), and chitooligosaccharides (COS) have been presented as suitable ingredients for the meat industry with the aim of obtaining healthier meat derivatives (e.g. with low fat or sugar content, reduced amount of additives, and desirable functional properties, etc.). However, studies considering application of such oligomers in the meat sector are scarce. In addition, a large number of issues remain to be solved related both to obtaining and characterizing the oligosaccharides available in the industry and to the effect that these ingredients have on the features of meat products (mainly physicochemical and sensory). The study of new oligosaccharides, the methodologies for obtaining them, and their application to new meat products should be promoted, as well as improving knowledge about their effects on the properties of functional meat foods.

ß-Galactosidase: a traditional enzyme given multiple roles through protein engineering.

ß-Galactosidases are crucial carbohydrate-active enzymes that naturally catalyze the hydrolysis of galactoside bonds in oligo- and disaccharides. These enzymes are commonly used to degrade lactose and produce low-lactose and lactose-free dairy products that are beneficial for lactose-intolerant people. ß-galactosidases exhibit transgalactosylation activity, and they have been employed in the synthesis of galactose-containing compounds such as galactooligosaccharides. However, most ß-galactosidases have intrinsic limitations, such as low transglycosylation efficiency, significant product inhibition effects, weak thermal stability, and a narrow substrate spectrum, which greatly hinder their applications. Enzyme engineering offers a solution for optimizing their catalytic performance. The study of the enzyme's structure paves the way toward explaining catalytic mechanisms and increasing the efficiency of enzyme engineering. In this review, the structure features of ß-galactosidases from different glycosyl hydrolase families and the catalytic mechanisms are summarized in detail to offer guidance for protein engineering. The properties and applications of ß-galactosidases are discussed. Additionally, the latest progress in ß-galactosidase engineering and the strategies employed are highlighted. Based on the combined analysis of structure information and catalytic mechanisms, the ultimate goal of this review is to furnish a thorough direction for ß-galactosidases engineering and promote their application in the food and dairy industries.

Prebiotics modulate the microbiota-gut-brain axis and ameliorate cognitive impairment in APP/PS1 mice.

PURPOSE: Prebiotics, including fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS), stimulate beneficial gut bacteria and may be helpful for patients with Alzheimer's disease (AD). This study aimed to compare the effects of FOS and GOS, alone or in combination, on AD mice and to identify their underlying mechanisms. METHODS: Six-month-old APP/PS1 mice and wild-type mice were orally administered FOS, GOS, FOS + GOS or water by gavage for 6 weeks and then subjected to relative assays, including behavioral tests, biochemical assays and 16S rRNA sequencing. RESULTS: Through behavioral tests, we found that GOS had the best effect on reversing cognitive impairment in APP/PS1 mice, followed by FOS + GOS, while FOS had no effect. Through biochemical techniques, we found that GOS and FOS + GOS had effects on multiple targets, including diminishing Aß burden and proinflammatory IL-1ß and IL-6 levels, and changing the concentrations of neurotransmitters GABA and 5-HT in the brain. In contrast, FOS had only a slight anti-inflammatory effect. Moreover, through 16S rRNA sequencing, we found that prebiotics changed composition of gut microbiota. Notably, GOS increased relative abundance of Lactobacillus, FOS increased that of Bifidobacterium, and FOS + GOS increased that of both. Furthermore, prebiotics downregulated the expression levels of proteins of the TLR4-Myd88-NF-κB pathway in the colons and cortexes, suggesting the involvement of gut-brain mechanism in alleviating neuroinflammation. CONCLUSION: Among the three prebiotics, GOS was the optimal one to alleviate cognitive impairment in APP/PS1 mice and the mechanism was attributed to its multi-target role in alleviating Aß pathology and neuroinflammation, changing neurotransmitter concentrations, and modulating gut microbiota.

Invited review: Properties of ß-galactosidases derived from Lactobacillaceae species and their capacity for galacto-oligosaccharide production.

