Production of polyunsaturated fatty acids in pork backfat fermented by Mucor circinelloides.

Pork backfat (PB) contains excessive saturated fatty acids (SFAs), but lacks polyunsaturated fatty acids (PUFAs). Excessive SFAs can be used as a substrate for the growth of certain microorganisms that convert them into PUFAs and monounsaturated fatty acids (MUFAs), and the added value of PB can be enhanced. In this study, Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189 were co-cultured for conversion of PB into fermented pork backfat (FPB) with high level of PUFAs. Our results showed that the content of γ-linolenic acid (GLA) and linoleic acid (LA) in the surface of FPB reached 9.04 ± 0.14 mg/g and 107.31 ± 5.16 mg/g for 7-day fermentation, respectively. To convert the internal SFAs of PB, ultrasound combined with papain was used to promote the penetrative growth of M. circinelloides into the internal PB, and the GLA level in the third layer of fat reached 2.58 ± 0.31 mg/g FPB. The internal growth of M. circinelloides in PB was promoted by adjusting the oxygen rate and ventilation rate through the wind velocity sensor. When the oxygen rate is 2 m/s and the ventilation rate is 18 m3/h, the GLA level in the third layer of fat reached 4.13 ± 1.01 mg/g FPB. To further improve the level of PUFAs in PB, FPB was produced by M. circinelloides at 18 °C. The GLA content on the surface of FPB reached 15.73 ± 1.13 mg/g FPB, and the GLA yield in the second and third layers of fat reached 8.68 ± 1.77 mg/g FPB and 6.13 ± 1.28 mg/g FPB, the LA yield in the second and third layers of fat reached 105.45 ± 5.01 mg/g FPB and 98.46 ± 4.14 mg/g FPB, respectively. These results suggested that excessive SFAs in PB can be converted into PUFAs and provided a new technique for improving PUFAs in FPB. KEY POINTS: • This article achieved the conversion of PUFAs in pork backfat by Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189. • This article solved the internal growth of M. circinelloides CBS277.49 in pork backfat by ultrasound combined with papain. • This article proposed an innovative of promoting the internal growth of M. circinelloides and increasing the PUFAs production by oxygen ventilation in pork backfat.

Catalytic mechanisms underlying fungal fatty acid desaturases activities.

Polyunsaturated fatty acids (PUFAs) have beneficial roles in a variety of human pathologies and disorders. Owing to the limited source of PUFAs in animals and plants, microorganisms, especially fungi, have become a new source of PUFAs. In fungi, fatty acid desaturases (F-FADS) are the main enzymes that convert saturated fatty acids (SFAs) into PUFAs. Their catalytic activities and substrate specificities, which are directly dependent on the structure of the FADS proteins, determine their efficiency to convert SFAs to PUFAs. Catalytic mechanisms underlying F-FADS activities can be determined from the findings of the relationship between their structure and function. In this review, the advances made in the past decade in terms of catalytic activities and substrate specificities of the fungal FADS cluster are summarized. The relationship between the key domain(s) and site(s) in F-FADS proteins and their catalytic activity is highlighted, and the FADS cluster is analyzed phylogenetically. In addition, subcellular localization of F-FADS is discussed. Finally, we provide prospective crystal structures of F-FADSs. The findings may provide a reference for the resolution of the crystal structures of F-FADS proteins and facilitate the increase in fungal PUFA production for human health.

Recent developments in horizontal gene transfer with the adaptive innovation of fermented foods.

Horizontal gene transfer (HGT) has contributed significantly to the adaptability of bacteria, yeast and mold in fermented foods, whose evidence has been found in several fermented foods. Although not every HGT has biological significance, it plays an important role in improving the quality of fermented foods. In this review, how HGT facilitated microbial domestication and adaptive evolution in fermented foods was discussed. HGT can assist in the industrial innovation of fermented foods, and this adaptive evolution strategy can improve the quality of fermented foods. Additionally, the mechanism underlying HGT in fermented foods were analyzed. Furthermore, the critical bottlenecks involved in optimizing HGT during the production of fermented foods and strategies for optimizing HGT were proposed. Finally, the prospect of HGT for promoting the industrial innovation of fermented foods was highlighted. The comprehensive report on HGT in fermented foods provides a new trend for domesticating preferable starters for food fermentation, thus optimizing the quality and improving the industrial production of fermented foods.

