Lactiplantibacillus plantarum: a new example of inclusion body producing bacteria.

BACKGROUND: Lactic Acid Bacteria such as Lactococcus lactis, Latilactobacillus sakei (basonym: Lactobacillus sakei) and Lactiplantibacillus plantarum (basonym: Lactobacillus plantarum) have gained importance as recombinant cell factories. Although it was believed that proteins produced in these lipopolysaccharides (LPS)-free microorganisms do not aggregate, it has been shown that L. lactis produce inclusion bodies (IBs) during the recombinant production process. These protein aggregates contain biologically active protein, which is slowly released, being a biomaterial with a broad range of applications including the obtainment of soluble protein. However, the aggregation phenomenon has not been characterized so far in L. plantarum. Thus, the current study aims to determine the formation of protein aggregates in L. plantarum and evaluate their possible applications. RESULTS: To evaluate the formation of IBs in L. plantarum, the catalytic domain of bovine metalloproteinase 9 (MMP-9cat) protein has been used as model protein, being a prone-to-aggregate (PTA) protein. The electron microscopy micrographs showed the presence of electron-dense structures in L. plantarum cytoplasm, which were further purified and analyzed. The ultrastructure of the isolated protein aggregates, which were smooth, round and with an average size of 250-300 nm, proved that L. plantarum also forms IBs under recombinant production processes of PTA proteins. Besides, the protein embedded in these aggregates was fully active and had the potential to be used as a source of soluble protein or as active nanoparticles. The activity determination of the soluble protein solubilized from these IBs using non-denaturing protocols proved that fully active protein could be obtained from these protein aggregates. CONCLUSIONS: These results proved that L. plantarum forms aggregates under recombinant production conditions. These aggregates showed the same properties as IBs formed in other expression systems such as Escherichia coli or L. lactis. Thus, this places this LPS-free microorganism as an interesting alternative to produce proteins of interest for the biopharmaceutical industry, which are obtained from the IBs in an important number of cases.

The future of recombinant host defense peptides.

The antimicrobial resistance crisis calls for the discovery and production of new antimicrobials. Host defense peptides (HDPs) are small proteins with potent antibacterial and immunomodulatory activities that are attractive for translational applications, with several already under clinical trials. Traditionally, antimicrobial peptides have been produced by chemical synthesis, which is expensive and requires the use of toxic reagents, hindering the large-scale development of HDPs. Alternatively, HDPs can be produced recombinantly to overcome these limitations. Their antimicrobial nature, however, can make them toxic to the hosts of recombinant production. In this review we explore the different strategies that are used to fine-tune their activities, bioengineer them, and optimize the recombinant production of HDPs in various cell factories.

Exploring the impact of the recombinant Escherichia coli strain on defensins antimicrobial activity: BL21 versus Origami strain.

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a ß-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality.

Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies.

A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies-IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations.

A new generation of recombinant polypeptides combines multiple protein domains for effective antimicrobial activity.

BACKGROUND: Although most of antimicrobial peptides (AMPs), being relatively short, are produced by chemical synthesis, several AMPs have been produced using recombinant technology. However, AMPs could be cytotoxic to the producer cell, and if small they can be easily degraded. The objective of this study was to produce a multidomain antimicrobial protein based on recombinant protein nanoclusters to increase the yield, stability and effectivity. RESULTS: A single antimicrobial polypeptide JAMF1 that combines three functional domains based on human α-defensin-5, human XII-A secreted phospholipase A2 (sPLA2), and a gelsolin-based bacterial-binding domain along with two aggregation-seeding domains based on leucine zippers was successfully produced with no toxic effects for the producer cell and mainly in a nanocluster structure. Both, the nanocluster and solubilized format of the protein showed a clear antimicrobial effect against a broad spectrum of Gram-negative and Gram-positive bacteria, including multi-resistant strains, with an optimal concentration between 1 and 10 µM. CONCLUSIONS: Our findings demonstrated that multidomain antimicrobial proteins forming nanoclusters can be efficiently produced in recombinant bacteria, being a novel and valuable strategy to create a versatile, highly stable and easily editable multidomain constructs with a broad-spectrum antimicrobial activity in both soluble and nanostructured format.