ß-galactosidase (enzymatic class 3.2.1.23) is one of the dairy industry's most important and widely used enzymes. The enzyme is part of a large family known to catalyze hydrolysis and transglycosylation reactions. Its hydrolytic activity is commonly used to decrease lactose content in dairy products, while its transglycosylase activity has recently been used to synthesize galacto-oligosaccharides (GOS). During the past couple of years, researchers have focused on studying ß-galactosidase isolated and purified from lactic acid bacteria. This review will focus on ß-galactosidase purified and characterized from what used to be the Lactobacillus genera. Furthermore, particular emphasis is given to its kinetics, biochemical characteristics, GOS production, market, and utilization by Lactobacilllaceae species.

Biofilm-Based Biocatalysis for Galactooligosaccharides Production by the Surface Display of ß-Galactosidase in Pichia pastoris.

Galactooligosaccharides (GOS) are one of the most important functional oligosaccharide prebiotics. The surface display of enzymes was considered one of the most excellent strategies to obtain these products. However, a rough industrial environment would affect the biocatalytic process. The catalytic process could be efficiently improved using biofilm-based fermentation with high resistance and activity. Therefore, the combination of the surface display of ß-galactosidase and biofilm formation in Pichia pastoris was constructed. The results showed that the catalytic conversion rate of GOS was up to 50.3% with the maximum enzyme activity of 5125 U/g by screening the anchorin, and the number of the continuous catalysis batches was up to 23 times. Thus, surface display based on biofilm-immobilized fermentation integrated catalysis and growth was a co-culture system, such that a dynamic equilibrium in the consolidated integrative process was achieved. This study provides the basis for developing biofilm-based surface display methods in P. pastoris during biochemical production processes.

Galactooligosaccharide Mediates NF-κB Pathway to Improve Intestinal Barrier Function and Intestinal Microbiota.

The use of antibiotics to treat diarrhea and other diseases early in life can lead to intestinal disorders in infants, which can cause a range of immune-related diseases. Intestinal microbiota diversity is closely related to dietary intake, with many oligosaccharides impacting intestinal microorganism structures and communities. Thus, oligosaccharide type and quantity are important for intestinal microbiota construction. Galactooligosaccharides (GOS) are functional oligosaccharides that can be supplemented with infant formula. Currently, information on GOS and its impact on intestinal microbiota diversity and disorders is lacking. Similarly, GOS is rarely reported within the context of intestinal barrier function. In this study, 16S rRNA sequencing, gas chromatography, and immunohistochemistry were used to investigate the effects of GOS on the intestinal microbiota and barrier pathways in antibiotic-treated mouse models. The results found that GOS promoted Bifidobacterium and Akkermansia proliferation, increased short-chain fatty acid levels, increased tight junction protein expression (occludin and ZO-1), increased secretory immunoglobulin A (SIgA) and albumin levels, significantly downregulated NF-κB expression, and reduced lipopolysaccharide (LPS), interleukin-IL-1ß (IL-1ß), and IL-6 levels. Also, a high GOS dose in ampicillin-supplemented animals provided resistance to intestinal damage.

Design of a Biocatalytic Flow Reactor Based on Hierarchically Structured Monolithic Silica for Producing Galactooligosaccharides (GOSs).

Climate change mitigation requires the development of greener chemical processes. In this context, biocatalysis is a pivotal key enabling technology. The advantages of biocatalysis include lower energy consumption levels, reduced hazardous waste production and safer processes. The possibility to carry out biocatalytic reactions under flow conditions provides the additional advantage to retain the biocatalyst and to reduce costly downstream processes. Herein, we report a method to produce galactooligosaccharides (GOSs) from a largely available feedstock (i.e. lactose from dairy production) using a flow reactor based on hierarchically structured monolithic silica. This reactor allows for fast and efficient biotransformation reaction in flow conditions.

Differential Effects of Early-Life and Postweaning Galacto-oligosaccharide Intervention on Colonic Bacterial Composition and Function in Weaning Piglets.

