Combinatorial Engineering of Escherichia coli for Enhancing 3-Fucosyllactose Production.

3-Fucosyllactose (3-FL) is an important fucosylated human milk oligosaccharide (HMO) with biological functions such as promoting immunity and brain development. Therefore, the construction of microbial cell factories is a promising approach to synthesizing 3-FL from renewable feedstocks. In this study, a combinatorial engineering strategy was used to achieve efficient de novo 3-FL production in Escherichia coli. α-1,3-Fucosyltransferase (futM2) from Bacteroides gallinaceum was introduced into E. coli and optimized to create a 3-FL-producing chassis strain. Subsequently, the 3-FL titer increased to 5.2 g/L by improving the utilization of the precursor lactose and down-regulating the endogenous competitive pathways. Furthermore, a synthetic membraneless organelle system based on intrinsically disordered proteins was designed to spatially regulate the pathway enzymes, producing 7.3 g/L 3-FL. The supply of the cofactors NADPH and GTP was also enhanced, after which the 3-FL titer of engineered strain E26 was improved to 8.2 g/L in a shake flask and 10.8 g/L in a 3 L fermenter. In this study, we developed a valuable approach for constructing an efficient 3-FL-producing cell factory and provided a versatile workflow for other chassis cells and HMOs.

Activation of a silent lactose utilization pathway in an evolved Listeria monocytogenes F2365 outbreak isolate.

Listeria monocytogenes, a widespread food-borne pathogen, utilizes diverse growth substrates including mono- and di-saccharides via PEP-phosphotransferase (PTS) systems. We evaluated a collection of L. monocytogenes isolates of different origins for their ability to utilize lactose, a disaccharide composed of galactose and glucose and the main carbon source in milk and dairy products. Notably, the dairy-associated outbreak strain F2365 could not utilize lactose efficiently, conceivably due to a frameshift mutation (lacR887del) resulting in a truncated LacR. Transcriptional activator LacR is involved in the expression of two PTS systems, encoded by the lpo operon lmo1718-1720 in combination with lmo2708 and the lmo2683-2685 operon, and linked to lactose and/or cellobiose metabolism in L. monocytogenes. Via experimental evolution of the ancestral strain F2365, an evolved isolate F2365 EV was obtained which showed enhanced growth and metabolism of lactose. Using the lactose-positive model strain L. monocytogenes EGDe as a control, HPLC experiments showed that EGDe and F2365 EV could consume lactose and utilize the glucose moiety, while the galactose moiety was exported from the cells. Genome sequencing of F2365 EV found the original lacR887del mutation was still present but an additional point mutation lmo2766C415T had occurred, resulting in an amino acid substitution in the putative regulator Lmo2766. The lmo2766 gene is located next to operon lmo2761-2765 with putative PTS genes in the genome. Notably, comparative RNAseq analysis confirmed that the lmo2761-2765 operon was strongly upregulated in F2365 EV in the presence of lactose but not in EGDe and F2365. Conversely, the LacR-regulated lpo operon, lmo2708, and lmo2683-2685 operon were only upregulated in EGDe. Additional growth and HPLC experiments, using mutants constructed in lactose-positive L. monocytogenes EGDe, showed reduced growth of the EGDe lacR887del mutant with no utilization of lactose, while the double mutant EGDe lacR887dellmo2766C415T showed enhanced growth and lactose utilization. Hence, these results demonstrate that an amino acid substitution in the Lmo2766 regulator activates a previously silent lactose utilization pathway encoded by PTS operon lmo2761-2765, facilitating the growth and metabolism of L. monocytogenes with lactose as a substrate. This finding enhances our understanding of the metabolic capabilities and adaptability of L. monocytogenes, offering a broader view of the lactose utilization capacity of this pathogen.

Freshwater microalgae Nannochloropsis limnetica for the production of ß-galactosidase from whey powder.