Exploring the use of leucine zippers for the generation of a new class of inclusion bodies for pharma and biotechnological applications.

BACKGROUND: Inclusion bodies (IBs) are biologically active protein aggregates forming natural nanoparticles with a high stability and a slow-release behavior. Because of their nature, IBs have been explored to be used as biocatalysts, in tissue engineering, and also for human and animal therapies. To improve the production and biological efficiency of this nanomaterial, a wide range of aggregation tags have been evaluated. However, so far, the presence in the IBs of bacterial impurities such as lipids and other proteins coexisting with the recombinant product has been poorly studied. These impurities could strongly limit the potential of IB applications, being necessary to control the composition of these bacterial nanoparticles. Thus, we have explored the use of leucine zippers as alternative tags to promote not only aggregation but also the generation of a new type of IB-like protein nanoparticles with improved physicochemical properties. RESULTS: Three different protein constructs, named GFP, J-GFP-F and J/F-GFP were engineered. J-GFP-F corresponded to a GFP flanked by two leucine zippers (Jun and Fos); J/F-GFP was formed coexpressing a GFP fused to Jun leucine zipper (J-GFP) and a GFP fused to a Fos leucine zipper (F-GFP); and, finally, GFP was used as a control without any tag. All of them were expressed in Escherichia coli and formed IBs, where the aggregation tendency was especially high for J/F-GFP. Moreover, those IBs formed by J-GFP-F and J/F-GFP constructs were smaller, rougher, and more amorphous than GFP ones, increasing surface/mass ratio and, therefore, surface for protein release. Although the lipid and carbohydrate content were not reduced with the addition of leucine zippers, interesting differences were observed in the protein specific activity and conformation with the addition of Jun and Fos. Moreover, J-GFP-F and J/F-GFP nanoparticles were purer than GFP IBs in terms of protein content. CONCLUSIONS: This study proved that the use of leucine zippers strategy allows the formation of IBs with an increased aggregation ratio and protein purity, as we observed with the J/F-GFP approach, and the formation of IBs with a higher specific activity, in the case of J-GFP-F IBs. Thus, overall, the use of leucine zippers seems to be a good system for the production of IBs with more promising characteristics useful for pharma or biotech applications.

Short communication: Recombinant mammary serum amyloid A3 as a potential strategy for preventing intramammary infections in dairy cows at dryoff.

Mammary serum amyloid A3 (M-SAA3) has shown potential in stimulating innate immunity during intramammary infections, at calving and at dryoff. In this study, we produced recombinant caprine M-SAA3 to test its ability to reduce intramammary infections with Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli, which are all common mastitis-producing pathogens. Recombinant production of M-SAA3 (followed by lipopolysaccharide removal to avoid lipopolysaccharide-nonspecific stimulation of the immune system) was successfully achieved. Mammary serum amyloid A3 stimulated the expression of IL-8 in a dose-dependent manner in primary mammary cultures. Although a direct killing effect on Staph. aureus by M-SAA3 was not detected, this acute phase protein was able to reduce Staph. aureus, Strep. uberis, and Strep. dysgalactiae infections by up to 50% and induced a reduction in E. coli counts of 67%. In general, the best concentration of caprine M-SAA3 for inhibiting infections was the lowest concentration tested (10 µg/mL), although higher concentrations (up to 160 µg/mL) increased its antimicrobial potential against some pathogens.

Getting value from the waste: recombinant production of a sweet protein by Lactococcus lactis grown on cheese whey.

