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.

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.

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).

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.

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.

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.

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.

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.

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).

Lactobacillus amylovorus Promotes Lactose Utilization in Small Intestine and Enhances Intestinal Barrier Function in Intrauterine Growth Restricted Piglets.

BACKGROUND: Intrauterine growth restriction (IUGR) resulted in high mortality and many physiological defects of piglets, causing huge economic loss in the swine industry. Lactobacillus amylovorus (L. amylovorus) was identified as one of the main differential bacteria between IUGR and normal piglets. However, the effects of L. amylovorus on the growth performance and intestinal health in IUGR piglets remained unclear. OBJECTIVES: This study aimed to investigate the promoting effects of L. amylovorus Mafic1501, a new strain isolated from normal piglets, on the growth performance and intestinal barrier functions in IUGR piglets. METHODS: Newborn mice or piglets were assigned into 3 groups: CON (normal birth weight, control), IUGR (low birth weight), and IUGR+L. amy (low birth weight), administered with sterile saline or L. amylovorus Mafic1501, respectively. Growth performance, lactose content in the digesta, intestinal lactose transporter, and barrier function parameters were profiled. IPEC-J2 cells were cultured to verify the effects of L. amylovorus Mafic1501 on lactose utilization and intestinal barrier functions. RESULTS: L. amylovorus Mafic1501 elevated body weight and average daily gain of IUGR mice and piglets (P < 0.05). The lactose content in the ileum was decreased, whereas gene expression of glucose transporter 2 (GLUT2) was increased by L. amylovorus Mafic1501 in IUGR piglets during suckling period (P < 0.05). Besides, L. amylovorus Mafic1501 promoted intestinal barrier functions by increasing the villus height and relative gene expressions of tight junctions (P < 0.05). L. amylovorus Mafic1501 and its culture supernatant decreased the lactose level in the medium and upregulated gene expressions of transporter GLUT2 and tight junction protein Claudin-1 of IPEC-J2 cells (P < 0.05). CONCLUSION: L. amylovorus Mafic1501 improved the growth performance of IUGR piglets by promoting the lactose utilization in small intestine and enhancing intestinal barrier functions. Our results provided the new evidence of L. amylovorus Mafic1501 for its application in the swine industry.

Effects of 2'-fucosyllactose on the composition and metabolic activity of intestinal microbiota from piglets after in vitro fermentation.

BACKGROUND: As indigestible carbohydrates, milk oligosaccharides possess various benefits for newborns, mainly through intestinal microbiota, among which 2'-fucosyllactose (2'-FL) is the most predominant milk oligosaccharide. However, knowledge about the fermentative characteristics of 2'-FL in the gut remains limited, especially in the small intestine. The aim of this study is to explore the differential fermentability of 2'-FL by the small and large intestinal microbiota of piglets using fructo-oligosaccharide (FOS) and lactose as controls in an in vitro batch fermentation experiment. During fermentation, microbial composition was characterized along with gas production and short-chain fatty acid production. RESULTS: 2'-Fucosyllactose showed differential fermentability in jejunal and colonic fermentation. Compared with the colon, 2'-FL produced less gas in the jejunum than in the FOS and lactose groups (P < 0.05). Meanwhile, 2'-FL exhibited a different influence on the microbial composition and metabolism in the jejunum and colon compared with FOS and lactose. In the jejunum, compared with the FOS and lactose groups, the 2'-FL group showed a higher abundance of Bacteroides, Prevotella, and Blautia, but a lower abundance of Streptococcus and Lactobacillus (P < 0.05), with a higher level of propionate and a lower level of lactate during fermentation (P < 0.05). In the colon, compared with the FOS and lactose groups, 2'-FL increased the abundance of Blautia, Faecalibacterium, and Lachnospiraceae FCS020, but decreased the abundance of Prevotella_9, Succinivibrio, and Megasphaera (P < 0.05) with an increase in acetate production (P < 0.05). CONCLUSION: Overall, the results suggested that the small intestinal microbiota had the potential to ferment milk oligosaccharides. Meanwhile, in comparison with FOS and lactose, 2'-FL selectively stimulated the growth of propionate-producing bacteria in the jejunum and acetate-producing bacteria in the colon. These results demonstrated the differences in fermentation properties of 2'-FL by small and large intestinal microbiota and provided new evidence for the application of 2'-FL in optimizing gut microbiota. © 2023 Society of Chemical Industry.

