Search Results (21)

As global milk production continues to rise, the disposal of expired milk contributes to environmental pollution and valuable resource wastage. This study presents the development of a novel L-arabinose isomerase, designated BmAIase12, and its application in the enzymatic recycling of expired milk. BmAIase12 exhibited a specific activity of 10.7 U/mg and showed optimal performance at 50 °C and pH 7.0. Furthermore, it exhibited higher activity than most other L-arabinose isomerases. It converted D-galactose into D-tagatose with a high conversion ratio of 53.3% after 48 h at 50 °C. The conversion efficiency of expired milk to D-tagatose was recorded at 40.62%, resulting in a maximum tagatose yield of 1.625 g/L. This was accomplished through the incorporation of β-galactosidase (120 U/mL) and Saccharomyces cerevisiae (30 mg/mL) to hydrolyze lactose and metabolize glucose, followed by the addition of 3 U/mL of BmAIase12. Ultimately, following purification, the purity of tagatose was determined to be 98%, and the final yield was 29.8%. These results suggest that BmAIase12 may serve as a promising enzyme for D-tagatose production due to its high conversion yield. Full article

Cold-adapted microorganisms possess cold-active enzymes with potential applications in different industries and research areas. In this study, two genes encoding β-d-galactosidases belonging to Glycoside Hydrolase families 2 and 42 from the psychrotolerant Arctic bacterium Arthrobacter sp. S3* were cloned, expressed in Escherichia coli and Komagataella phaffii, purified and characterized. The GH2 β-d-galactosidase is a tetramer with a molecular weight of 450 kDa, while the GH42 β-d-galactosidase is a 233 kDa trimer. The Bgal2 was optimally active at pH 7.5 and 22 °C and maintained 57% of maximum activity at 10 °C, whereas the Bgal42 was optimally active at pH 7.0 and 40 °C and exhibited 44% of maximum activity at 10 °C. Both enzymes hydrolyzed lactose and showed transglycosylation activity. We also found that 2 U/mL of the Bgal2 hydrolyzed 85% of lactose in milk within 10 h at 10 °C. The enzyme synthesized galactooligosaccharides, heterooligosaccharides, alkyl galactopyranosides and glycosylated salicin. The Bgal42 synthesized galactooligosaccharides and 20 U/mL of the enzyme hydrolyzed 72% of milk lactose within 24 h at 10 °C. The properties of Arthrobacter sp. S3* Bgal2 make it a candidate for lactose hydrolysis in the dairy industry and a promising tool for the glycosylation of various acceptors in the biomedical sector. Full article

The production performance of dairy cattle is closely related to their metabolic state. This study aims to provide a comprehensive understanding of the production performance and metabolic features of Sanhe dairy cattle across different parities, with a specific focus on evaluating variations in milk traits and metabolites in both milk and serum. Sanhe dairy cattle from parities 1 to 4 (S1, n = 10; S2, n = 9; S3, n = 10; and S4, n = 10) at mid-lactation were maintained under the same feeding and management conditions. The milk traits, hydrolyzed milk amino acid levels, serum biochemical parameters, and serum free amino acid levels of the Sanhe dairy cattle were determined. Multiparous Sanhe dairy cattle (S2, S3, and S4) had a greater milk protein content, lower milk lactose content, and lower solids-not-fat content than primiparous Sanhe dairy cattle (S1). Moreover, S1 had a higher ratio of essential to total amino acids (EAAs/TAAs) in both the serum and milk. The serum biochemical results showed the lower glucose and total protein levels in S1 cattle were associated with milk quality. Furthermore, ultra-high-resolution high-performance liquid chromatography with tandem MS analysis (UPLC-MS/MS) identified 86 and 105 differential metabolites in the serum and milk, respectively, and these were mainly involved in amino acid, carbohydrate, and lipid metabolism. S1 and S2/S3/S4 had significantly different metabolic patterns in the serum and milk, and more vitamin B-related metabolites were significantly higher identified in S1 than in multiparous cattle. Among 36 shared differential metabolites in the serum and milk, 10 and 7 metabolites were significantly and strongly correlated with differential physiological indices, respectively. The differential metabolites identified were enriched in key metabolic pathways, illustrating the metabolic characteristics of the serum and milk from Sanhe dairy cattle of different parities. L-phenylalanine, dehydroepiandrosterone, and linoleic acid in the milk and N-acetylornithine in the serum could be used as potential marker metabolites to distinguish between Sanhe dairy cattle with parities of 1–4. In addition, a metabolic map of the serum and milk from the three aspects of carbohydrates, amino acids, and lipids was created for the further analysis and exploration of their relationships. These results reveal significant variations in milk traits and metabolites across different parities of Sanhe dairy cattle, highlighting the influence of parity on the metabolic profiles and production performance. Tailored nutritional strategies based on parity-specific metabolic profiles are recommended to optimize milk production and quality in Sanhe cattle. Full article