This study investigated the first-ever reported use of freshwater Nannochloropsis for the bioremediation of dairy processing side streams and co-generation of valuable products, such as ß-galactosidase enzyme. In this study, N. limnetica was found to grow rapidly on both autoclaved and non-autoclaved whey-powder media (referred to dairy processing by-product or DPBP) without the need of salinity adjustment or nutrient additions, achieving a biomass concentration of 1.05-1.36 g L-1 after 8 days. The species secreted extracellular ß-galactosidase (up to 40.84 ± 0.23 U L-1) in order to hydrolyse lactose in DPBP media into monosaccharides prior to absorption into biomass, demonstrating a mixotrophic pathway for lactose assimilation. The species was highly effective as a bioremediation agent, being able to remove > 80% of total nitrogen and phosphate in the DPBP medium within two days across all cultures. Population analysis using flow cytometry and multi-channel/multi-staining methods revealed that the culture grown on non-autoclaved medium contained a high initial bacterial load, comprising both contaminating bacteria in the medium and phycosphere bacteria associated with the microalgae. In both autoclaved and non-autoclaved DPBP media, Nannochloropsis cells were able to establish a stable microalgae-bacteria interaction, suppressing bacterial takeover and emerging as dominant population (53-80% of total cells) in the cultures. The extent of microalgal dominance, however, was less prominent in the non-autoclaved media. High initial bacterial loads in these cultures had mixed effects on microalgal performance, promoting ß-galactosidase synthesis on the one hand while competing for nutrients and retarding microalgal growth on the other. These results alluded to the need of effective pre-treatment step to manage bacterial population in microalgal cultures on DPBP. Overall, N. limnetica cultures displayed competitive ß-galactosidase productivity and propensity for efficient nutrient removal on DPBP medium, demonstrating their promising nature for use in the valorisation of dairy side streams.

Potentially functional lactose-free ice cream with Lacticaseibacillus casei CSL3, ginger, and honey.

AIMS: To develop and characterize a functional lactose-free ice cream with added ginger and honey, evaluate the survival of Lacticaseibacillus casei CSL3 under frozen storage and the simulated gastrointestinal tract (GIT), as well as antioxidant activity and product acceptability. METHODS AND RESULTS: The survival of Lacticaseibacillus casei CSL3 was evaluated for 180 days, under frozen storage, and GIT at 60 days. At 15 days of storage, proximal composition, antioxidant activity, color, pH, acidity, fusion, density, overrun, and sensory analysis were performed. Ice cream was an effective food matrix for maintaining the viability of CSL3, with concentrations > 7 log CFU g- 1 during storage and GIT. In addition, the analysis showed overrun and prebiotic characteristics through high values of antioxidant activity and phenolic compounds, good acceptability, and purchase intention. CONCLUSIONS: The product has satisfactory market potential (acceptance rate of 95.19% and purchase intention rate > 96%), and it could become another means of inserting probiotics in food.

Engineering Bacillus subtilis for highly efficient production of functional disaccharide lactulose from lactose.

Bioconversion of lactose to functional lactose derivatives attracts increasing attention. Lactulose is an important high-value lactose derivative, which has been widely used in pharmaceutical, nutraceutical, and food industries. Lactulose can be enzymatically produced from lactose by cellobiose 2-epimerase (CEase). Several studies have already focused on the food-grade expression of CEase, but they are all aimed at the biosynthesis of epilactose. Herein, we reported for the first time the biosynthesis of lactulose using the recombinant food-grade Bacillus subtilis. Lactulose biosynthesis was optimized by varying lactulose-producing CEases and expression vectors. Caldicellulosiruptor saccharolyticus CEase and pP43NMK were determined to be the optimal CEase and expression vector. Fine-tuning of CEase expression was investigated by screening a beneficial N-terminal coding sequence. After fed-batch cultivation, the highest fermentation isomerization activity reached 11.6 U/mL. Lactulose was successfully produced by the broth of the engineered B. subtilis with a yield of 52.1 %.

Multidrug-resistance and extended-spectrum beta-lactamase-producing lactose-fermenting enterobacteriaceae in the human-dairy interface in northwest Ethiopia.

