Search Results (27)

The aim of this article was to present the biological activity of milk components, particularly lactoferrin (LF), and techniques for its extraction and purification. Dairy products have long been recognized for their significant contributions to human health and nutrition. Recent studies indicate that dairy consumption offers various health benefits, particularly concerning bone health, metabolic wellness, and cardiovascular health. LF, abundantly present in milk, exhibits a range of health-promoting properties that are increasingly recognized for their significance in nutrition and disease prevention. The production of LF can be approached through two main avenues: extraction from milk and recombinant expression systems. Both methods present unique advantages and challenges that influence the efficiency of LF production on an industrial scale. Moreover, advances in purification and drying techniques are crucial to enhance the overall efficiency of LF production. Recent studies have focused on methods such as monolithic ion-exchange chromatography and membrane technologies to improve yield and reduce costs of LF extraction. These innovations not only facilitate the extraction but also preserve the structural integrity and the functional properties of LF. The article presents the discussion of the applications of the LF in the dairy industry, indicating its growing importance as a functional ingredient in health products. Full article

Foot-and-mouth disease virus (FMDV) and Senecavirus A (SVA) have similar pathogenic characteristics, and both are important pathogens that harm the livestock industry. Studies have shown that lactoferrin peptides can inhibit the replication of various viruses and enhance the body’s immune functions. Based on this, in the present study, we aimed to investigate the effects of bovine lactoferricin-lactoferrampin (LFCA) on replicating FMDV and SVA and to analyze its role in the cellular antioxidant response caused by viral infection; in addition, we fed mice with constructed recombinant Lactobacillus reuteri expressing LFCA. Treatment with LFCA at different stages significantly inhibited the replication of both SVA and FMDV. Pretreatment before SVA infection achieved an inhibition rate of up to 94.9%, while treatment during the FMDV replication stage achieved an inhibition rate of 74.3%. After infection with either virus, intracellular ROS and MDA levels were significantly reduced, as was GSH-Px activity. However, SOD activity showed no significant difference, compared with the virus-exposed group, and remained at a high level, suggesting an increased cellular antioxidant capacity. LFCA treatment significantly increased the transcription levels of the Nrf2, Ho-1, and Nqo1 genes. In mouse experiments, the LFCA-treated group showed significantly lower viral loads in lung and intestinal tissues, compared with the SVA infection group, validating LFCA’s protective effect against SVA infection. These findings demonstrate the potential of LFCA as an antiviral drug. Full article

Semen is a complex fluid that contains spermatozoa and also functions as a dynamic bacterial microecosystem, comprising probiotics, pathogenic bacteria, and natural antimicrobial substances. Probiotic bacteria, such as Lactobacillus and Bifidobacterium, along with pathogenic bacteria like Pseudomonas aeruginosa and Escherichia coli, play significant roles in semen preservation and reproductive health. Studies have explored the impact of pathogenic bacteria on sperm quality, providing insights into the bacterial populations in mammalian semen and their influence on sperm function. These reviews highlight the delicate balance between beneficial and harmful bacteria, alongside the role of natural antimicrobial substances that help maintain this equilibrium. Moreover, we discuss the presence and roles of antimicrobial substances in semen, such as lysozyme, secretory leukocyte peptidase inhibitors, lactoferrin, and antimicrobial peptides, as well as emerging antibacterial substances like amyloid proteins. Understanding the interactions among probiotics, pathogens, and antimicrobial agents is crucial for elucidating semen preservation and fertility mechanisms. Additionally, the potential for adding probiotic bacteria with recombinant antibacterial properties presents a promising avenue for the development of new semen extenders. This review offers updated insights to understand the equilibrium of the bacterial microecosystem in semen and points toward innovative approaches for improving semen preservation. Full article

Bovine lactoferrin is a natural iron-binding glycoprotein known for its antimicrobial, antiviral, antitumor, anti-inflammatory, and immunomodulatory properties. In this study, we artificially recombined a fragment of bovine lactoferrin with immunomodulatory and antimicrobial properties to create a novel peptide named LF-MQL. The primary objective was to investigate the effects of LF-MQL on the intestinal tract and immune cells in animals. First, we assessed the in vitro activation effects of LF-MQL on mouse peritoneal macrophages. The results indicated that LF-MQL enhanced the macrophage phagocytic activity and increased IL-1β mRNA expression without significantly affecting IL-6 mRNA levels. Next, we examined the effects of LF-MQL on mucosal immunity by administering LF-MQL orally at doses of 300 mg/kg, 30 mg/kg, and 3 mg/kg to mice. The results demonstrated that different doses of LF-MQL modulated IL-6 and IL-10 mRNA levels in the small intestine. Low doses enhanced the intestinal immune response, while higher doses reduced the inflammatory response. In conclusion, LF-MQL exerts immunomodulatory effects rather than simply boosting immune activity in animal models. Full article