Linoleic acid enrichment of cheese by okara flour and Geotrichum candidum overexpressing Δ12 fatty acid desaturase.

BACKGROUND: Mold-ripened cheeses have low levels of unsaturated fatty acids (UFAs). Geotrichum candidum is an adjunct culture for the development of Geotrichum-ripened cheese but has a low ability to produce high levels of UFAs. Δ12 fatty acid desaturase (FADS12) is a pivotal enzyme that converts oleic acid (OA) to linoleic acid (LA) and plays a vital role in UFA biosynthesis. By investigating FADS12 catalytic activity from various species with OA substrates, we found that FADS12 from Mucor circinelloides (McFADS12) had the highest catalytic activity for OA. RESULTS: In the current study, a plasmid harboring McFADS12 was constructed and overexpressed in G. candidum. Our results showed that LA production increased to 31.1 ± 1.4% in engineered G. candidum - three times higher than that in wild-type G. candidum. To enhance LA production, an exogenous substrate (OA) was supplemented, and the yield of LA was increased to 154 ± 6 mg L-1 in engineered G. candidum. Engineered G. candidum was used as an adjunct culture for Geotrichum-ripened cheese production. The LA level reached 74.3 ± 5.4 g kg-1 cheese, whereas the level of saturated fatty acids (SFAs) decreased by 9.9 ± 0.5%. In addition, the soybean byproduct (okara) was introduced into the engineered G. candidum growth and the level of LA increased to 126 ± 4 g kg-1 cheese and the percentage of UFAs:SFAs increased from 0.8:1 to 1.3:1. CONCLUSION: This study offers a suitable technology for converting SFAs to UFAs in Geotrichum-ripened cheeses and provides a novel trend for converting soybean waste into a value-added product. © 2022 Society of Chemical Industry.

Production of eicosapentaenoic acid by application of a delta-6 desaturase with the highest ALA catalytic activity in algae.

Dunaliella salina is a unicellular green alga with a high α-linolenic acid (ALA) level, but a low eicosapentaenoic acid (EPA) level. In a previous analysis of the catalytic activity of delta 6 fatty acid desaturase (FADS6) from various species, FADS6 from Thalassiosira pseudonana (TpFADS6), a marine diatom, showed the highest catalytic activity for ALA. In this study, to enhance EPA production in D. salina, FADS6 from D. salina (DsFADS6) was identified, and substrate specificities for DsFADS6 and TpFADS6 were characterized. Furthermore, a plasmid harboring the TpFADS6 gene was constructed and overexpressed in D. salina. Our results revealed that EPA production reached 21.3 ± 1.5 mg/L in D. salina transformants. To further increase EPA production, myoinositol (MI) was used as a growth-promoting agent; it increased the dry cell weight of D. salina transformants, and EPA production reached 91.3 ± 11.6 mg/L. The combination of 12% CO2 aeration with glucose/KNO3 in the medium improved EPA production to 192.9 ± 25.7 mg/L in the Ds-TpFADS6 transformant. We confirmed that the increase in ALA was optimal at 8 °C; the EPA percentage reached 41.12 ± 4.78%. The EPA yield was further increased to 554.3 ± 95.6 mg/L by supplementation with 4 g/L perilla seed meal (PeSM), 500 mg/L MI, and 12% CO2 aeration with glucose/KNO3 at varying temperatures. EPA production and the percentage of EPA in D. salina were 343.8-fold and 25-fold higher than those in wild-type D. salina, respectively. IMPORTANCE: FADS6 from Thalassiosira pseudonana, which demonstrates high catalytic activity toward α-linolenic acid, was used to enhance EPA production by Dunaliella salina. Transformation of FADS6 from Thalassiosira pseudonana into Dunaliella salina with myoinositol, CO2, low temperatures, and perilla seed meal supplementation substantially increased EPA production in Dunaliella salina to 554.3 ± 95.6 mg/L. Accordingly, D. salina could be a potential alternative source of EPA and is suitable for its large-scale production.

Application of a delta-6 desaturase with α-linolenic acid preference on eicosapentaenoic acid production in Mortierella alpina.