BACKGROUND: Recent biotechnological advancements have allowed for the adoption of Lactococcus lactis, a typical component of starter cultures used in food industry, as the host for the production of food-grade recombinant targets. Among several advantages, L. lactis has the important feature of growing on lactose, the main carbohydrate in milk and a majoritarian component of dairy wastes, such as cheese whey. RESULTS: We have used recombinant L. lactis NZ9000 carrying the nisin inducible pNZ8148 vector to produce MNEI, a small sweet protein derived from monellin, with potential for food industry applications as a high intensity sweetener. We have been able to sustain this production using a medium based on the cheese whey from the production of ricotta cheese, with minimal pre-treatment of the waste. As a proof of concept, we have also tested these conditions for the production of MMP-9, a protein that had been previously successfully obtained from L. lactis cultures in standard growth conditions. CONCLUSIONS: Other than presenting a new system for the recombinant production of MNEI, more compliant with its potential applications in food industry, our results introduce a strategy to valorize dairy effluents through the synthesis of high added value recombinant proteins. Interestingly, the possibility of using this whey-derived medium relied greatly on the choice of the appropriate codon usage for the target gene. In fact, when a gene optimized for L. lactis was used, the production of MNEI proceeded with good yields. On the other hand, when an E. coli optimized gene was employed, protein synthesis was greatly reduced, to the point of being completely abated in the cheese whey-based medium. The production of MMP-9 was comparable to what observed in the reference conditions.

Trends in recombinant protein use in animal production.

Recombinant technologies have made possible the production of a broad catalogue of proteins of interest, including those used for animal production. The most widely studied proteins for the animal sector are those with an important role in reproduction, feed efficiency, and health. Nowadays, mammalian cells and fungi are the preferred choice for recombinant production of hormones for reproductive purposes and fibrolytic enzymes to enhance animal performance, respectively. However, the development of low-cost products is a priority, particularly in livestock. The study of cell factories such as yeast and bacteria has notably increased in the last decades to make the new developed reproductive hormones and fibrolytic enzymes a real alternative to the marketed ones. Important efforts have also been invested to developing new recombinant strategies for prevention and therapy, including passive immunization and modulation of the immune system. This offers the possibility to reduce the use of antibiotics by controlling physiological processes and improve the efficacy of preventing infections. Thus, nowadays different recombinant fibrolytic enzymes, hormones, and therapeutic molecules with optimized properties have been successfully produced through cost-effective processes using microbial cell factories. However, despite the important achievements for reducing protein production expenses, alternative strategies to further reduce these costs are still required. In this context, it is necessary to make a giant leap towards the use of novel strategies, such as nanotechnology, that combined with recombinant technology would make recombinant molecules affordable for animal industry.

A combination of lactic acid bacteria regulates Escherichia coli infection and inflammation of the bovine endometrium.

Uterine function in cattle is compromised by bacterial contamination and inflammation after calving. The objective of this study was to select a combination of lactic acid bacteria (LAB) to decrease endometrium inflammation and Escherichia coli infection. Primary endometrial epithelial cells were cultured in vitro to select the most favorable LAB combination modulating basal tissue inflammation and E. coli infection. Supernatants were obtained to determine expression of pro-inflammatory cytokines, and E. coli infection was evaluated after harvesting the tissue and plate counting. The selected LAB combination was tested in uterus explants to assess its capacity to modulate basal and acute inflammation (associated with E. coli infection). The combination of Lactobacillus rhamnosus, Pediococcus acidilactici, and Lactobacillus reuteri at a ratio of 25:25:2, respectively, reduced E. coli infection in vitro with (89.77%) or without basal tissue inflammation (95.10%) compared with single LAB strains. Lactic acid bacteria treatment reduced CXCL8 and IL1B expression 4.7- and 2.2-fold, respectively, under acute inflammation. Ex vivo, the tested LAB combination reduced acute inflammation under E. coli infection, decreasing IL-8, IL-1ß, and IL-6 up to 2.2-, 2.5-, and 2.2-fold, respectively. In the total inflammation model, the LAB combination decreased IL-8 1.6-fold and IL-6 1.2-fold. Ultrastructural evaluation of the tissue suggested no direct interaction between the LAB and E. coli, although pathological effects of E. coli in endometrial cells were greatly diminished or even reversed by the LAB combination. This study shows the promising potential of LAB probiotics for therapeutic use against endometrial inflammation and infection.

Influence of milk processing temperature on growth performance, nitrogen retention, and hindgut's inflammatory status and bacterial populations in a calf model.