BACKGROUND: Antimicrobial resistance (AMR) is among the top public health concerns in the globe. Estimating the prevalence of multidrug resistance (MDR), MDR index (MDR-I) and extended-spectrum beta-lactamase (ESBL)-producing lactose fermenting Enterobacteriaceae (LFE) is important in designing strategies to combat AMR. Thus, this study was designed to determine the status of MDR, MDR-I and ESBL-producing LFE isolated from the human-dairy interface in the northwestern part of Ethiopia, where such information is lacking. METHODOLOGY: A cross-sectional study was conducted from June 2022 to August 2023 by analyzing 362 samples consisting of raw pooled milk (58), milk container swabs (58), milker's hand swabs (58), farm sewage (57), milker's stool (47), and cow's feces (84). The samples were analyzed using standard bacteriological methods. The antimicrobial susceptibility patterns and ESBL production ability of the LFE isolates were screened using the Kirby-Bauer disk diffusion method, and candidate isolates passing the screening criteria were phenotypically confirmed by using cefotaxime (30 µg) and cefotaxime /clavulanic acid (30 µg/10 µg) combined-disk diffusion test. The isolates were further characterized genotypically using multiplex polymerase chain reaction targeting the three ESBL-encoding- genes namely blaTEM, blaSHV, and blaCTX-M. RESULTS: A total of 375 bacterial isolates were identified and the proportion of MDR and ESBL-producing bacterial isolates were 70.7 and 21.3%, respectively. The MDR-I varied from 0.0 to 0.81 with an average of 0.30. The ESBL production was detected in all sample types. Genotypically, the majority of the isolates (97.5%), which were positive on the phenotypic test, were carrying one or more of the three genes. CONCLUSION: A high proportion of the bacterial isolates were MDR; had high MDR-I and were positive for ESBL production. The findings provide evidence that the human-dairy interface is one of the important reservoirs of AMR traits. Therefore, the implementation of AMR mitigation strategies is highly needed in the area.

Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome.

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity.

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.

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.

A catalytic membrane approach as a way to obtain sweet and unsweet lactose-free milk.

The growing need in the current market for innovative solutions to obtain lactose-free (L-F) milk is caused by the annual increase in the prevalence of lactose intolerance inside as well as the newborn, children, and adults. Various configurations of enzymes can yield two distinct L-F products: sweet (ß-galactosidase) and unsweet (ß-galactosidase and glucose oxidase) L-F milk. In addition, the reduction of sweetness through glucose decomposition should be performed in a one-pot mode with catalase to eliminate product inhibition caused by H2O2. Both L-F products enjoy popularity among a rapidly expanding group of consumers. Although enzyme immobilization techniques are well known in industrial processes, new carriers and economic strategies are still being searched. Polymeric carriers, due to the variety of functional groups and non-toxicity, are attractive propositions for individual and co-immobilization of food enzymes. In the presented work, two strategies (with free and immobilized enzymes; ß-galactosidase NOLA, glucose oxidase from Aspergillus niger, and catalase from Serratia sp.) for obtaining sweet and unsweet L-F milk under low-temperature conditions were proposed. For free enzymes, achieving the critical assumption, lactose hydrolysis and glucose decomposition occurred after 1 and 4.3 h, respectively. The tested catalytic membranes were created on regenerated cellulose and polyamide. In both cases, the time required for lactose and glucose bioconversion was extended compared to free enzymes. However, these preparations could be reused for up to five (ß-galactosidase) and ten cycles (glucose oxidase with catalase).

Regulation of lactose, glucose and sucrose metabolisms in S. thermophilus.

Streptococcus thermophilus is a bacterium widely used in the production of yogurts and cheeses, where it efficiently ferments lactose, the saccharide naturally present in milk. It is also employed as a starter in dairy- or plant-based fermented foods that contain saccharides other than lactose (e.g., sucrose, glucose). However, little is known about how saccharide use is regulated, in particular when saccharides are mixed. Here, we determine the effect of the 5 sugars that S. thermophilus is able to use, at different concentration and when they are mixed on the promoter activities of the C-metabolism genes. Using a transcriptional fusion approach, we discovered that lactose and glucose modulated the activity of the lacS and scrA promoters in a concentration-dependent manner. When mixed with lactose, glucose also repressed the two promoter activities; when mixed with sucrose, lactose still repressed scrA promoter activity. We determined that catabolite control protein A (CcpA) played a key role in these dynamics. We also showed that promoter activity was linked with glycolytic flux, which varied depending on saccharide type and concentration. Overall, this study identified key mechanisms in carbohydrate metabolism - autoregulation and partial hierarchical control - and demonstrated that they are partly mediated by CcpA.