BACKGROUND: Delta-6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid (LA) or α-linolenic acid (ALA) in the biosynthesis of polyunsaturated fatty acids (PUFAs). In previous work, we analyzed the substrate specificity of two FADS6 enzymes from Mortierella alpina ATCC 32222 (MaFADS6) and Micromonas pusilla CCMP1545 (MpFADS6), which showed preference for LA and ALA, respectively. We also clarified the PUFA profiles in M. alpina, where these lipids were synthesized mainly via the ω6 pathway and rarely via the ω3 pathway and as a result contained low ALA and eicosapentaenoic acid (EPA) levels. RESULT: To enhance EPA production in M. alpina by favoring the ω3 pathway, a plasmid harboring the MpFADS6 gene was constructed and overexpressed in a uracil-auxotrophic strain of M. alpina using the Agrobacterium tumefaciens-mediated transformation (ATMT) method. Our results revealed that the EPA production reached 80.0 ± 15.0 and 90.4 ± 9.7 mg/L in MpFADS6 transformants grown at 28 and at 12 °C, respectively. To raise the level of ALA, free form fatty acid was used as exogenous substrate, which increased the EPA production up to 114.5 ± 12.4 mg/L. To reduce the cost of EPA production in M. alpina, peony seed oil (PSO) and peony seed meal (PSM) were used as source of ALA, and EPA production was improved to 149.3 ± 7.8 and 515.29 ± 32.66 mg/L by supplementing with 0.1 % PSO and 50 g/L PSM, respectively. The EPA yield was further increased to 588.5 ± 29.6 mg/L in a 5-L bioreactor, which resulted in a 26.2-fold increase compared to EPA production in wild-type M. alpina. In this work, we have significantly enhanced EPA production through overexpression of a FADS6 desaturase with preference for ALA, combined with supplementation of its substrate. CONCLUSION: An ALA-preferring FADS6 from M. pusilla CCMP1545 was applied to enhance EPA production in M. alpina. By exogenous addition of peony seed oil or peony seed meal, EPA production was further increased in flasks and fermenters. This research also highlights the value of peony seed meal which can be converted to a high value-added product containing EPA, and as a way to increase the EPA/AA ratio in M. alpina.

Molecular mechanism of substrate specificity for delta 6 desaturase from Mortierella alpina and Micromonas pusilla.

The ω6 and ω3 pathways are two major pathways in the biosynthesis of PUFAs. In both of these, delta 6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid or α-linolenic acid. Microbial species have different propensity for accumulating ω6- or ω3-series PUFAs, which may be determined by the substrate preference of FADS6 enzyme. In the present study, we analyzed the molecular mechanism of FADS6 substrate specificity. FADS6 cDNAs were cloned from Mortierella alpina (ATCC 32222) and Micromonas pusilla (CCMP1545) that synthesized high levels of arachidonic acid and EPA, respectively. M. alpina FADS6 (MaFADS6-I) showed substrate preference for LA; whereas, M. pusilla FADS6 (MpFADS6) preferred ALA. To understand the structural basis of substrate specificity, MaFADS6-I and MpFADS6 sequences were divided into five sections and a domain swapping approach was used to examine the role of each section in substrate preference. Our results showed that sequences between the histidine boxes I and II played a pivotal role in substrate preference. Based on our domain swapping results, nine amino acid (aa) residues were targeted for further analysis by site-directed mutagenesis. G194L, E222S, M227K, and V399I/I400E substitutions interfered with substrate recognition, which suggests that the corresponding aa residues play an important role in this process.

Structural properties and biological activities of the extracellular polysaccharide of Bacillus subtilis LZ13-4.