This research communication describes a study aimed at evaluating the effects of heat treatment of milk on growth performance, N retention, and hindgut's inflammatory status and bacterial populations using young dairy calves as a model. Twenty-one Holstein calves were randomly allocated to one of three treatments: raw milk (RM), pasteurised milk (PAST), or UHT milk (UHT). Calves were submitted to a N balance study, and a biopsy from the distal colon and a faecal sample were obtained from 5 animals per treatment to determine expression of several genes and potential changes in the hindgut's bacterial population. Milk furosine content was 33-fold greater in UHT than in RM and PAST milks. Calves receiving RM grew more than those fed UHT, and urinary N excretion was greatest in calves fed UHT. Quantification of Lactobacillus was lower in calves consuming PAST or UHT, and Gram negative bacteria were greater in UHT than in PAST calves. The expression of IL-8 in the hindgut's mucosa was lowest and that of IL-10 tended to be lowest in RM calves, and expression of claudin-4 tended to be greatest in UHT calves. In conclusion, the nutritional value of UHT-treated milk may be hampered because it compromises growth and increases N excretion in young calves and may have deleterious effects on the gut's bacterial population and inflammation status.

LysJEP8: A promising novel endolysin for combating multidrug-resistant Gram-negative bacteria.

Antimicrobial resistance (AMR) is an escalating global health crisis, driven by the overuse and misuse of antibiotics. Multidrug-resistant Gram-negative bacteria, such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, are particularly concerning due to their high morbidity and mortality rates. In this context, endolysins, derived from bacteriophages, offer a promising alternative to traditional antibiotics. This study introduces LysJEP8, a novel endolysin derived from Escherichia phage JEP8, which exhibits remarkable antimicrobial activity against key Gram-negative members of the ESKAPE group. Comparative assessments highlight LysJEP8's superior performance in reducing bacterial survival rates compared to previously described endolysins, with the most significant impact observed against P. aeruginosa, and notable effects on A. baumannii and K. pneumoniae. The study found that LysJEP8, as predicted by in silico analysis, worked best at lower pH values but lost its effectiveness at salt concentrations close to physiological levels. Importantly, LysJEP8 exhibited remarkable efficacy in the disruption of P. aeruginosa biofilms. This research underscores the potential of LysJEP8 as a valuable candidate for the development of innovative antibacterial agents, particularly against Gram-negative pathogens, and highlights opportunities for further engineering and optimization to address AMR effectively.

Designing eHealth interventions for children with complex care needs requires continuous stakeholder collaboration and co-creation.

Objective: Hospital-to-home (H2H) transitions challenge families of children with medical complexity (CMC) and healthcare professionals (HCP). This study aimed to gain deeper insights into the H2H transition process and to work towards eHealth interventions for its improvement, by applying an iterative methodology involving both CMC families and HCP as end-users. Methods: For 20-weeks, the Dutch Transitional Care Unit consortium collaborated with the Amsterdam University of Applied Sciences, HCP, and CMC families. The agile SCREAM approach was used, merging Design Thinking methods into five iterative sprints to stimulate creativity, ideation, and design. Continuous communication allowed rapid adaptation to new information and the refinement of solutions for subsequent sprints. Results: This iterative process revealed three domains of care - care coordination, social wellbeing, and emotional support - that were important to all stakeholders. These domains informed the development of our final prototype, 'Our Care Team', an application tailored to meet the H2H transition needs for CMC families and HCP. Conclusion: Complex processes like the H2H transition for CMC families require adaptive interventions that empower all stakeholders in their respective roles, to promote transitional care that is anticipatory, rather than reactive. Innovation: A collaborative methodology is needed, that optimizes existing resources and knowledge, fosters innovation through collaboration while using creative digital design principles. This way, we might be able to design eHealth solutions with end-users, not just for them.

Immunomodulation Evidence of Nanostructured Recombinant Proteins in Salmonid Cells.