Optimizing microbioreactor cultivation strategies for Trichoderma reesei: from batch to fed-batch operations.

BACKGROUND: Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. RESULTS: This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) - 1 , the lowest rate of 0.3 g (l h) - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in ß -glucosidase activity compared to batch processes using the same type and amount of carbon sources. CONCLUSION: We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology.

Phylogeny and structural modeling of the transcription factor CsqR (YihW) from Escherichia coli.

CsqR (YihW) is a local transcription factor that controls expression of yih genes involved in degradation of sulfoquinovose in Escherichia coli. We recently showed that expression of the respective gene cassette might be regulated by lactose. Here, we explore the phylogenetic and functional traits of CsqR. Phylogenetic analysis revealed that CsqR had a conserved Met25. Western blot demonstrated that CsqR was synthesized in the bacterial cell as two protein forms, 28.5 (CsqR-l) and 26 kDa (CsqR-s), the latter corresponding to start of translation at Met25. CsqR-s was dramatically activated during growth with sulfoquinovose as a sole carbon source, and displaced CsqR-l in the stationary phase during growth on rich medium. Molecular dynamic simulations revealed two possible states of the CsqR-s structure, with the interdomain linker being represented by either a disordered loop or an ɑ-helix. This helix allowed the hinge-like motion of the N-terminal domain resulting in a switch of CsqR-s between two conformational states, "open" and "compact". We then modeled the interaction of both CsqR forms with putative effectors sulfoquinovose, sulforhamnose, sulfoquinovosyl glycerol, and lactose, and revealed that they all preferred the same pocket in CsqR-l, while in CsqR-s there were two possible options dependent on the linker structure.

Added Value of 13C Analysis in Breath Tests in H2-Negative Subjects to Diagnose Lactose Malabsorption: A Proof of Concept Study.

INTRODUCTION: Diagnosing lactose malabsorption is usually based on hydrogen excretion in breath after a lactose challenge. However, a proportion of subjects with lactose malabsorption will not present a rise in hydrogen. Measuring excretion of methane or stable isotope labeled 13CO2 after ingestion of 13C-lactose has been proposed to mitigate this problem. OBJECTIVE: The aim of the study was to assess the performance of measuring methane and 13CO2 in individuals with normal hydrogen excretion compared to a genetic lactase non-persistence test. METHODS: Individuals referred for lactose breath testing and healthy controls were included. Participants received 13C-enriched lactose, performed breath testing, and underwent genotyping for a marker of lactase non-persistence (13910C*T). Using genotype as gold standard, the performance of measuring methane and 13CO2 excretion was assessed. RESULTS: 151 subjects participated in the study, 50 of which presented a lactase non-persistent genotype. Of these, 72% were correctly diagnosed through hydrogen excretion of ≥ 20 ppm above baseline. In subjects with normal hydrogen excretion, cumulative 13C excretion had an area under the curve (AUC) of the receiver operating characteristics (ROC) curve of 0.852. Sensitivity was 93% and specificity was 51% for the current cutoff of 14.5%. The optimal cutoff was 12.65% (sensitivity 93%, specificity 70%). The ROC curve of peak methane had an AUC of 0.542 (sensitivity of 14%, specificity of 91% for cutoff ≥ 10 ppm). CONCLUSIONS: In individuals with genetically demonstrated lactase non-persistence and negative hydrogen breath test, the use of 13C-lactose with measurement of 13CO2 excretion and hydrogen is a well-performing test to detect the lactose malabsorption and performs better than methane in our cohort.

Characterization and application of active human α2,6-sialyltransferases ST6GalNAc V and ST6GalNAc VI recombined in Escherichia coli.