The remarkable functional characteristics of Bacillus subtilis extracellular polysaccharides (BSPS) are of great interest. Therefore, in the present study, BSPS was isolated and characterized to obtain two fractions, BSPS-1 and BSPS-2, respectively, and to investigate their biological activities. BSPS-1 contained fructose, glucose, and galactose (molar ratio: 25.27:43.37:31.36), while BSPS-2 contained fructose with only trace amounts of glucose, galactose, and mannose (molar ratio: 55.08:19.03:19.21:6.68), and their respective average molecular weights were 16.9 kDa and 202.67 kDa. With a 93.55 % clearance of ABTS•+ at a concentration of 2 mg/mL of BSPS-1, the antioxidant activity revealed that BSPS-1 had greater antioxidant activity than BSPS-2 and that both were concentration-dependent. The inhibitory effect on HepG2 cells demonstrated that BSPS-1 and BSPS-2 significantly inhibited the proliferation of HepG2 and increased the expression of apoptotic proteins, causing apoptosis. The inhibition rate on HepG2 cells was dose-dependent and reached 52.7 % and 40.3 % after 48 h of action. BSPS-2 and 800 µg/mL BSPS-1 growth was inhibited in the G1/G0 phase, while 200 and 400 µg/mL BSPS-1 growth was inhibited in the S phase. In conclusion, the study of the BSPS's structure and properties can offer a theoretical foundation for real-world industrial applications.

Therapeutic Potential of Bacteroides fragilis SNBF-1 as a Next-Generation Probiotic: In Vitro Efficacy in Lipid and Carbohydrate Metabolism and Antioxidant Activity.

This study explores the potential of aerotolerant Bacteroides fragilis (B. fragilis) strains as next-generation probiotics (NGPs), focusing on their adaptability in the gastrointestinal environment, safety profile, and probiotic functions. From 23 healthy infant fecal samples, we successfully isolated 56 beneficial B. fragilis strains. Notably, the SNBF-1 strain demonstrated superior cholesterol removal efficiency in HepG2 cells, outshining all other strains by achieving a remarkable reduction in cholesterol by 55.38 ± 2.26%. Comprehensive genotype and phenotype analyses were conducted, including sugar utilization and antibiotic sensitivity tests, leading to the development of an optimized growth medium for SNBF-1. SNBF-1 also demonstrated robust and consistent antioxidant activity, particularly in cell-free extracts, as evidenced by an average oxygen radical absorbance capacity value of 1.061 and a 2,2-diphenyl-1-picrylhydrazyl scavenging ability of 94.53 ± 7.31%. The regulation of carbohydrate metabolism by SNBF-1 was assessed in the insulin-resistant HepG2 cell line. In enzyme inhibition assays, SNBF-1 showed significant α-amylase and α-glucosidase inhibition, with rates of 87.04 ± 2.03% and 37.82 ± 1.36%, respectively. Furthermore, the cell-free supernatant (CFS) of SNBF-1 enhanced glucose consumption and glycogen synthesis in insulin-resistant HepG2 cells, indicating improved cellular energy metabolism. This was consistent with the observation that the CFS of SNBF-1 increased the proliferation of HepG2 cells by 123.77 ± 0.82% compared to that of the control. Overall, this research significantly enhances our understanding of NGPs and their potential therapeutic applications in modulating the gut microbiome.

Advances in fermented foods revealed by multi-omics: A new direction toward precisely clarifying the roles of microorganisms.

Fermented foods generally comprise a complex micro-ecosystem with beneficial microbiota, functional products, and special flavors and qualities that are welcomed globally. Single-omics analysis allows for a comprehensive characterization of the main microbial factors influencing the function, flavor, and quality of fermented foods. However, the species, relative abundance, viability, growth patterns, and metabolic processes of microorganisms vary with changes in processing and environmental conditions during fermentation. Furthermore, the mechanisms underlying the complex interaction among microorganisms are still difficult to completely understand and analyze. Recently, multi-omics analysis and the integration of multiple types of omics data allowed researchers to more comprehensively explore microbial communities and understand the precise relationship between fermented foods and their functions, flavors, and qualities. Multi-omics approaches might help clarify the mechanisms underpinning the fermentation processes, metabolites, and functional components of these communities. This review clarified the recent advances in the roles of microorganisms in fermented foods based on multi-omics data. Current research achievements may allow for the precise control of the whole industrial processing technology of fermented foods, meeting consumers' expectations of healthy products.

Potential of Probiotics as an Adjunct for Patients with Major Depressive Disorder.