Observational study on the associations between milk yield, composition, and coagulation properties with blood biomarkers of health in Holstein cows.

The considerable increase in the production capacity of individual cows owing to both selective breeding and innovations in the dairy sector has posed challenges to management practices in terms of maintaining the nutritional and metabolic health status of dairy cows. In this observational study, we investigated the associations between milk yield, composition, and technological traits and a set of 21 blood biomarkers related to energy metabolism, liver function or hepatic damage, oxidative stress, and inflammation or innate immunity in a population of 1,369 high-yielding Holstein-Friesian dairy cows. The milk traits investigated in this study included 4 production traits (milk yield, fat yield, protein yield, daily milk energy output), 5 traits related to milk composition (fat, protein, casein, and lactose percentages and urea), 11 milk technological traits (5 milk coagulation properties and 6 curd-firming traits). All milk traits (i.e., production, composition, and technological traits) were analyzed according to a linear mixed model that included the days in milk, the parity order, and the blood metabolites (tested one at a time) as fixed effects and the herd and date of sampling as random effects. Our findings revealed that milk yield and daily milk energy output were positively and linearly associated with total cholesterol, nonesterified fatty acids, urea, aspartate aminotransferase, γ-glutamyl transferase, total bilirubin, albumin, and ferric-reducing antioxidant power, whereas they were negatively associated with glucose, creatinine, alkaline phosphatase, total reactive oxygen metabolites, and proinflammatory proteins (ceruloplasmin, haptoglobin, and myeloperoxidase). Regarding composition traits, the protein percentage was negatively associated with nonesterified fatty acids and ß-hydroxybutyrate (BHB), while the fat percentage was positively associated with BHB, and negatively associated with paraoxonase. Moreover, we found that the lactose percentage increased with increasing cholesterol and albumin and decreased with increasing ceruloplasmin, haptoglobin, and myeloperoxidase. Milk urea increased with an increase in cholesterol, blood urea, nonesterified fatty acids, and BHB, and decreased with an increase in proinflammatory proteins. Finally, no association was found between the blood metabolites and milk coagulation properties and curd-firming traits. In conclusion, this study showed that variations in blood metabolites had strong associations with milk productivity traits, the lactose percentage, and milk urea, but no relationships with technological traits of milk. Specifically, increasing levels of proinflammatory and oxidative stress metabolites, such as ceruloplasmin, haptoglobin, myeloperoxidase, and total reactive oxygen metabolites, were shown to be associated with reductions in milk yield, daily milk energy output, lactose percentage, and milk urea. These results highlight the close connection between the metabolic and innate immunity status and production performance. This connection is not limited to specific clinical diseases or to the transition phase but manifests throughout the entire lactation. These outcomes emphasize the importance of identifying cows with subacute inflammatory and oxidative stress as a means of reducing metabolic impairments and avoiding milk fluctuations.

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.

Chitosan-based oral hydrogel formulations of ß-galactosidase to improve enzyme supplementation therapy for lactose intolerance.

ß-Galactosidase supplementation plays an important role in the life of people with lactose intolerance. However, these formulations are rendered ineffective by the low pH and pepsin in the stomach and pancreatic proteases in the intestine. Therefore, it is necessary to develop oral transport systems for carrying this enzyme in the active form up to the intestine, where the lactose digestion occurs. In this research, a new hydrogel was developed that could potentially be used for enzyme supplement therapy. In this regard, the chitosan-based ß-Gal formulations described in the manuscript are an alternative long-acting preparation to the so far available preparations that allow for enzyme protection and mucosal targeting. These hydrogels were prepared from chitosan and polyethylene glycol and contained a covalently immobilized ß-galactosidase from Aspergillus oryzae. The ß-galactosidase in the hydrogel was protected from degradation in a gastric medium at a pH of 2.5 and retained 75 % of its original activity under subsequent intestinal conditions. In the case of a simulated gastric fluid with a pH of 1.5, a copolymer containing methacrylic acid functional groups was sufficient to protect the hybrid hydrogel from the extremely acidic pH. In addition, the surface of the hydrogel was chemically modified with thiol and amidine groups, which increased the binding to intestinal mucin by 20 % compared with the unmodified hydrogel. These results represent a promising approach for oral transport as a reservoir for ß-galactosidase in the small intestine to reduce the symptoms of hypolactasia.