Due to its very early introduction, cow’s milk is one of the first foods that can cause adverse reactions in human beings. Lactose intolerance (LI) and cow’s milk allergy (CMA) are the most common adverse reactions to cow’s milk. While LI is due to insufficient small intestinal lactase activity and/or a large quantity of ingested lactose, CMA is an aberrant immune reaction to cow’s milk proteins, particularly casein or β-lactoglobulin. However, the clinical manifestations of LI and CMA, particularly their gastrointestinal signs and symptoms, are very similar, which might lead to misdiagnosis or delayed diagnosis as well as nutritional risks due to inappropriate dietary interventions or unnecessary dietary restriction. Formula-fed infants with LI should be treated with formula with reduced or no lactose, while those with CMA should be treated with formula containing extensive hydrolyzed cow’s milk protein or amino acids. This review is therefore written to assist clinicians to better understand the pathophysiologies of LI and CMA as well as to recognize the similarities and differences between clinical manifestations of LI and CMA. Full article

The performance of dairy cows is closely tied to the metabolic state, and this performance varies depending on the number of times the cows have given birth. However, there is still a lack of research on the relationship between the metabolic state of Holstein cows and the performance of lactation across multiple parities. In this study, biochemical analyses and metabolomics studies were performed on the serum and milk from Holstein cows of parities 1–4 (H1, N = 10; H2, N = 7; H3, N = 9; H4, N = 9) in mid-lactation (DIM of 141 ± 4 days) to investigate the link between performance and metabolic changes. The results of the milk quality analysis showed that the lactose levels were highest in H1 (p = 0.036). The total protein content in the serum increased with increasing parity (p = 0.013). Additionally, the lipase activity was found to be lowest in H1 (p = 0.022). There was no difference in the composition of the hydrolyzed amino acids in the milk among H1 to H4. However, the free amino acids histidine and glutamate in the serum were lowest in H1 and highest in H3 (p < 0.001), while glycine was higher in H4 (p = 0.031). The metabolomics analysis revealed that 53 and 118 differential metabolites were identified in the milk and serum, respectively. The differential metabolites in the cows’ milk were classified into seven categories based on KEGG. Most of the differential metabolites in the cows’ milk were found to be more abundant in H1, and these metabolites were enriched in two impact pathways. The differential metabolites in the serum could be classified into nine categories and enriched in six metabolic pathways. A total of six shared metabolites were identified in the serum and milk, among which cholesterol and citric acid were closely related to amino acid metabolism in the serum. These findings indicate a significant influence of blood metabolites on the energy and amino acid metabolism during the milk production process in the Holstein cows across 1–4 lactations, and that an in-depth understanding of the metabolic changes that occur in Holstein cows during different lactations is essential for precision farming, and that it is worthwhile to further investigate these key metabolites that have an impact through controlled experiments. Full article

The bioactive conjugated linolenic acid (CLNA) can be microbiologically produced by different probiotic strains when in the presence of α-linolenic acid (α-LNA). Food matrices are a good vector, such as has been previously demonstrated with fermented milk enriched with microbial CLNA by Bifidobacterium breve DSM 20091 from lipase-hydrolyzed flaxseed oil. The aim of the present work was to further assess the nutritional, biochemical and organoleptic properties of the developed dairy product, as well as its storage stability throughout 28 days at 4 °C, proving its suitability for consumption. Milk lactose hydrolyzed into glucose (0.89 g/100 g) and galactose (0.88 g/100 g), which were further metabolized into lactic (0.42 g/100 g), acetic (0.44 g/100 g) and propionic (0.85 g/100 g) acids. Titratable acidity reached 0.69% and pH 4.93. Compared with the control (no CLNA), fat content was slightly higher (2.0 g/100 g). Acetic acid was the major volatile (83.32%), lacking important dairy flavor contributors, like acetaldehyde. Sensory analysis revealed predominant astringency and bitterness. No microbial concerns arose during storage, but the CLNA content increased, and some saturated fatty acids seemed to oxidize. In conclusion, the CLNA-enriched fermented milk revealed reasonable compositional properties, yet further improvements are needed for optimal consumer acceptance and a prolonged shelf-life. Full article