Major depressive disorder (MDD) is an enfeebling disease with a lifetime incidence of 20%. While accumulating studies implicate a correlation between the disease and gut microbiota, data show that not every patient responded to probiotic treatments. To comprehensively assess the potential role of probiotics in MDD, this study first summarizes the current pathological hypothesis of the disease from a life-stage perspective, focuses on the potential role of "depression gut microbiota." Currently available managements are then briefly summarized and novel bio-materials having potential therapeutic effects on MDD are also evaluated. To harness the positive effect of probiotics, prebiotics, and postbiotics, clinical evidence and their applications on MDD patients are listed. Factors that may counteract the pre/probiotic applications, such as diet, physiology, gender difference, and use of antibiotics and antidepressants are also discussed. The endocannabinoid (eCBs) system may be promising targets for probiotic therapy. More evidence is needed to demonstrate the hierarchical factors in the complex network driving the disease, and probiotic can be one promising adjunct for patients with MDD.

Synergistic effect of docosahexaenoic acid or conjugated linoleic acid with caffeic acid on ameliorating oxidative stress of HepG2 cells.

Exploring the synergistic effect of docosahexaenoic acid (DHA) or conjugated linoleic acid (CLA) with caffeic acid (CA) on ameliorating oxidative stress, thereby introducing CA to DHA or CLA will contribute significantly to enhance the bioactivity. We observed that DHA or CLA with CA promoted the recovery of intact individual morphology and the decline of cavities inside the nucleus and apoptosis under the observation of confocal laser scanning microscopy and fluorescent inverted microscope. The activity of intracellular antioxidant enzymes catalase (CAT) and glutathione peroxidase (GSH-Px), lactate dehydrogenase (LDH) leakage, pyruvate and malondialdehyde and reactive oxygen species (ROS), cellular morphology, and cell cycle were analyzed. Our results showed that DHA or CLA with CA enhanced the activity of CAT and GSH-Px, decreased LDH leakage and the number of apoptotic, significantly inhibited (ROS-induced cellular injury. Cell arrest in G1 and G2 phase during cell mitosis was reduced by the measurement of flow cytometry. DHA or CLA combined with CA could markedly strengthen the free radical scavenging and endogenous antioxidant defense capacity on HepG2 cells. This study provides a new direction in the application of synergies to antioxidant compounds. PRACTICAL APPLICATION: Caffeic acid (CA) can synergize with docosahexaenoic acid (DHA) or conjugated linoleic acid (CLA) to enhance antioxidant capacity. This study highlighted an effect of ameliorating oxidative stress injury DHA or CLA with CA on HepG2 cells. The data indicated that DHA or CLA with CA might be used to relieve oxidative stress damage.

High-throughput sequencing provides new insights into the roles and implications of core microbiota present in pasteurized milk.

Residual microorganisms in dairy products are closely related to their quality deterioration and safety. Based on the minimum sterilization conditions required by Grade A Pasteurized Milk Ordinance, this study explored the microbiota present in milk products that were high temperature short time pasteurized at 72, 75, 80, 83, or 85 °C for 15 s, 20 s, and 30 s separately. Based on high-throughput sequencing results, 6 phyla and 18 genera were identified as dominant microbiota. Proteobacteria and Firmicutes were the maior bacteria in phyla, and each comprising more than 50%. Pseudomonas was account for more than 42% of all the genera detected in all samples. Moreover, the changes in flavor substances in pasteurized milk, including 16 free amino acids, 9 fatty acids, and 17 volatile compounds, were detected using principal component and multi factor analyses. The Pearson correlation coefficient analysis identified six bacteria genera as the core functional microbiota that significantly affected the flavor compounds and the safety and quality of pasteurized milk. Interestingly, Pseudomonas, Omithimimicrobium, Cyanobacteria and Corynebacterium had positive correlations with the flavor substances, whereas Streptococcus and Paeniclostridium had significant negative correlations with these substances. The results may help enhance the quality control of dairy products and can be used as indicators of microbial contamination of pasteurized dairy products.

Determination of allosteric and active sites responsible for catalytic activity of delta 12 fatty acid desaturase from Geotrichum candidum and Mortierella alpina by domain swapping.

