Biomolecular investigations into BBI reveal an enzymatic mechanism for PUFA isomerisation in bifidobacterium CFA bioconversion strains.

Multiple species of Bifidobacterium exhibit the ability to bioconvert conjugated fatty acids (CFAs), which is considered an important pathway for these strains to promote host health. However, there has been limited progress in understanding the enzymatic mechanism of CFA bioconversion by bifidobacteria, despite the increasing number of studies identifying CFA-producing strains. The protein responsible for polyunsaturated fatty acid (PUFA) isomerization in B. breve CCFM683 has recently been discovered and named BBI, providing a starting point for exploring Bifidobacterium isomerases (BIs). This study presents the sequence classification of membrane-bound isomerases from four common Bifidobacterium species that produce CFA. Heterologous expression, purification, and enzymatic studies of the typical sequences revealed that all possess a single c9, t11 isomer as the product and share common features in terms of enzymatic properties and catalytic kinetics. Using molecular docking and alanine scanning, Lys84, Tyr198, Asn202, and Leu245 located in the binding pocket were identified as critical to the catalytic activity, a finding further confirmed by site-directed mutagenesis-based screening assays. Overall, these findings provide insightful knowledge concerning the molecular mechanisms of BIs. This will open up additional opportunities for the use of bifidobacteria and CFAs in probiotic foods and precision nutrition.

Advances in research on microbial conjugated linoleic acid bioconversion.

Conjugated linoleic acid (CLA) is a functional food ingredient with prebiotic properties that provides health benefits for various human pathologies and disorders. However, limited natural CLA sources in animals and plants have led microorganisms like Lactobacillus and Bifidobacterium to emerge as new CLA sources. Microbial conversion of linoleic acid to CLA is mediated by linoleic acid isomerase and multicomponent enzymatic systems, with CLA production efficiency dependent on microbial species and strains. Additionally, complex factors like LA concentration, growth status, culture substrates, precursor type, prebiotic additives, and co-cultured microbe identity strongly influence CLA production and isomer composition. This review summarizes advances in the past decade regarding microbial CLA production, including bacteria and fungi. We highlight CLA production and potential regulatory mechanisms and discuss using microorganisms to enhance CLA content and nutritional value of fermented products. We also identify primary microbial CLA production bottlenecks and provide strategies to address these challenges and enhance production through functional gene and enzyme mining and downstream processing. This review aims to provide a reference for microbial CLA production and broaden the understanding of the potential probiotic role of microbial CLA producers.

Computational Analysis and Heterologous Expression of BBI-like Proteins from Food-Grade Bifidobacterium Species Reveal Possibly a Key Factor in Conjugated Linoleic Acid Bioconversion.

Bifidobacteria are well-known probiotics that can produce multiple health-promoting bioactivities, including the bioconversion of conjugated linoleic acid (CLA). However, there is a lack of insights into the genetic diversity of functional proteins in Bifidobacterium at the species level, particularly owing to the vastly different CLA conversion capabilities of these strains. Here, we performed a comprehensive bioinformatics analysis and the in vitro expression of bbi-like sequences that are widely distributed among CLA-producing Bifidobacterium strains. The BBI-like protein sequences from all four species of bifidobacterial CLA-producing strains were predicted to be stable integral membrane proteins with a transmembrane topology number of seven or nine. All BBI-like proteins were found to be expressed in the Escherichia coli BL21(DE3) hosts with a pure c9, t11-CLA-producing activity. Furthermore, their activities significantly differed in the same genetic background, and their sequence differences were indicated to be important potential factors contributing to the high activity levels in CLA-producing Bifidobacterium breve strains. The use of microorganisms, such as food-grade or industrial-grade strains, to obtain single CLA isomers will accelerate CLA-related food and nutrition research and further enrich the scientific theory of bifidobacteria as probiotics.

Heterologous expression of a novel linoleic acid isomerase BBI, and effect of fusion tags on its performance.

Functional proteins with the ability to isomerise free linoleic acid (LA) to conjugated linoleic acid (CLA) are termed linoleic acid isomerases (LAI). BBI is a novel LAI from Bifidobacterium breve with unique advantages in the production of a single CLA isomer; however, its complex membrane-bound form hampers over-expression of the protein in its natural host. To overcome this challenge, heterologous expression of BBI in Pichia pastoris was studied. Further, to investigate the influence of His-tags on the heterologous expression of BBI, three P. pastoris recombinant strains carrying either a C-terminal His-tag, an N-terminal His-tag, or none were constructed. The expression of recombinant proteins was analysed by dot and western blotting, and the enzyme activity was determined by GC-MS. The results show that the three P. pastoris recombinant strains successfully expressed the recombinant protein and had LAI activity. Compared with those BBIs without a His-tag or carrying a His-tag on the C-terminus, the BBI carrying an N-terminal His-tag had reduced expression and enzyme activity and that was also explained by the protein modelling analysis. Moreover, this study highlights the advantages of using P. pastoris for BBI expression to achieve efficient production of c9, t11-CLA monomers; the highest conversion rate of the substrate LA was over 80%, resulting in the production of 0.81 mg of c9, t11-CLA per mg of crude enzyme.