Lactoferrin (LF) is a major component of human milk. LF supplementation (currently bovine) supports the immune system and helps maintain iron homeostasis in adults. No recombinant human lactoferrin (rhLF) is available for commercial food use. To determine the extent to which rhLF (Effera™) produced by Komagataella phaffii digests similarly to hmLF, a validated in vitro digestion protocol was carried out. Bovine LF (bLF) was used as an additional control, as it is approved for use in various food categories. This study compared the extent of intact protein retention and the profile of peptides released in hmLF, bLF and rhLF (each with low and high iron saturation) across simulated adult gastric and intestinal digestion using gel electrophoresis, ELISA and LC-MS. Intact LF retention across digestion was similar across LF types, but the highest iron-saturated hmLF had greater retention in the simulated gastric fluid than all other sample types. Peptides identified in digested hmLF samples strongly correlated with digested rhLF samples (0.86 < r < 0.92 in the gastric phase and 0.63 < r < 0.70 in the intestinal phase), whereas digested bLF samples were significantly different. These findings support the potential for rhLF as a food ingredient for human consumption. Full article

The effect of lactoferrin on skin was simulated using a recombinant human epidermal model. The anti-inflammatory and soothing effect of lactoferrin was verified using IL-1α and TSLP Elisa assay. The effects of lactoferrin on the expression of related genes and proteins were detected using qPCR and immunofluorescence staining. The results showed that lactoferrin can effectively enhance the Transepidermal Electrical Resistance (TEER) and inhibit the secretion of inflammatory cytokine IL-1α and TSLP. In addition, it was confirmed using qPCR that lactoferrin had high expression levels on AQP3, FLG, IVL, CLDN1 and HAS1 genes. Immunofluorescence staining confirmed that lactoferrin had high fluorescence intensity and expression in AQP3, Filaggrin and Involucrin. The results showed that lactoferrin improved the skin barrier at higher than 1.5 mg/mL. At the same time, it can have anti-inflammatory and moisturizing effects. This study provides a strong basis for the application of lactoferrin in cosmetics and daily chemical products. Full article

This study investigated the impact of incorporating various inactivated probiotic formulations, with or without recombinant lactoferrin (LF) expression, into a standard chow diet on metabolic-related disorders in obese mice. After inducing obesity through a 13-week high-fat diet followed by a standard chow diet, mice received daily oral administrations of different probiotics for 6 weeks using the oral gavage approach. These probiotic formulations consisted of a placebo (MRS), heat-inactivated Lactobacillus gasseri HM1 (HK-HM1), heat-killed LF-expression HM1 (HK-HM1/LF), sonication-killed HM1 (SK-HM1), and sonication-killed LF-expression HM1 (SK-HM1/LF). The study successfully induced obesity, resulting in worsened glucose tolerance and insulin sensitivity. Interestingly, the regular diet alone improved glucose tolerance, and the addition of inactivated probiotics further enhanced this effect, with SK-HM1/LF demonstrating the most noticeable improvement. However, while regular dietary intervention alone improved insulin sensitivity, probiotic supplementation did not provide additional benefits in this aspect. Inflammation in perirenal and epididymal fat tissues was partially alleviated by the regular diet and further improved by probiotics, particularly by SK-HM1, which showed the most significant reduction. Additionally, HK-HM1 and HK-HM1/LF supplements could contribute to the improvement of serum total triglycerides or total cholesterol, respectively. Overall, incorporating inactivated probiotics into a regular diet may enhance metabolic indices, and recombinant LF may offer potential benefits for improving glucose tolerance. Full article

DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a variant of CE7 (Colicin E7 DNase) from Escherichia coli. The resulting novel recombinant open reading frame was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 10⁵ nM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required. Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics. Full article