Cheese lacks essential fatty acids (EFAs). Delta 12 fatty acid desaturase (FADS12) is a critical enzyme required for EFA biosynthesis in fermentation of the predominant strains of cheese. Previously, we identified the FADS12 gene and characterized its function for the first time in Geotrichum candidum, a dominant strain used to manufacture soft cheese with white rind. In this study, we analyzed the molecular mechanism of FADS12 function by swapping domains from Mortierella alpina and G. candidum that had, respectively, high and low oleic acid conversion rates. The results revealed three regions that are essential to this process, including regions from the end of the second transmembrane domain to the beginning of the third transmembrane domain, from the end of the third transmembrane domain to the beginning of the fourth transmembrane domain, and from the 30-amino acid from the end of the sixth transmembrane domain to the C-terminal end region. Based on our domain swapping analyses, nine pairs of amino acids including H112, S118, H156, Q161, K301, R306, E307, A309 and S323 in MaFADS12 (K123, A129, N167, M172, T302, D307, I308, E310 and D324 in GcFADS12) were identified as having a significantly effect on FADS12 catalytic efficiency, and linoleic acid and its analogues (12,13-cyclopropenoid fatty acid) were found to inhibit the catalytic activity of FADS12 and related recombinant enzymes. Furthermore, the molecular mechanism of FADS12 inhibition was analyzed. The results revealed two allosteric domains, including one domain from the N-terminal region to the beginning of the first transmembrane domain and another from the 31st amino acid from the end of the sixth transmembrane domain to the C terminus. Y4 and F398 amino acid residues from MaFADS12 and eight pairs of amino acids including G56, L60, L344, G10, Q13, S24, K326 and L344 in MaFADS12 (while Y66, F70, F345, F20, Y23, Y34, F327 and F345 in GcFADS12) played a pivotal role in FADS12 inhibition. Finally, we found that both allosteric and active sites were responsible for the catalytic activity of FADS12 at various temperatures, pH, and times. This study offers a solid theoretical basis to develop preconditioning methods to increase the rate at which GcFADS12 converts oleic and linoleic acids to produce higher levels of EFAs in cheese.

Δ12 fatty acid desaturase gene from Geotrichum candidum in cheese: molecular cloning and functional characterization.

Soft cheese with white rind lacks essential fatty acids (EFAs), and as a result its long-term consumption may lead to various kinds of cardiovascular and cerebrovascular diseases, such as hyperlipidemia, hypertension, and atherosclerosis. Geotrichum candidum is a dimorphic yeast that plays an important role in the ripening of mold cheese. A gene coding for Δ12 fatty acid desaturase, a critical bifunctional enzyme desaturating oleic acid (OA) and linoleic acid (LA) to produce LA and α-linolenic acid (ALA), respectively, was isolated from G. candidum, and then cloned and heterologously expressed in Saccharomyces cerevisiae. This gene, named GcFADS12, had an open reading frame of 1257 bp and codes for a protein of 419 amino acids with a predicted molecular mass of 47.5 kDa. Characterization showed that GcFADS12 had the ability to convert OA to LA and LA to ALA, and the conversion rates for OA and LA were 20.40 ± 0.66% and 6.40 ± 0.57%, respectively. We also found that the protein product of GcFADS12 catalyzes the conversion of the intermediate product (LA) to ALA by addition of OA as the sole substrate. The catalytic activity of GcFADS12 on OA and LA was unaffected by fatty acid concentrations. Kinetic analysis revealed that GcFADS12 had stronger affinity for the OA than for the LA substrate. This study offers a solid basis for improving the production of EFAs by G. candidum in cheese.

Characterization and comparison of metaproteomes in traditional and commercial dajiang, a fermented soybean paste in northeast China.

Dajiang is a popular Chinese fermented soybean condiment. Here, a comparative metaproteomic analysis of traditional and commercial dajiang was performed during fermentation. A total of 4250 and 1421 peptide sequences were obtained from 3493 and 1987 proteins in traditional and commercial dajiang, respectively. 4299 differentially expressed microbial proteins show a high metabolic heterogeneity between the two types of dajiang. The KEGG annotation indicated that there were some pathways related to human diseases, which suggest that some microbes in traditional dajiang fermentation may have greater food safety hazards. In combination with qualitative metabolomic analysis, we further traced metabolic intermediates and key enzymes in several main fermentation pathways of dajiang to be mainly affiliated with Penicillium, Tetracoccus and Bacillus in traditional samples, as well as Aspergilus in commercial samples. These results could provide information for the selection of strains that are more suitable to produce high quality dajiang and other fermented products.