Eukaryotic sialyltransferases play key roles in many physiological and pathological events. The expression of active human recombinant sialyltransferases in bacteria is still challenging. In the current study, the genes encoding human N-acetylgalactosaminide α2,6-sialyltransferase V (hST6GalNAc V) and N-acetylgalactosaminide α2,6-sialyltransferase VI (hST6GalNAc VI) lacking the N-terminal transmembrane domains were cloned into the expression vectors, pET-32a and pET-22b, respectively. Soluble and active forms of recombinant hST6GalNAc V and hST6GalNAc VI when coexpressed with the chaperone plasmid pGro7 were successfully achieved in Escherichia coli. Further, lactose (Lac), Lacto-N-triose II (LNT II), lacto-N-tetraose (LNT), and sialyllacto-N-tetraose a (LSTa) were used as acceptor substrates to investigate their activities and substrate specificities. Unexpectedly, both can transfer sialic acid onto all those substrates. Compared with hST6GalNAc V expressed in the mammalian cells, the recombinant two α2,6-sialyltransferases in bacteria displayed flexible substrate specificities and lower enzymatic efficiency. In addition, an important human milk oligosaccharide disialyllacto-N-tetraose (DSLNT) can be synthesized by both human α2,6-sialyltransferases expressed in E. coli using LSTa as an acceptor substrate. To the best of our knowledge, these two active human α2,6-sialyltransferases enzymes were expressed in bacteria for the first time. They showed a high potential to be applied in biotechnology and investigating the molecular mechanisms of biological and pathological interactions related to sialylated glycoconjugates.

Efficient Biosynthesis of Lacto-N-Biose I, a Building Block of Type I Human Milk Oligosaccharides, by a Metabolically Engineered Escherichia coli.

Lacto-N-biose I (LNB), termed a Type 1 disaccharide, is an important building block of human milk oligosaccharides. It shows promising prebiotic activity by stimulating the proliferation of many gut-associated bifidobacteria and thus displays good potential in infant foods or supplements. Enzymatic and microbial approaches to LNB synthesis have been studied, almost all of which involve glycosylation of LNB phosphorylase as the final step. Herein, we report a new and easier microbial LNB synthesis strategy through the route "lactose → lacto-N-triose II (LNTri II) → lacto-N-tetraose (LNT) → LNB". A previously constructed LNT-producing Escherichia coli BL21(DE3) strain was engineered for LNB biosynthesis by introducing Bifidobacterium bifidum LnbB. LNB was efficiently produced, accompanied by lactose regeneration. Genomic integration of key pathway genes related to LNTri II and LNT synthesis was performed to enhance LNB titers. The final engineered strain produced 3.54 and 26.88 g/L LNB by shake-flask and fed-batch cultivation, respectively.

Production of D -tagatose, bioethanol, and microbial protein from the dairy industry by-product whey powder using an integrated bioprocess.

We designed and constructed a green and sustainable bioprocess to efficiently coproduce D -tagatose, bioethanol, and microbial protein from whey powder. First, a one-pot biosynthesis process involving lactose hydrolysis and D -galactose redox reactions for D -tagatose production was established in vitro via a three-enzyme cascade. Second, a nicotinamide adenine dinucleotide phosphate-dependent galactitol dehydrogenase mutant, D36A/I37R, based on the nicotinamide adenine dinucleotide-dependent polyol dehydrogenase from Paracoccus denitrificans was created through rational design and screening. Moreover, an NADPH recycling module was created in the oxidoreductive pathway, and the tagatose yield increased by 3.35-fold compared with that achieved through the pathway without the cofactor cycle. The reaction process was accelerated using an enzyme assembly with a glycine-serine linker, and the tagatose production rate was 9.28-fold higher than the initial yield. Finally, Saccharomyces cerevisiae was introduced into the reaction solution, and 266.5 g of D -tagatose, 162.6 g of bioethanol, and 215.4 g of dry yeast (including 38% protein) were obtained from 1 kg of whey powder (including 810 g lactose). This study provides a promising sustainable process for functional food (D -tagatose) production. Moreover, this process fully utilized whey powder, demonstrating good atom economy.