2′-fucosyllactose (2′FL) is an important nutrient in human milk that stimulates beneficial microbiota and prevents infection. α-L-fucosidase is a promising component for 2′FL synthesis. In this study, a soil-oriented α-L-fucosidase-producing strain from Enterococcus gallinarum ZS1 was isolated. Escherichia coli was employed as a host for cloning and expressing the α-L-fucosidase gene (entfuc). The EntFuc was predicted as a member of the GH29 family with a molecular mass of 58 kDa. The optimal pH and temperature for the activity of EntFuc were pH 7.0 and 30 °C, respectively. The enzyme exhibited a strictly specific activity for 4-Nitrophenyl-α-L-fucopyranoside (pNP-Fuc) and had a negligible effect on hydrolyzing 2′FL. EntFuc could catalyze the synthesis of 2′FL via transfucosylation action from pNP-Fuc and lactose. The yield of 2′FL reached 35% under optimal conditions. This study indicated that EntFuc with a high conversion rate is a promising enzyme source for the biosynthesis of 2′FL. Full article

During the thermal processing of dairy products, the Maillard reaction occurs between milk proteins and lactose, resulting in the formation of various products including glycated proteins. In this study, lactose-glycated casein was generated through the Maillard reaction between casein and lactose and then hydrolyzed by a trypsin preparation. The anti-inflammatory effect of the resultant glycated casein hydrolysate (GCH) was investigated using the lipopolysaccharide (LPS)-sitmulated rat intestinal epithelial (IEC-6) cells as a cell model and corresponding casein hydrolysate (CH) as a control. The results indicated that the preformed glycation enabled lactose conjugation to casein, which endowed GCH with a lactose content of 12.61 g/kg protein together with a lower activity than CH to enhance the viability value of the IEC-6 cells. The cells with LPS stimulation showed significant inflammatory responses, while a pre-treatment of the cells with GCH before LPS stimulation consistently led to a decreased secretion of three pro-inflammatory mediators, namely, IL-6, IL-1β and tumor necrosis factor-α (TNF-α) but an increased secretion of two anti-inflammatory mediators, including IL-10 and transforming growth factor-β (TGF-β), demonstrating the anti-inflammatory potential of GCH in LPS-stimulated cells. In addition, GCH up-regulated the expression of TLR4, p-p38, and p-p65 proteins in the stimulated cells, resulting in the suppression of NF-κB and MAPK signaling pathways. Collectively, GCH was mostly less efficient than CH to exert these assessed anti-inflammatory activities in the cells and more importantly, GCH also showed an ability to cause cell inflammation by promoting IL-6 secretion and up-regulating the expression of TLR4 and p-p65. The casein lactose-glycation of the Maillard-type was thereby concluded to attenuate the anti-inflammatory potential of the resultant casein hydrolysate. It is highlighted that the casein lactose-glycation of the Maillard-type might cause a negative impact on the bioactivity of casein in the intestine, because the glycated casein after digestion could release GCH with reduced anti-inflammatory activity. Full article

