Cloning and characterization of a novel mesophilic xylanase gene Fgxyn3 from Fusarium graminearum Z-1.

In order to search for high specific activity and the resistant xylanases to XIP-I and provide more alternative xylanases for industrial production, a strain of Fusarium graminearum from Triticum aestivum grains infected with filamentous fungus produced xylanases was isolated and identified. Three xylanase genes from Fusarium graminearum Z-1 were cloned and successfully expressed in E. coli and P. pastoris, respectively. The specific activities of Fgxyn1, EFgxyn2 and EFgxyn3 for birchwood xylan were 38.79, 0.85 and 243.83 U/mg in E. coli, and 40.11, 0 and 910.37 U/mg in P. pastoris, respectively. EFgxyn3 and PFgxyn3 had the similar optimum pH at 6.0 and pH stability at 5.0-9.0. However, they had different optimum temperature and thermal stability, with 30 °C for EFgxyn3 and 40 °C for PFgxyn3, and 4-35 °C for EFgxyn3 and 4-40 °C for PFgxyn3, respectively. The substrate spectrum and the kinetic parameters showed that the two xylanases also exhibited the highest xylanase activity and catalytic efficiency (kcat/km) toward birchwood xylan, with 243.83 U/mg and 61.44 mL/mg/s for EFgxyn3 and 910.37 U/mg and 910.37 mL/mg/s for PFgxyn3, respectively. This study provided a novel mesophilic xylanase with high specific activity and catalytic efficiency, thus making it a promising candidate for extensive applications in animal feed and food industry. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03973-0.

Directed Modification of a GHF11 Thermostable Xylanase AusM for Enhancing Inhibitory Resistance towards SyXIP-I and Application of AusMPKK in Bread Making.

To reduce the inhibition sensitivity of a thermoresistant xylanase AusM to xylanase inhibitor protein (XIP)-type in wheat flour, the site-directed mutagenesis was conducted based on the computer-aided redesign. First, fourteen single-site variants and one three-amino acid replacement variant in the thumb region of an AusM-encoding gene (AusM) were constructed and expressed in E. coli BL21(DE3), respectively, as predicted theoretically. At a molar ratio of 100:1 between SyXIP-I/xylanase, the majority of mutants were nearly completely inactivated by the inhibitor SyXIP-I, whereas AusMN127A retained 62.7% of its initial activity and AusMPKK retained 100% of its initial activity. The optimal temperature of the best mutant AusMPKK was 60 °C, as opposed to 60-65 °C for AusM, while it exhibited improved thermostability, retaining approximately 60% of its residual activity after heating at 80 °C for 60 min. Furthermore, AusMPKK at a dosage of 1000 U/kg was more effective than AusM at 4000 U/kg in increasing specific bread loaf volume and reducing hardness during bread production and storage. Directed evolution of AusM significantly reduces inhibition sensitivity, and the mutant enzyme AusMPKK is conducive to improving bread quality and extending its shelf life.

Characteristics of the microstructure and the key components of white kidney bean sourdough bread induced by mixed-strain fermentation and its influence on gut microbiota.

In this study, the effect of mixed-strain fermentation using Kluyveromyces marxianus with either Lactobacillus plantarum or Pediococcus pentosaceus on the physiochemical and nutritional properties of white kidney bean flour sourdough was investigated. The results indicated that mixed-strain fermentation reduced the anti-nutritional factors produced from the white kidney bean flour, especially in the sourdough fermented by L. plantarum and K. marxianus (WKS-LK) compared to that by P. pentosaceus and K. marxianus (WKS-JK). Meanwhile, the content of lactic acid and acetic acid and the proportion of peptides with molecular weights ranging from <500 to 5000 Da were increased in the sourdoughs (WKS-LK > WKS-JK). Compared to the control (WK), microstructural characteristics of the dough seemed to be improved in WKS-LK followed by WKS-JK in terms of their corresponding gluten network consistency. Moreover, mixed fermentation led to a reduced starch digestibility accompanied by a higher content of resistant starch and slowly digestible starch. In contrast, protein digestibility was enhanced in WKS-LK and WKS-JK sourdough breads. More importantly, the changes in gut microbiota composition, short-chain fatty acid (SCFA) production, systemic inflammation, glucose tolerance and liver tissue histopathology following 21-day consumption of the sourdough bread were also evaluated via an animal model. The intake of sourdough breads reduced the abundance of the pathogenic microbiota Escherichia shigella. In contrast, the corresponding abundance of Rikenellaceae, Akkermansiaceae, Erysipelotrichaceae, Prevotellaceae and Eubacterium coprostanoligenes was increased, followed by enhanced SCFA generation, with the highest in WKS-LK and then WKS-JK. Meanwhile, a reduced level of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α in the serum and improved glucose tolerance and liver tissue histopathology following the bread consumption were also achieved in the order of WKS-LK, then WKS-JK mice compared to WK.

Thermostability modification of ß-mannanase from Aspergillus niger via flexibility modification engineering.