Research progress on conjugated linoleic acid bio-conversion in Bifidobacterium.

Conjugated linoleic acid (CLA), a group of 18 carbon conjugated dienoic acids, has been considered a promising food supplement owing to its various physiological benefits to human health. Owing to a high isomer selectivity of the product and a simple isolation and purification process, microbial CLA has become a research hotspot. Many food-grade bacteria such as Lactobacillus and Bifidobacterium have been reported to possess CLA-production ability. Particularly, Bifidobacterium has high bio-conversion rate and enhanced CLA production, and is one of the best and most promising CLA producers among microorganisms. Consequently, this article aimed to review the current knowledge about Bifidobacterium CLA producers, the complex factors regulating CLA production in Bifidobacterium, the role of CLA production in Bifidobacterium, and the potential mechanism underlying Bifidobacterium CLA production. In summary, the above information offers novel insights into the production of food-grade CLA as well as the rational design of health-promoting fermented foods or synbiotics.

Linoleic Acid Triggered a Metabolomic Stress Condition in Three Species of Bifidobacteria Characterized by Different Conjugated Linoleic Acid-Producing Abilities.

Abundant conjugated linoleic acid (CLA) producers exist among Bifidobacterium species. This CLA production is related to the mitigation of LA toxicity. However, there is still a lack of information on the metabolic response underlying this detoxification strategy. In this study, six bifidobacteria strains belonging to three different species were used to characterize growth and CLA accumulation in the presence of LA. A combination of non-targeted metabolomics techniques and biochemical indicators were used to explore metabolic profile changes in response to LA and the expression of important factors driving CLA production in Bifidobacterium species. The results suggested that free LA had growth inhibitory effects on bifidobacteria, resulting in a global metabolic stress response that caused metabolic reprogramming on all tested strains and promoted malondialdehyde production, inducing a redox imbalance. In particular, a strong decrease in reduced glutathione level was observed in Bifidobacterium breve CCFM683 [log2(FC) = -3.29]. Furthermore, LA-induced oxidative stress is an important factor driving high CLA production in certain strains.

Effect of the Combination of Vanillin and Chitosan Coating on the Microbial Diversity and Shelf-Life of Refrigerated Turbot (Scophthalmus maximus) Filets.

The effect of the combination of vanillin and chitosan (VC) coating on the microbiota composition and shelf-life of turbot (Scophthalmus maximus) filets during a 15-day storage period at 4 ± 1°day was investigated in this study. The control and coated fish samples were analyzed periodically for sensory and chemical attributes [total volatile basic nitrogen (TVB-N), thiobarbituric acid reactive substances (TBARS), and pH] and the presence of dominant spoilage microbiota. The results suggested that the sensory and the chemical quality of turbot filets effectively improved after treatment with vanillin (final concentration 2 mg/ml) combined with 1% chitosan, and the shelf-life was prolonged for 6 to 7 days compared with the control group. Furthermore, high-throughput sequencing showed that Proteobacteria (52.2%) and Firmicutes (29.8%) were the dominant bacteria at the phylum level in fresh turbot filets, while Pseudomonadaceae (40.2%) and Lactobacillaceae (39.4%) were the dominant bacteria at the family level in deteriorated turbot filets. However, after VC treatment, the relative abundance of Pseudomonadaceae and Lactobacillaceae decreased significantly due to the growth inhibition of potential bacteria, specifically spoilage bacteria, along with the rich bacterial diversity at the end of storage. Therefore, our data indicated that VC treatment might be effective in decreasing bacteria-induced quality deterioration and in extending the shelf-life of refrigerated turbot filets.

Methyl anthranilate: A novel quorum sensing inhibitor and anti-biofilm agent against Aeromonas sobria.