Much attention has recently been paid to β-Galactosidases (β-D-galactoside galactohidrolase; EC 3.2.1.23), commonly known as lactases, due to the lactose intolerance of the human population and the importance of dairy products in the human diet. This enzyme, produced by microorganisms, is being used in the dairy industry for hydrolyzing the lactose found in milk to produce lactose-free milk (LFM). Conventionally, β-galactosidases catalyze the hydrolysis of lactose to produce glucose and galactose in LFM; however, they can also catalyze transgalactosylation reactions that produce a wide range of galactooligosaccharides (GOS), which are functional prebiotic molecules that confer health benefits to human health. In this field, different works aims to identify novel microbial sources of β-galactosidase for removing lactose from milk with the relative GOS production. Lactase extracted from thermophilic microorganisms seems to be more suitable for the transgalactosylation process at relatively high temperatures, as it inhibits microbial contamination. Different immobilization methods, such as adsorption, covalent attachment, chemical aggregation, entrapment and micro-encapsulation, have been used to synthesize lactose-derived oligosaccharides with immobilized β-galactosidases. In this mini-review, particular emphasis has been given to the immobilization techniques and bioreactor configurations developed for GOS synthesis in milk, in order to provide a more detailed overview of the biocatalytic production of milk oligosaccharides at industrial level. Full article

Background: Cow’s milk allergy (CMA) and cow’s milk intolerance (CMI) are the major cow’s milk disorders observed in infants and young children. This study investigates, for the first time, physician knowledge regarding CMA and CMI prevalence, diagnosis, and management in the Middle East and North Africa (MENA) region. In addition, we explore the role of goat milk-based formula as an alternative in infants suffering from CMI. Method: This cross-sectional survey was conducted from December 2020 to February 2021. A convenience sample of 2500 MENA-based physicians received the questionnaire, developed by a working group of pediatric experts. Results: 1868 physicians completed the questionnaire, including pediatric specialists (80.8%), training physicians (0.2%), dermatologists (0.1%), family/general physicians (12.9%), neonatologists (3.6%), neurosurgeons (0.2%), allergy nurse specialists (0.3%), pharmacists (2.1%), and public health workers (0.1%). Differentiation between CMA and CMI was recognized by the majority of respondents (80.7%), for which the majority of respondents (35.4%) identified that the elimination and challenge test was the best test to differentiate CMA from CMI, whereas 30.7% and 5.4% preferred the immunoglobulin E (IgE) test and skin prick test, respectively. In addition, 28.5% of respondents reported that there is no confirmatory test to differentiate CMA from CMI. The majority of respondents (47.3%) reported that amino acid-based formula (AAF)/ extensively hydrolyzed formula (EHF) is the cornerstone for the management of CMA. However, most respondents (33.7%) reported that lactose avoidance was best for the management of CMI. Overall, 65% of the respondents were aware of nutritionally adapted goat’s milk formula as an alternative to cow’s milk products and 37% would recommend its routine use in infants (≤2 years of age). Conclusion: The results of this survey demonstrate that the majority of physicians are aware of the underlying pathophysiology and management of CMA and CMI. However, a significant proportion of physicians do not follow the clinical guidelines concerning CMA/CMI diagnosis and management. Notably, this survey identified that goat’s milk formulas may offer a suitable alternative to AAF/EHF in infants with CMI as they contain β-casein protein which is easily digestible. In addition, goat’s milk formulas contain higher levels of oligosaccharides and medium-chained fatty acids compared with standard cow’s milk formulas, yet further clinical trials are warranted to support the inclusion of goat’s milk formulas in clinical guidelines. Full article

β-Galactosidase has been greatly used in the dairy industry. This study investigated a novel thermostable β-galactosidase (lacZBa) from Bacillus aryabhattai GEL-09 and evaluated the hydrolytic performance of this enzyme. Firstly, the lacZBa-encoding gene was cloned and overexpressed in Escherichia coli BL21(DE3). Phylogenetic analyses revealed that lacZBa belonged to the glycoside hydrolase family 42. Using SDS-PAGE, we determined that the molecular weight of lacZBa was ~75 kDa. Purified lacZBa exhibited a maximum activity at 45 °C, pH 6.0, and could be activated following incubation at 45 °C for several minutes. The half-life of lacZBa at 45 °C and 50 °C was 264 h and 36 h, respectively. While Co²⁺, Mn²⁺, Zn²⁺, Fe²⁺, Mg²⁺, and Ca²⁺ enhanced enzymatic activity, Cu²⁺ and ethylenediaminetetraacetic acid inhibited enzymatic activity. Moreover, lacZBa could hydrolyze lactose and oNPG with K_m values of 85.09 and 14.38 mM. Molecular docking results revealed that lacZBa efficiently recognized and catalyzed lactose. Additionally, the hydrolysis of lactose by lacZBa was studied in lactose solution and commercial milk. Lactose was completely hydrolyzed within 4 h with 8 U/mL of lacZBa at 45 °C. These results suggested that lacZBa identified in this study has potential applications in the dairy industry. Full article