Introduction: ß-Mannanases can hydrolyze mannans, which are widely available in nature. However, the optimum temperature of most ß-mannanases is too low to be directly utilized in industry. Methods: To further improve the thermostability of Anman (mannanase from Aspergillus niger CBS513.88), B-factor and Gibbs unfolding free energy change were used to modify the flexible of Anman, and then combined with multiple sequence alignment and consensus mutation to generate an excellent mutant. At last, we analyzed the intermolecular forces between Anman and the mutant by molecular dynamics simulation. Results: The thermostability of combined mutant mut5 (E15C/S65P/A84P/A195P/T298P) was increased by 70% than the wild-type Amman at 70°C, and the melting temperature (Tm) and half-life (t1/2) values were increased by 2°C and 7.8-folds, respectively. Molecular dynamics simulation showed reduced flexibility and additional chemical bonds in the region near the mutation site. Discussion: These results indicate that we obtained a Anman mutant that is more suitable for industrial application, and they also confirm that a combination of rational and semi-rational techniques is helpful for screening mutant sites.

Encapsulation of Folic Acid and α-Tocopherol in Lysozyme Particles and Their Bioaccessibility in the Presence of DNA.

Protein particles have been reported as the potential carriers for the co-encapsulation of bioactive components. In this study, lysozyme, a basic protein, was used to simultaneously encapsulate folic acid and α-tocopherol at pH 4.0. The encapsulation efficiency and loading capacity of folic acid or α-tocopherol increased with its respective concentration. Folic acid had no influence on the encapsulation of α-tocopherol. However, the encapsulation of folic acid was improved by α-tocopherol below 40 µg/mL but reduced by α-tocopherol at higher concentrations. The encapsulation by lysozyme shielded folic acid, α-tocopherol, or both partially from the attack of 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical cation. No masking effect of lysozyme encapsulation on α-tocopherol was found in DPPH antioxidant activity assay. Furthermore, the DNA coating was used to improve the dispersion of lysozyme with folic acid and α-tocopherol. The lysozyme/DNA particles with folic acid and α-tocopherol showed a homogenous size distribution of 180-220 nm with ζ-potential values between -33 and -36 mV. The release and bioaccessibility of folic acid in lysozyme/DNA with α-tocopherol were similar to that of folic acid alone, while the release of α-tocopherol was delayed and its bioaccessibility was improved by encapsulation in lysozyme/DNA with folic acid. The data gathered here would provide guidance for the use of lysozyme-based co-encapsulating carriers in the development of functional foods.

Potential Health Benefits of Yeast-Leavened Bread Containing LAB Pediococcus pentosaceus Fermented Pitaya (Hylocereus undatus): Both In Vitro and In Vivo Aspects.

This study aimed to investigate the effect of the incorporation of 0-25% pitaya (Hylocereus undatus) fermented by Pediococcus pentosaceus on physicochemical and bioactive properties of yeast-leavened wheat-mung bean bread. The results revealed that ß-glucosidase activity increased during dough proofing, which may contribute to changes in dietary fiber. Compared to wheat bread, experimental bread had an increased content of soluble dietary fiber (SDF), total phenolic, total flavonoid, and slowly digestible starch, especially in wheat-mung bean bread prepared with 15% pitaya fermentates (WMB-15F). The effect of bread consumption on systemic inflammation, glucose tolerance, and blood lipid profiles was also evaluated via a mice model. The results indicated that levels of pro-inflammatory cytokines declined and glucose tolerance improved, while LDL and HDL were positively modified compared to control. Furthermore, an increased abundance of Lactobacillus, Lachnospiraceae, and Bifidobacterium spp. was observed in WMB-15F mice. Acetic acid was the dominant short-chain fatty acids (SCFAs) in feces and serum in all groups. Total SCFAs in circulation were highest in WMB-15F mice compared to other groups. In summary, an increased abundance of beneficial gut microbiota and promoted SCFA production might be highly associated with increased SDF and the release of key phenolic compounds during dough proofing, which exerts health benefits aroused from the consumption of yeast-leavened bread.

Suitability of pitaya fruit fermented by sourdough LAB strains for bread making: its impact on dough physicochemical, rheo-fermentation properties and antioxidant, antifungal and quality performance of bread.

The objective of this study was to investigate the suitability of incorporating pitaya fruit fermented by antifungal LAB strains Lactiplantibacillus plantarum and Pediococcus pentosaceus at 1: 30 °C for 24h or 2: 31 °C for 19.5h as an ingredient with respect to bread making performance and bio-preservation effect. Underlying mechanisms related to gluten protein hydrolysis, starch hydrolysis, and yeast activity in dough were explored. The antioxidant activity, antifungal activity and bread making performance of the resulted breads were analyzed. Also, the antifungal phenolic acids in the breads were identified and quantified. Incorporation of fermented substrates in dough increased yeast activity and gas production capacity, but decreased gas retention capacity. This was attributed to increased dough acidity after incorporating fruit substrates. As a result, reducing sugar and free sulfhydryl (SH) groups increased in these doughs which indicated higher starch and gluten protein hydrolysis, respectively. However, SH groups increased at lower rate in presence of substrates fermented by L. plantarum and P. pentosaceus at condition 2 than 1. This could be due to improvement of gluten network as revealed by decreased α-helix (%) and increased ß-turn (%) in secondary gluten structures in these doughs which subsequently resulted in more homogeneous microstructural properties than in presence of unfermented substrate compared to wheat dough. Subsequently, bread specific volume increased (6.6-20.0%) in presence of fermented substrates, especially fermented by L. plantarum at (2). Moreover, bread incorporated with fermented substrates (P. pentosaceus than L. plantarum at 1 than 2) had enhanced antioxidant activities, lower fungal growth rates based on challenge tests and mold free shelf life. Antifungal phenolic acids such as gallic acids, caffeic acid, protocatechuic acid were only detected in bread incorporated with fruit substrates, and their total content higher in fermented substrates.