Lactoferrin (LF) stands as one of the extensively investigated iron-binding glycoproteins within milk, exhibiting diverse biological functionalities. The global demand for LF has experienced consistent growth. Biotechnological strategies aimed at enhancing LF productivity through microbial expression systems offer substantial cost-effective advantages and exhibit fewer constraints compared to traditional animal bioreactor technologies. This study devised a novel recombinant plasmid, wherein the AOX1 promoter was replaced with a glucose-inducible G1 promoter (P_G1) to govern the expression of recombinant porcine LF (rpLF) in Pichia pastoris GS115. High-copy-number P_G1-rpLF yeast clones were meticulously selected, and subsequent induction with 0.05 g/L glucose demonstrated robust secretion of rpLF. Scaling up production transpired in a 5 L fermenter, yielding an estimated rpLF productivity of approximately 2.8 g/L by the conclusion of glycerol-fed fermentation. A three-step purification process involving tangential-flow ultrafiltration yielded approximately 6.55 g of rpLF crude (approximately 85% purity). Notably, exceptional purity of rpLF was achieved through sequential heparin and size-exclusion column purification. Comparatively, the present glucose-inducible system outperformed our previous methanol-induced system, which yielded a level of 87 mg/L of extracellular rpLF secretion. Furthermore, yeast-produced rpLF demonstrated affinity for ferric ions (Fe³⁺) and exhibited growth inhibition against various pathogenic microbes (E. coli, S. aureus, and C. albicans) and human cancer cells (A549, MDA-MB-231, and Hep3B), similar to commercial bovine LF (bLF). Intriguingly, the hydrolysate of rpLF (rpLFH) manifested heightened antimicrobial and anticancer effects compared to its intact form. In conclusion, this study presents an efficient glucose-inducible yeast expression system for large-scale production and purification of active rpLF protein with the potential for veterinary or medical applications. Full article

The production of human recombinant proteins to be used for therapeutic or nutritional purposes must focus on obtaining a molecule that is as close as possible to the native human protein. This biotechnological tool has been documented in various studies published in recent decades, with lactoferrin being one of those that has generated the most interest, being a promising option for recombinant technology. However, stability studies including thermodynamic parameters have not been reported for recombinant lactoferrin (Lf). The objective of this work was to obtain the human recombinant protein using the yeast Komagataella phaffii to study structural changes modifying pH and temperature using circular dichroism spectroscopy (CD). Thermodynamic parameters such as ΔH, ΔS and Tm were calculated and compared with commercial human lactoferrin. We propose the potential use of CD and thermodynamic parameters as a criterion in the production of recombinant proteins to be used in the production of specialized recombinant proteins. Full article

The multifunctional antibacterial peptide lactoferricin-lactoferrampin (LFCA) is derived from bovine lactoferrin. Optimization of the fermentation process should be studied since different microorganisms have their own favorable conditions and processes for growth and the production of metabolites. In this study, the culture conditions of a recombinant strain, pPG-LFCA-E/LR-CO21 (LR-LFCA), expressing LFCA was optimized, utilizing the high-density fermentation process to augment the biomass of LimosiLactobacillus reuteri and the expression of LFCA. Furthermore, an assessment of the protective effect of LR-LFCA on intestinal inflammation induced by lipopolysaccharide (LPS) was conducted to evaluate the impact of LR-LFCA on the disease resistance of piglets. The findings of this study indicate that LR-LFCA fermentation conditions optimally include 2% inoculation volume, 36.5 °C fermentation temperature, 9% dissolved oxygen concentration, 200 revolutions/minute stirring speed, pH 6, 10 mL/h glucose flow, and 50% glucose concentration. The inclusion of fermented LR-LFCA in the diet resulted in an elevation of immunoglobulin levels, significant upregulation of tight junction proteins ZO-1 and occludin, reinforcement of the intestinal barrier function, and significant amelioration of the aberrant alterations in blood physiological parameters induced by LPS. These results offer a theoretical framework for the implementation of this micro-ecological preparation in the field of piglet production to enhance intestinal well-being. Full article

Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes. Full article

Lactoferrin (LF) is a glycoprotein found in mammalian milk, and lactoferricin is a peptide derived from LF hydrolysate. Both LF and lactoferricin (LFcin) have diverse functions that could benefit mammals. Bovine LF (BLF) and BLFcin exhibit a wide range of antimicrobial activities, but most probiotic strains are relatively resistant to their antibacterial effects. BLF and BLF hydrolysate can promote the growth of specific probiotics depending on the culture conditions, the dose of BLF or BLF-related peptides, and the probiotic strains used. BLF supplementation has been shown to modulate several central molecular pathways or genes in Lacticaseibacillus rhamnosus GG under cold conditions, which may explain the prebiotic roles of BLF. LF alone or in combination with selected probiotics can help control bacterial infections or metabolic disorders, both in animal studies and in human clinical trials. Various LF-expressing probiotics, including those expressing BLF, human LF, or porcine LF, have been developed to facilitate the combination of LFs with specific probiotics. Supplementation with LF-expressing probiotics has positive effects in animal studies. Interestingly, inactivated LF-expressing probiotics significantly improved diet-induced nonalcoholic fatty liver disease (NAFLD) in a mouse model. This review highlights the accumulated evidence supporting the use of LF in combination with selected LF-resistant probiotics or LF-expressing probiotics in the field. Full article

Background: Immunoglobulin-E(IgE)-mediated hypersensitivity to cow’s milk allergens is a frequent cause of severe and life-threatening anaphylactic reactions. Besides case histories and controlled food challenges, the detection of the IgE antibodies specific to cow’s milk allergens is important for the diagnosis of cow-milk-specific IgE sensitization. Cow´s milk allergen molecules provide useful information for the refined detection of cow-milk-specific IgE sensitization. Methods: A micro-array based on ImmunoCAP ISAC technology was developed and designated milk allergen micro-array (MAMA), containing a complete panel of purified natural and recombinant cow’s milk allergens (caseins, α-lactalbumin, β-lactoglobulin, bovine serum albumin-BSA and lactoferrin), recombinant BSA fragments, and α-casein-, α-lactalbumin- and β-lactoglobulin-derived synthetic peptides. Sera from 80 children with confirmed symptoms related to cow’s milk intake (without anaphylaxis: n = 39; anaphylaxis with a Sampson grade of 1–3: n = 21; and anaphylaxis with a Sampson grade of 4–5: n = 20) were studied. The alterations in the specific IgE levels were analyzed in a subgroup of eleven patients, i.e., five who did not and six who did acquire natural tolerance. Results: The use of MAMA allowed a component-resolved diagnosis of IgE sensitization in each of the children suffering from cow’s-milk-related anaphylaxis according to Sampson grades 1–5 requiring only 20–30 microliters of serum. IgE sensitization to caseins and casein-derived peptides was found in each of the children with Sampson grades of 4–5. Among the grade 1–3 patients, nine patients showed negative reactivity to caseins but showed IgE reactivity to alpha-lactalbumin (n = 7) or beta-lactoglobulin (n = 2). For certain children, an IgE sensitization to cryptic peptide epitopes without detectable allergen-specific IgE was found. Twenty-four children with cow-milk-specific anaphylaxis showed additional IgE sensitizations to BSA, but they were all sensitized to either caseins, alpha-lactalbumin, or beta-lactoglobulin. A total of 17 of the 39 children without anaphylaxis lacked specific IgE reactivity to any of the tested components. The children developing tolerance showed a reduction in allergen and/or peptide-specific IgE levels, whereas those remaining sensitive did not. Conclusions: The use of MAMA allows for the detection, using only a few microliters of serum, of IgE sensitization to multiple cow’s milk allergens and allergen-derived peptides in cow-milk-allergic children with cow-milk-related anaphylaxis. Full article

In this article, we review the benefits of applying bovine colostrum (BC) and lactoferrin (LF) in animal models and clinical trials that include corticosteroid application and psychic stress, treatment with non-steroid anti-inflammatory drugs (NSAIDs) and antibiotics. A majority of the reported investigations were performed with native bovine or recombinant human LF, applied alone or in combination with probiotics, as nutraceutics and diet supplements. Apart from reducing adverse side effects of the applied therapeutics, BC and LF augmented their efficacy and improved the wellness of patients. In conclusion, LF and complete native colostrum, preferably administered with probiotic bacteria, are highly recommended for inclusion in therapeutic protocols in NSAIDs and corticosteroid anti-inflammatory, as well as antibiotic, therapies. These colostrum-based products can also be of value for individuals subjected to prolonged psychophysical stress (mediated by endogenous corticosteroids), especially at high ambient temperatures (soldiers and emergency services), as well as physically active people and training athletes. They are also recommended for patients during recovery from trauma and surgery, which are always associated with severe psychophysical stress. Full article