Quorum sensing (QS), bacterial cell-to-cell communication, is a gene regulatory mechanism that regulates virulence potential and biofilm formation in many pathogens. Aeromonas sobria, a common aquaculture pathogen, was isolated and identified by our laboratory from the deteriorated turbot, and its potential for virulence factors and biofilm production was regulated by QS system. In view of the interference with QS system, this study was aimed to investigate the effect of methyl anthranilate at sub-Minimum Inhibitory Concentrations (sub-MICs) on QS-regulated phenotypes in A. sobria. The results suggested that 0.5 µL/mL of methyl anthranilate evidently reduced biofilm formation (51.44%), swinging motility (74.86%), swarming motility (71.63%), protease activity (43.08%), and acyl-homoserine lactone (AHL) production. Furthermore, the real-time quantitative PCR (RT-qPCR) and in silico analysis showed that methyl anthranilate might inhibit QS system in A. sobria by interfering with the biosynthesis of AHL, as well as competitively binding with receptor protein. Therefore, our data indicated the feasibility of methyl anthranilate as a promising QS inhibitor and anti-biofilm agent for improving food safety.

Involvement of Exogenous N-Acyl-Homoserine Lactones in Spoilage Potential of Pseudomonas fluorescens Isolated From Refrigerated Turbot.

Some bacteria can modulate their spoilage potential by responding to environmental signaling molecules via the quorum sensing (QS) system. However, the ability of Pseudomonas fluorescens, the specific spoilage organism (SSO) of turbot, to response to environmental signaling molecules remains unclear. This study investigated the effects of six synthetic N-acyl homoserine lactones (AHLs) on typical behaviors mediated by QS in P. fluorescens, such as biofilm formation and extracellular protease activity. Total volatile basic nitrogen (TVB-N) was used as a spoilage indicator to evaluate quality changes in AHL-treated turbot filets during storage. The results confirm the enhancing effect of environmental AHLs on QS-dependent factors of P. fluorescens and quality deterioration of turbot filets, with C4-HSL and C14-HSL being the most effective. Moreover, the content decrease of exogenous AHLs was also validated by gas chromatography-mass spectrometry analysis. Further, changes in rhlR transcription levels in P. fluorescens suggest that this bacterium can sense environmental AHLs. Finally, molecular docking analysis demonstrates the potential interactions of RhlR protein with various exogenous AHLs. These findings strongly implicate environmental AHLs in turbot spoilage caused by P. fluorescens, suggesting preservation of turbot should not exclusively consider the elimination of SSO-secreted AHLs.

Inhibition of quorum sensing-controlled virulence factors and biofilm formation in Pseudomonas fluorescens by cinnamaldehyde.

Pseudomonas fluorescens, an important food spoiling bacteria, uses quorum sensing to control biofilm formation and motility. To date, only a few compounds targeting the LuxR-based quorum sensing system of P. fluorescens have been identified. In the present study, the quorum sensing inhibitory effect of cinnamaldehyde at sublethal concentrations was investigated in terms of inhibition of the extracellular protease, biofilm formation, and swimming and swarming motility. The total volatile basic nitrogen value was also measured to evaluate the effect of cinnamaldehyde on quality preservation of turbot fillets stored at 4 ±â€¯1 °C for 15 days. The results showed that cinnamaldehyde significantly inhibited quorum sensing-dependent factors in P. fluorescens and extended the storage life of turbot. Unexpectedly, cinnamaldehyde did not interfere with production of AHLs (N-acylhomoserine lactones) by P. fluorescens, as shown by measurement of AHL production using GC-MS. Molecular docking analysis revealed that cinnamaldehyde can interact with the LuxR-type protein of P. fluorescens, which could constitute the molecular basis of the quorum sensing inhibition observed. These findings strongly suggest that cinnamaldehyde is a quorum sensing inhibitor with great potential for the preservation of aquatic products to guarantee food safety.

Reducing Quorum Sensing-Mediated Virulence Factor Expression and Biofilm Formation in Hafnia alvei by Using the Potential Quorum Sensing Inhibitor L-Carvone.

Quorum sensing (QS), one of the most remarkable microbiological discoveries, is considered a global gene regulatory mechanism for various traits in bacteria, including virulence and spoilage. Hafnia alvei, an opportunistic pathogen and a dominant psychrophile, uses the lux-type QS system to regulate the production of virulence factors and biofilms, which are harmful to the food industry. Based on the QS interference approach, this study aimed to reveal the efficacy of L-carvone at sublethal concentrations on QS-regulated virulence factors and biofilm formation in H. alvei. QS inhibitory activity was demonstrated by the reduction in swinging motility (61.49%), swarming motility (74.94%), biofilm formation (52.41%) and acyl-homoserine lactone (AHL) production (0.5 µL/mL). Additionally, in silico analysis and RT-qPCR studies for AHL synthase HalI and QS transcriptional regulator HalR revealed a plausible molecular mechanism for QS inhibition by L-carvone. These findings suggest that L-carvone (a main component of spearmint essential oils) could be used as a novel quorum sensing inhibitor to control H. alvei in the food industry.