The potential of lactose to inhibit cereulide biosynthesis of emetic Bacillus cereus in milk.

This study aims to investigate the potential role of lactose on cereulide biosynthesis by emetic Bacillus cereus in dairy matrices. The cereulide yields in whole milk and lactose-free milk were investigated using the emetic reference strain F4810/72. To eliminate the influence of complex food substrates, the LB medium model was further used to characterize the effect of lactose on cereulide produced by F4810/72 and five other emetic B. cereus strains. Results showed that the lactose-free milk displayed a 13-fold higher amount of cereulide than whole milk, but the cereulide level could be reduced by 91 % when the lactose content was restored. The significant inhibition of lactose on cereulide yields of all tested B. cereus strains was observed in LB medium, showing a dose-dependent manner with inhibition rates ranging of 89-98 %. The growth curves and lactose utilization patterns of all strains demonstrated that B. cereus cannot utilize lactose as a carbon source and lactose might act as a signal molecule to regulate cereulide production. Moreover, lactose strongly repressed the expression of cereulide synthetase genes (ces), possibly by inhibiting the key regulator Spo0A at the transcriptional level. Our findings highlight the potential of lactose as an effective strategy to control cereulide production in food.

Changes in gut microbiota and lactose intolerance symptoms before and after daily lactose supplementation in individuals with the lactase nonpersistent genotype.

BACKGROUND: Approximately 70%-100% of the Asian adult population is lactase nonpersistent (LNP). The literature shows that many individuals with the LNP-genotype can consume ≤12 g of lactose without experiencing gastrointestinal discomfort. Repetitive consumption of lactose may reduce intolerance symptoms via adaptation of the gut microbiota. OBJECTIVE: This study aimed to assess the effects of daily consumption of incremental lactose doses on microbiota composition and function, and intolerance symptoms. METHODS: Twenty-five healthy adults of Asian origin, carrying the LNP-genotype were included in this 12-wk before and after intervention trial. Participants consumed gradually increasing lactose doses from 3 to 6 g to 12 g twice daily, each daily dose of 6 g, 12 g, or 24 g being provided for 4 consecutive weeks. Participants handed-in repeated stool samples and underwent a 25 g lactose challenge hydrogen breath test (HBT) before and after the 12-wk intervention. Daily gastrointestinal symptoms and total symptom scores (TSSs) during the lactose challenge were recorded. RESULTS: A significant increase from 5.5% ± 7.6% to 10.4% ± 9.6% was observed in Bifidobacterium relative abundance after the intervention (P = 0.009), accompanied by a 2-fold increase (570 ± 269 U/g; P < 0.001) in fecal ß-galactosidase activity compared with baseline (272 ± 158 U/g). A 1.5-fold decrease (incremental area under the curve; P = 0.01) in expired hydrogen was observed during the second HBT (38 ± 35 ppm·min), compared with the baseline HBT (57 ± 38 ppm·min). There was a nonsignificant decrease in TSS (10.6 ± 8.3 before compared with 8.1 ± 7.2 after intervention; P = 0.09). Daily consumption of lactose was well tolerated, with mild to no gastrointestinal complaints reported during the intervention. CONCLUSIONS: Increased levels of Bifidobacterium indicate an adaptation of the gut microbiota upon repetitive consumption of incremental doses of lactose, which was well tolerated as demonstrated by reduced expired hydrogen concentrations during the second 25-g lactose HBT. Bifidobacteria metabolize lactose without gas production thereby potentially reducing intestinal gas formation in the gut of individuals with the LNP-genotype. This increased lactose tolerance possibly lifts the necessity to remove nutrient-rich dairy foods completely from the diet. The trial is registered at the International Clinical Trials Registry Platform: NL9516. The effect of dietary lactose in lactase nonpersistent individuals on gut microbiota.

A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways.

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.