Recent studies have demonstrated that immune-related recombinant proteins can enhance immune function, increasing host survival against infectious diseases in salmonids. This research evaluated inclusion bodies (IBs) of antimicrobial peptides (CAMPIB and HAMPIB) and a cytokine (IL1ßIB and TNFαIB) as potential immunostimulants in farmed salmonids. For this purpose, we produced five IBs (including iRFPIB as a control), and we evaluated their ability to modulate immune marker gene expression of three IBs in the RTS11 cell line by RT-qPCR. Additionally, we characterized the scale-up of IBs production by comparing two different scale systems. The results showed that CAMPIB can increase the upregulation of tnfα, il1ß, il8, and il10, HAMPIB significantly increases the upregulation of tnfα, inos, and il10, and IL1ßIB significantly upregulated the expression of tnfα, il1ß, and cox2. A comparison of IL1ßIB production showed that the yield was greater in shake flasks than in bioreactors (39 ± 1.15 mg/L and 14.5 ± 4.08 mg/L), and larger nanoparticles were produced in shake flasks (540 ± 129 nm and 427 ± 134 nm, p < 0.0001, respectively). However, compared with its shake flask counterpart, the IL1ßIB produced in a bioreactor has an increased immunomodulatory ability. Further studies are needed to understand the immune response pathways activated by IBs and the optimal production conditions in bioreactors, such as a defined medium, fed-batch production, and mechanical bacterial lysis, to increase yield.

Antimicrobial Applications of Inclusion Bodies.

A broad number of inclusion bodies (IBs) potential uses, including biocatalysis, biocompatible nanomaterials, and nanopills for biomedicine, have been described so far. Recently, it has also been shown that they can also be used as antimicrobial agents. Here, we describe the protocol used to produce and purify IBs with antimicrobial activity at desirable yields and also an optimized and simple methodology to determine the antimicrobial activity of IBs against bacterial strains.

Design strategies for positively charged endolysins: Insights into Artilysin development.

Endolysins are bacteriophage-encoded enzymes that can specifically degrade the peptidoglycan layer of bacterial cell wall, making them an attractive tool for the development of novel antibacterial agents. The use of genetic engineering techniques for the production and modification of endolysins offers the opportunity to customize their properties and activity against specific bacterial targets, paving the way for the development of personalized therapies for bacterial infections. Gram-negative bacteria possess an outer membrane that can hinder the action of recombinantly produced endolysins. However, certain endolysins are capable of crossing the outer membrane by virtue of segments that share properties resembling those of cationic peptides. These regions increase the affinity of the endolysin towards the bacterial surface and assist in the permeabilization of the membrane. In order to improve the bactericidal effectiveness of endolysins, approaches have been implemented to increase their net charge, including the development of Artilysins containing positively charged amino acids at one end. At present, there are no specific guidelines outlining the steps for implementing these modifications. There is an ongoing debate surrounding the optimal location of positive charge, the need for a linker region, and the specific amino acid composition of peptides for modifying endolysins. The aim of this study is to provide clarity on these topics by analyzing and comparing the most effective modifications found in previous literature.

Citrus Flavonoids Supplementation as an Alternative to Replace Zinc Oxide in Weanling Pigs' Diets Minimizing the Use of Antibiotics.

Since citrus flavonoids have antioxidant and anti-inflammatory properties, it was hypothesized that these compounds would become a suitable alternative to the use of therapeutic doses of zinc oxide at weaning. A total of 252 weaned pigs ([LargeWhite × Landrace] × Pietrain) were distributed according to BW (5.7 kg ± 0.76) into 18 pens (6 pens per diet, 14 pigs/pen). Three experimental diets for the prestarter (0-14 d postweaning) and starter (15-35 d postweaning) period were prepared: (i) a nonmedicated (CON) diet, (ii) a CON diet supplemented with zinc oxide at 2500 mg/kg, amoxicillin at 0.3 mg/kg and apramycin at 0.1 mg/kg (ZnO), and (iii) CON diet with the addition of a commercial citrus flavonoid extract at 0.3 mg/kg and amoxicillin at 0.3 mg/kg (FLAV). Pig BW, ADG, ADFI, and FCR were assessed on d7, d14, and d35. Samples of intestinal tissue, cecal content, and serum were collected on day seven (18 piglets). FLAV treatment achieved greater BW and ADG during the starter and for the entire experimental period compared with the CON diet (p < 0.05), whereas ZnO pigs evidenced intermediate results. Jejunum tissue analysis showed that pigs fed the FLAV diet overexpressed genes related to barrier function, digestive enzymes, and nutrient transport compared to those pigs fed the CON diet (p < 0.05). An increase in the abundance of bacterial genera such as Succinivibrio, Turicibacter, and Mitsuokella (p < 0.05) was observed in the FLAV compared with the CON and ZnO piglets. ZnO and FLAV increased the expression of TAS2R39, while ZnO pigs also expressed greater TAS2R16 than CON (p < 0.05) in the intestine. FLAV treatment improved the gut function, possibly explaining a higher performance at the end of the nursery period. Consequently, citrus flavonoids supplementation, together with amoxicillin, is a promising alternative to the use of zinc oxide plus amoxicillin and apramycin in weanling pigs, minimizing the use of antibiotics.