Recently, we proved that the early-life galacto-oligosaccharides (GOS) intervention could improve the colonic function by altering the bacterial composition in suckling piglets. However, whether the early-life GOS (ELG) intervention could have a long influence on the colonic microbiota and whether the combined ELG and postweaning GOS (PWG) intervention would have an interacting effect on maintaining colonic health in weaning piglets remain to be explored. In this study, we illustrated the differential effects of the ELG and PWG interventions on colonic microbiota and colonic function of weaning piglets. Our results showed that the ELG and PWG interventions decreased the frequency of diarrhea in weaning piglets while the PWG intervention increased colonic indexes. After 16S rRNA gene MiSeq sequencing of the gut bacteria belonging to different colonic niches (mucosa and digesta), the increase in the α-diversity of the colonic mucosal bacteria during PWG intervention was revealed. In addition, we found that both the ELG and PWG interventions enriched the relative abundances of short-chain fatty acid (SCFA) producers in different colonic niches and increased the total SCFA concentration in colonic digesta. These changes selectively modulated the mRNA expression levels of pattern recognition receptors and barrier proteins in the colonic mucosa. Of note, the combined effect of ELG and PWG effectively enhanced colonic SCFA producer enrichment and upregulated the butyrate concentration. Meanwhile, the expression levels of MyD88-NF-κB signaling and the proinflammatory cytokines were markedly reduced under the combined effect of ELG and PWG. IMPORTANCE Reducing the disorders of the gut ecosystem is an effective way to relieve weaning stresses of piglets and minimize economic losses in the modern swine industry. To this end, prebiotics have been often added to their diet during the weaning transition. In the present study, we demonstrated that the ELG and PWG interventions showed different effects on the bacterial composition of different colonic niches and on colonic function in the weaning piglets. Especially under the combined effect of ELG and PWG intervention, the expression levels of MyD88-NF-κB and the proinflammatory cytokines decreased with increasing concentrations of butyrate, which is an important microbial metabolite involved in the colon of weaning piglets. These findings further provided new insights into nutritional interventions that alleviate intestinal ecosystem dysbiosis and gut dysfunction in the piglets during the weaning transition.

Prebiotic Galacto-Oligosaccharides and Fructo-Oligosaccharides, but Not Acacia Gum, Increase Iron Absorption from a Single High-Dose Ferrous Fumarate Supplement in Iron-Depleted Women.

BACKGROUND: Prebiotic galacto-oligosaccharides (GOS) increase iron absorption from fortification-level iron doses given as ferrous fumarate (FeFum) in women and children. Whether GOS or other fibers, such as prebiotic fructo-oligosaccharides (FOS) and acacia gum, increase iron absorption from higher supplemental doses of FeFum is unclear. OBJECTIVES: In iron-depleted [serum ferritin (SF) <25 µg/L] women, we tested if oral coadministration of 15 g GOS, FOS, or acacia gum increased iron absorption from a 100 mg Fe supplement given as FeFum. METHODS: In a randomized, single-blind, crossover study, 30 women (median age: 26.2 y; median SF: 12.9 µg/L) consumed a 100 mg Fe tablet labeled with 4 mg 57Fe or 58Fe, given with either 1) 15 g GOS; 2) 15 g FOS; 3) 15 g acacia gum; or 4) 6.1 g lactose and 1.5 g sucrose (control; matching the amounts of sucrose and lactose present in the GOS powder providing 15 g GOS), dissolved in water. The primary outcome, fractional iron absorption (FIA), was assessed by erythrocyte isotopic incorporation 14 d after administration. Data were analyzed using a linear mixed-effect model. We also tested, in vitro, iron solubility at different pH and dialyzability from the different supplement combinations administered in vivo. RESULTS: FIA from FeFum given with GOS and FOS was significantly higher (+45% and +51%, respectively; P < 0.001 for both) than control; median [IQR] total iron absorption was 34.6 mg [28.4-49.1 mg], 36.1 mg [29.0-46.2 mg], and 23.9 mg [20.5-34.0 mg], respectively. Acacia gum did not significantly affect FIA from FeFum (P = 0.688). In vitro, iron dialyzability of FeFum + GOS was 46% higher than that of FeFum alone (P = 0.003). CONCLUSIONS: In iron-depleted women, both GOS and FOS coadministration with FeFum increased iron absorption by ∼50% from a 100 mg oral iron dose, resulting in an additional 10-12 mg of absorbed iron. Thus, GOS and FOS may be promising new enhancers of supplemental iron absorption. This trial was registered at clinicaltrials.gov as NCT04194255.