The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10–30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT. Full article

In this study, the preparation of a milk whey-based beverage with the addition of different concentrations of hydrolyzed collagen (0.3%, 0.5%, 0.75%, and 1%) was carried out. The control was considered at a concentration of 0%. Physicochemical properties, viscosity, antioxidant activity, and microbiological parameters were evaluated. The 1% collagen treatment showed the highest protein content (9.75 ± 0.20 g/L), as well as radical inhibition for ATBS (48.30%) and DPPH (30.06%). There were no significant differences (p ≥ 0.05) in the fat and lactose parameters. However, the pH in the control treatment was lower compared to beverages treated with hydrolyzed collagen. Fourier transform-infrared spectroscopy showed spectra characteristic of lactose and collagen amides. The viscosity increased significantly as the concentration of hydrolyzed collagen increased. The addition of hydrolyzed collagen increased the bioavailability, nutritional value, and the antioxidant activity of the beverage. Hydrolyzed collagen acted as an antimicrobial agent, as there was no presence of microorganism pathogens observed in the treated beverages. Full article

A great number of factors can influence milk fermentation for yoghurt production such as fermentation conditions, starter cultures and milk characteristics. It is important for dairy companies to know the best combinations of these parameters for a controlled fermentation and for the desired qualities of yoghurt. This study investigates the use of a ¹H-NMR metabolomics approach to monitor the changes in milk during fermentation from time 0 to 24 h, taking samples every hour in the first 8 h and then at the end-point at 24 h. Three different starter cultures (L. delbrueckii ssp. bulgaricus, S. thermophilus and their combination) were used and two different heat treatments (99 or 105 °C) were applied to milk. The results clearly show the breakdown of proteins and lactose as well as the concomitant increase in acetate, lactate and citrate during fermentation. Formate is found at different initial concentrations depending on the heat treatment of the milk and its different time trajectory depends on the starter cultures: Lactobacillus cannot produce formate, but needs it for growth, whilst Streptococcus is able to produce formate from pyruvate, therefore promoting the symbiotic relationship between the two strains. On the other hand, Lactobacillus can hydrolyze milk proteins into amino acids, enriching the quality of the final product. In this way, better insight into the protocooperation of lactic acid bacteria strains and information on the impact of a greater heat treatment in the initial matrix were obtained. The global chemical view on the fermentations provided using NMR is key information for yoghurt producers and companies producing starter cultures. Full article

The endoenzyme β-galactosidase (β-d-galactoside galactohydrolase; EC 3.2.1.23) has been used at industrial scales for the preparation of lactose-free milk and for the conversion of lactose to galacto-oligosaccharides (GOS) prebiotics. In this study, using Plackett–Burman (PB) design and the response surface methodology (RSM), the batch growth conditions for the production of β-galactosidase in DeMan-Rogosa-Sharpe (MRS) media have been studied and optimized for Lactobacillus leichmannii 313 (ATCC 7830™) for the first time. The incubation temperature (30  <  T  <  55 °C), starting pH (5.5  <  pH  <  7.5), and carbon source (glucose, lactose, galactose, fructose, and sucrose) were selected as the significant variables for optimization. The maximum crude β-galactosidase production (measured by specific activity) was 4.5 U/mg proteins and was obtained after 12 h of fermentation. The results of the PB design and further optimization by RSM showed that the initial pH of 7.0 and 15.29 g/L of lactose were the levels that gave the optimum observed and predicted β-galactosidase activities of 23.13 U/mg and 23.40 U/mg, respectively. Through RSM optimization, β-galactosidase production increased significantly (over five-fold) in optimized medium (23.13 U/mg), compared with unoptimized medium (4.5 U/mg). Moreover, the crude enzyme obtained was able to hydrolyze lactose and also produce galacto-oligosaccharides. Because its ability to produce β-galactosidase was significantly improved through optimization by RSM, L. leichmannii 313 can serve as a potential source of β-galactosidase for food applications at an industrial scale. Full article