Relationship Between Maternal Age and Macronutrient Content of Colostrum.

BACKGROUND: Little is known about the relationship between maternal age and the macronutrient content of colostrum. RESEARCH AIMS: This study aimed to evaluate the relationship between maternal age and human milk macronutrient content by comparing the concentrations of lactose, proteins, and lipids in the colostrum of women with younger, moderate, and advanced maternal age. METHODS: An observational, cross-sectional study was designed to compare the macronutrient concentrations in the colostrum of women aged < 20 years, 20 to 34 years, and > 34 years (younger, moderate, and advanced maternal age, respectively; n = 33 per group). For each participant, 3 ml of colostrum was collected by manual extraction from the right breast at 10 am, 39-48 hr after delivery, and analyzed using a Miris Human Milk Analyzer. Macronutrient concentrations were compared between the groups using analysis of variance. P < 0.05 was considered significant. RESULTS: Mothers with moderate maternal age had a higher colostrum lipid concentration than those with younger or advanced maternal age (2.3 mg, SD = 1.4 mg vs. 1.5 mg, SD = 1.0 mg vs. 1.6 mg, SD = 0.9 mg, respectively; p = 0.007). Lactose and protein contents in the analyzed samples did not differ among the three study groups. CONCLUSION: This study lends support to the potential variation of lipids in colostrum by maternal age and suggests individual adaptation to the nutritional components of milk to the needs of the infant may be beneficial.

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).

Postprandial metabolism and gut permeability in calves fed milk replacer with different macronutrient profiles or a whole milk powder.

Significant differences exist in the composition of current milk replacers (MR) and bovine whole milk. This study investigated how the macronutrient profile of 3 different MR formulations containing varying amounts of fat, lactose, and protein, and a whole milk powder (WP), affect postprandial metabolism and gut permeability in male Holstein calves. Sixty-four calves (45.4 ± 4.19 kg [mean ± SD] and 1.8 ± 0.62 d of age) were blocked in order of arrival to the facility and within each block, calves were randomly assigned to 1 of 4 treatments. Treatments included a high-fat MR (HF: 25.0% dry matter [DM] fat, 22.5% protein, 38.6% lactose; n = 14), a high-lactose MR (HL: 44.6% lactose, 22.5% protein, 18.0% fat; n = 17), a high-protein MR (HP: 26.0% protein, 18.0% fat, 41.5% lactose; n = 17), and WP (26.0% fat, 24.5% protein, 38.0% lactose; n = 16). Calves were fed 3.0 L (135 g/L) 3 times daily at 0600, 1200, and 1800 h with a teat bucket. Milk intake was recorded daily for the first 28 d after arrival, and blood sampling and body weight measurements were performed at arrival and on d 7, 14, 21, and 27. Gut permeability was estimated from fractional urinary excretion of indigestible markers (Cr-EDTA, lactulose, and d-mannitol) administered as a single dose on d 21 instead of the morning milk meal. Digestibility was determined simultaneously from a total collection of feces over 24 h. Postprandial dynamics were measured on d 28 by sequential blood sampling over 7.5 h. Dry matter intake of MR over 28 d was slightly greater in calves fed HL and HP than in WP. Recovery of Cr-EDTA and d-mannitol over a 24-h urine collection was greater in calves fed WP and HP than HL calves. Apparent total-tract digestibility of crude ash, protein, and fat did not differ among treatments; however, DM digestibility was lower in calves fed WP than in other treatment groups. In addition, abomasal emptying, as indicated by the area under the curve (AUC) for acetaminophen, was slower in calves fed WP than in calves fed HF and HL. The AUC for postprandial plasma glucose was lower in calves fed HL than WP and HF and lower in calves fed HP than WP. The AUC for postprandial serum insulin was greater in calves fed HP than WP and HF, whereas calves fed HL did not differ from the other treatments. Postprandial triglycerides were greater in calves fed WP, and postprandial adiponectin was higher in calves fed HL than other treatments. The high content of lactose and protein in MR had a major effect on postprandial metabolism. This raises the possibility of optimizing MR formulations to maintain metabolic homeostasis and influence development.