Prebiotic Galacto-Oligosaccharides and Fructo-Oligosaccharides, but Not Acacia Gum, Increase Iron Absorption from a Single High-Dose Ferrous Fumarate Supplement in Iron-Depleted Women.

BACKGROUND: Prebiotic galacto-oligosaccharides (GOS) increase iron absorption from fortification-level iron doses given as ferrous fumarate (FeFum) in women and children. Whether GOS or other fibers, such as prebiotic fructo-oligosaccharides (FOS) and acacia gum, increase iron absorption from higher supplemental doses of FeFum is unclear. OBJECTIVES: In iron-depleted [serum ferritin (SF) <25 µg/L] women, we tested if oral coadministration of 15 g GOS, FOS, or acacia gum increased iron absorption from a 100 mg Fe supplement given as FeFum. METHODS: In a randomized, single-blind, crossover study, 30 women (median age: 26.2 y; median SF: 12.9 µg/L) consumed a 100 mg Fe tablet labeled with 4 mg 57Fe or 58Fe, given with either 1) 15 g GOS; 2) 15 g FOS; 3) 15 g acacia gum; or 4) 6.1 g lactose and 1.5 g sucrose (control; matching the amounts of sucrose and lactose present in the GOS powder providing 15 g GOS), dissolved in water. The primary outcome, fractional iron absorption (FIA), was assessed by erythrocyte isotopic incorporation 14 d after administration. Data were analyzed using a linear mixed-effect model. We also tested, in vitro, iron solubility at different pH and dialyzability from the different supplement combinations administered in vivo. RESULTS: FIA from FeFum given with GOS and FOS was significantly higher (+45% and +51%, respectively; P < 0.001 for both) than control; median [IQR] total iron absorption was 34.6 mg [28.4-49.1 mg], 36.1 mg [29.0-46.2 mg], and 23.9 mg [20.5-34.0 mg], respectively. Acacia gum did not significantly affect FIA from FeFum (P = 0.688). In vitro, iron dialyzability of FeFum + GOS was 46% higher than that of FeFum alone (P = 0.003). CONCLUSIONS: In iron-depleted women, both GOS and FOS coadministration with FeFum increased iron absorption by ∼50% from a 100 mg oral iron dose, resulting in an additional 10-12 mg of absorbed iron. Thus, GOS and FOS may be promising new enhancers of supplemental iron absorption.This trial was registered at clinicaltrials.gov as NCT04194255.

An acid-tolerant and cold-active ß-galactosidase potentially suitable to process milk and whey samples.

A novel ß-galactosidase gene (galM) was cloned from an aquatic habitat metagenome. The analysis of its translated sequence (GalM) revealed its phylogenetic closeness towards Verrucomicrobia sp. The sequence comparison and homology structure analysis designated it a member of GH42 family. The three-dimensional homology model of GalM depicted a typical (ß/α)8 TIM-barrel containing the catalytic core. The gene (galM) was expressed in a heterologous host, Escherichia coli, and the purified protein (GalM) was subjected to biochemical characterization. It displayed ß-galactosidase activity in a wide range of pH (2.0 to 9.0) and temperature (4 to 60 °C). The heat exposed protein showed considerable stability at 40 and 50 °C, with the half-life of about 100 h and 35 h, respectively. The presence of Na, Mg, K, Ca, and Mn metals was favorable to the catalytic efficiency of GalM, which is a desirable catalytic feature, as these metals exist in milk. It showed remarkable tolerance of glucose and galactose in the reaction. Furthermore, GalM discerned transglycosylation activity that is useful in galacto-oligosaccharides' production. These biochemical properties specify the suitability of this biocatalyst for milk and whey processing applications. KEY POINTS: • A novel ß-galactosidase gene was identified and characterized from an aquatic habitat. • It was active in extreme acidic to mild alkaline pH and at cold to moderate temperatures. • The ß-galactosidase was capable to hydrolyze lactose in milk and whey.