A Novel Generation of Tailored Antimicrobial Drugs Based on Recombinant Multidomain Proteins.

Antibiotic resistance has exponentially increased during the last years. It is necessary to develop new antimicrobial drugs to prevent and treat infectious diseases caused by multidrug- or extensively-drug resistant (MDR/XDR)-bacteria. Host Defense Peptides (HDPs) have a versatile role, acting as antimicrobial peptides and regulators of several innate immunity functions. The results shown by previous studies using synthetic HDPs are only the tip of the iceberg, since the synergistic potential of HDPs and their production as recombinant proteins are fields practically unexplored. The present study aims to move a step forward through the development of a new generation of tailored antimicrobials, using a rational design of recombinant multidomain proteins based on HDPs. This strategy is based on a two-phase process, starting with the construction of the first generation molecules using single HDPs and further selecting those HDPs with higher bactericidal efficiencies to be combined in the second generation of broad-spectrum antimicrobials. As a proof of concept, we have designed three new antimicrobials, named D5L37ßD3, D5L37D5L37 and D5LAL37ßD3. After an in-depth exploration, we found D5L37D5L37 to be the most promising one, since it was equally effective against four relevant pathogens in healthcare-associated infections, such as methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis (MRSE) and MDR Pseudomonas aeruginosa, being MRSA, MRSE and P. aeruginosa MDR strains. The low MIC values and versatile activity against planktonic and biofilm forms reinforce the use of this platform to isolate and produce unlimited HDP combinations as new antimicrobial drugs by effective means.

Supplementing Citrus aurantium Flavonoid Extract in High-Fat Finishing Diets Improves Animal Behavior and Rumen Health and Modifies Rumen and Duodenum Epithelium Gene Expression in Holstein Bulls.

One hundred and forty-six bulls (178.2 ± 6.64 kg BW and 146.0 ± 0.60 d of age) were randomly allocated to one of eight pens and assigned to control (C) or citrus flavonoid (BF) treatments (Citrus aurantium, Bioflavex CA, HTBA, S.L.U., Barcelona, Spain, 0.4 kg per ton of Bioflavex CA). At the finishing phase, the dietary fat content of the concentrate was increased (58 to 84 g/kg DM). Concentrate intake was recorded daily, and BW and animal behavior by visual scan, fortnightly. After 168 d, bulls were slaughtered, carcass data were recorded, and rumen and duodenum epithelium samples were collected. Performance data were not affected by treatment, except for the growing phase where concentrate intake (p < 0.05) was lesser in the BF compared with the C bulls. Agonistic and sexual behaviors were more frequent (p < 0.01) in the C than in the BF bulls. In the rumen epithelium, in contrast to duodenum, gene expression of some bitter taste receptors (7, 16, 39) and other genes related to behavior and inflammation was higher (p < 0.05) in the BF compared with the C bulls. Supplementing citrus flavonoids in high-fat finishing diets to Holstein bulls reduces growing concentrate consumption and improves animal welfare.

Nondenaturing Solubilization of Inclusion Bodies from Lactic Acid Bacteria.

Since inclusion bodies (IBs) contain an important amount of properly folded and active proteins, their solubilization using nondenaturing conditions to obtain aggregation-prone proteins has gained interest. Through these conditions, the refolding step is no longer required, which avoids the usual protein yield loss after this process. Here, we reveal a simple methodology to obtain pure and active difficult-to-produce proteins using two LPS-free expression systems: Lactococcus lactis and Lactobacillus plantarum. This protocol has proven to be successful to obtain proteins which are labile and prone-to-attach (difficult to be purified from other cytoplasmic proteins) and prone-to-aggregate (difficult to be obtained in their soluble form).