An Updated Review on Prebiotics: Insights on Potentials of Food Seeds Waste as Source of Potential Prebiotics.

Prebiotics are a group of biological nutrients that are capable of being degraded by microflora in the gastrointestinal tract (GIT), primarily Lactobacilli and Bifidobacteria. When prebiotics are ingested, either as a food additive or as a supplement, the colonic microflora degrade them, producing short-chain fatty acids (SCFA), which are simultaneously released in the colon and absorbed into the blood circulatory system. The two major groups of prebiotics that have been extensively studied in relation to human health are fructo-oligosaccharides (FOS) and galactooligosaccharides (GOS). The candidature of a compound to be regarded as a prebiotic is a function of how much of dietary fiber it contains. The seeds of fruits such as date palms have been reported to contain dietary fiber. An increasing awareness of the consumption of fruits and seeds as part of the daily diet, as well as poor storage systems for seeds, have generated an enormous amount of seed waste, which is traditionally discarded in landfills or incinerated. This cultural practice is hazardous to the environment because seed waste is rich in organic compounds that can produce hazardous gases. Therefore, this review discusses the potential use of seed wastes in prebiotic production, consequently reducing the environmental hazards posed by these wastes.

Production and Digestibility Studies of ß-Galactosyl Xylitol Derivatives Using Heterogeneous Catalysts of LacA ß-Galactosidase from Lactobacillus Plantarum WCFS1.

The synthesis of ß-galactosyl xylitol derivatives using immobilized LacA ß-galactosidase from Lactobacillus plantarum WCFS1 is presented. These compounds have the potential to replace traditional sugars by their properties as sweetener and taking the advantages of a low digestibility. The enzyme was immobilized on different supports, obtaining immobilized preparations with different activity and stability. The immobilization on agarose-IDA-Zn-CHO in the presence of galactose allowed for the conserving of 78% of the offered activity. This preparation was 3.8 times more stable than soluble. Since the enzyme has polyhistidine tags, this support allowed the immobilization, purification and stabilization in one step. The immobilized preparation was used in synthesis obtaining two main products and a total of around 68 g/L of ß-galactosyl xylitol derivatives and improving the synthesis/hydrolysis ratio by around 30% compared to that of the soluble enzyme. The catalyst was recycled 10 times, preserving an activity higher than 50%. The in vitro intestinal digestibility of the main ß-galactosyl xylitol derivatives was lower than that of lactose, being around 6 and 15% for the galacto-xylitol derivatives compared to 55% of lactose after 120 min of digestion. The optimal amount immobilized constitutes a very useful tool to synthetize ß-galactosyl xylitol derivatives since it can be used as a catalyst with high yield and being recycled for at least 10 more cycles.

Prebiotic effect of galacto-oligosaccharides on the skin microbiota and determination of their diffusion properties.

OBJECTIVE: Recently, prebiotics are attracting plenty of attention in the field of skin care, since it is found that they are able to support the balance of beneficial and harmful microorganisms on the skin, and accordingly prevent several skin conditions associated with microbial imbalance. Topical application of prebiotics, although insufficiently investigated, holds great promise in improving skin health. The purpose of this research was to determine the prebiotic potential of galacto-oligosaccharides (GOS) for skin microbiota and suitability for incorporation in different topical formulations, and finally, provide insights into their diffusion properties. METHODS: The prebiotic effect of GOS was evaluated through the influence on the growth of Staphylococcus epidermidis and Staphylococcus aureus, the most common resident and pathogenic bacterium of the skin microbiota, respectively. Also, with the future use of GOS in cosmetic products in mind, the diffusion of GOS molecules from two different topical formulations, hydrogel and oil-in-water (O/W) gel emulsion, was monitored employing Franz diffusion cell and two systems-with cellulose acetate membrane and transdermal diffusion test model, Strat-M® membrane. Course of fermentation and the amount of diffused GOS molecules were monitored using high-performance liquid chromatography (HPLC). RESULTS: The in vitro results revealed that GOS at a concentration of 5% (w/v) has a pronounced stimulatory effect on S. epidermidis, while simultaneously showing an inhibitory effect on S. aureus, both in nutrient broth and cosmetic formulations. GOS trisaccharide and tetrasaccharide diffusion coefficients from O/W gel emulsion were calculated to be 5.61·10-6  cm2  s-1 and 1.41·10-8  cm2  s-1 , respectively. The diffusion coefficient of GOS trisaccharides from hydrogel was 3.22·10-6  cm2  s-1 , while it was not determined for tetrasaccharides due to low diffused concentration. Transdermal diffusion tests revealed that GOS incorporated in two formulations stays at the surface of the skin even after 24 h. CONCLUSION: When applied in adequate concentration, GOS has the potential to be used as a skin prebiotic. Novel GOS enriched formulations, Aristoflex® AVC-based hydrogel and Heliogel™-based O/W gel emulsion, provided efficient diffusion and delivery of prebiotic GOS molecules to the skin surface.

OBJECTIF: Récemment, les prébiotiques attirent beaucoup d'attention dans le domaine des soins de la peau, car il a été constaté qu'ils sont capables de soutenir l'équilibre des micro-organismes bénéfiques et nocifs sur la peau et, par conséquent, de prévenir plusieurs affections cutanées associées à un déséquilibre microbien. L'application topique de prébiotiques, bien qu'insuffisamment étudiée, est très prometteuse pour améliorer la santé de la peau. Le but de cette recherche était de déterminer le potentiel prébiotique des galacto-oligosaccharides (GOS) pour le microbiote cutané et leur aptitude à être incorporés dans différentes formulations topiques, et enfin, de fournir des informations sur leurs propriétés de diffusion. MÉTHODES: L'effet prébiotique du GOS a été évalué à travers l'influence sur la croissance de Staphylococcus epidermidis et de Staphylococcus aureus, les bactéries résidentes et pathogènes les plus courantes du microbiote cutané, respectivement. De plus, en gardant à l'esprit l'utilisation future du GOS dans les produits cosmétiques, la diffusion des molécules de GOS à partir de deux formulations topiques différentes, l'hydrogel et l'émulsion de gel huile-dans-eau (H/E), a été surveillée à l'aide d'une cellule de diffusion de Franz et de deux systèmes - avec de la cellulose membrane en acétate et modèle de test de diffusion transdermique, membrane Strat-M®. Le cours de la fermentation et la quantité de molécules de GOS diffusées ont été surveillés en utilisant la chromatographie liquide à haute performance (HPLC). RESULTATS: Les résultats in vitro ont révélé que le GOS à une concentration de 5% (p/v) a un effet stimulant prononcé sur S. epidermidis, tout en montrant simultanément un effet inhibiteur sur S. aureus, à la fois dans les bouillons nutritifs et les formulations cosmétiques. Les coefficients de diffusion GOS trisaccharide et tétrasaccharide de l'émulsion de gel H/E ont été calculés comme étant de 5,61·10−6 cm2 s−1 et 1,41·10-8 cm2 s−1 , respectivement. Le coefficient de diffusion des trisaccharides GOS à partir de l'hydrogel était de 3,22·10−6 cm2 s−1 , alors qu'il n'a pas été déterminé pour les tétrasaccharides en raison de la faible concentration diffusée. Des tests de diffusion transdermique ont révélé que le GOS incorporé dans deux formulations reste à la surface de la peau même après 24 h. CONCLUSION: Lorsqu'il est appliqué à une concentration adéquate, le GOS a le potentiel d'être utilisé comme prébiotique cutané. De nouvelles formulations enrichies en GOS, l'hydrogel à base d'Aristoflex® AVC et l'émulsion de gel H/E à base d'Heliogel™, ont permis une diffusion et une délivrance efficaces des molécules GOS prébiotiques à la surface de la peau.