Improving the inhibitory resistance of xylanase FgXyn11C from Fusarium graminearum to SyXIP-I by site-directed mutagenesis.

The aim of this study was to improve the inhibitory resistance of xylanase FgXyn11C from Fusarium graminearum to XIP in cereal flour. Site saturation mutagenesis was performed using computer-aided redesign. Firstly, based on multiple primary structure alignments, the amino acid residues in the active site architecture were identified, and specific residue T144 in the thumb region of FgXyn11C was selected for site-saturation mutagenesis. After screening, FgXyn11CT144F was selected as the best mutant, as it displayed the highest enzymatic activity and resistance simultaneously compared to other mutants. The specific activity of FgXyn11CT144F was 208.8 U/mg and it exhibited complete resistance to SyXIP-I. Compared with the wild-type, FgXyn11CT144F displayed similar activity and the most resistant against SyXIP-I. The optimal temperature and pH of the wild-type and purified FgXyn11CT144F were similar at pH 5.0 and 30 °C. Our findings provided preliminary insight into how the specific residue at position 144 in the thumb region of FgXyn11C influenced the enzymatic properties and interacted with SyXIP-I. The inhibition sensitivity of FgXyn11C was reduced through directed evolution, leading to creation of the mutant enzyme FgXyn11CT144F. The FgXyn11CT144F resistance to SyXIP-I has potential application and can also provide references for engineering other resistant xylanases of the GHF11.

Expression and characterization of ß-1,3-1,4-glucanase of Aspergillus usamii in Escherichia coli and its application in sourdough bread making.

A ß-1,3-1,4-glucanase gene (Auglu12A) from Aspergillus usamii was successfully expressed in Escherichia coli BL21(DE3). The recombinant enzyme, reAuglu12A was efficiently purified using the one-step nickel-nitrilotriacetic acid affinity chromatography. The specific activity of reAuglu12A was 694.8 U/mg, with an optimal temperature of 55°C and pH of 5.0. The reAuglu12A exhibited stability at temperatures up to 60°C and within the pH range of 4.0-5.5. The reAuglu12A hydrolytic activity was increased in the presence of metal ions, especially K+ and Na+ , whereas it exhibited a Km and Vmax of 8.35 mg/mL and 1254.02 µmol/min/mg, respectively, toward barley ß-glucan at pH 5.0 and 55°C. The addition of reAuglu12A significantly increased the specific volume (p < 0.05) and reduced crumb firmness and chewiness (p < 0.05) of wheat-barley sourdough bread during a 7-day storage period compared to the control. Overall, the quality of wheat-barley sourdough bread was improved after incorporation of reAuglu12A (especially at 3000 U/300 g). These changes were attributed to the synergistic effect of acidification by sourdough and its metabolites which provided a conducive environment for the optimal action of reAuglu12A in the degradation of ß-glucans of barley flour in sourdough. This stabilized the dough structure, thereby enhancing the quality, texture, and shelf life of the bread. These findings suggest that reAuglu12A holds promise as a candidate for ß-glucanase application in the baking industry.

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.

Rheo-Fermentation Dough Properties, Bread-Making Quality and Aroma Characteristics of Red Bean (Vigna angularis) Sourdough Induced by LAB Weissella confusa QS813 Strain Fermentation.

This study investigated the impact of in situ-formed exopolysaccharides (EPS) in red bean (Vigna angularis) sourdough fermented by Weissella confusa QS813 on dough rheo-fermentation properties, bread-making quality and aroma characteristics of red bean sourdough bread. The EPS formed in red bean sourdough and sourdough-induced acidification improved the maximum dough fermentation height, gas retention coefficient and viscoelastic properties of dough. Doughs had a lower increase rate of total SDS-soluble gluten proteins, a low decline in GMP content and similar free sulfhydryl content to wheat dough. Resultantly, breads showed declines in baking loss and hardness, increase in specific volume and lower moisture loss and staling rate after 7 days of storage. Finally, despite a reduction in the total content of aroma compounds, new aroma compounds such as acetic acid and higher contents of 3-methyl-1-butanol and 2,3-butanediol were enriched in red bean sourdough bread. Sourdough acidification probably promoted interaction of EPS with gluten or red bean proteins through bond interactions to form structures which stabilized gluten in dough and increased water-binding ability in red bean sourdough bread. This study provided a better understanding of the role of EPS in sourdough in improving bread quality and of promising strategies to address consumer demand for nutritious and clean-label products.

The Effects of Volatile Organic Compounds (VOCs) on the Formation of Heterocyclic Amines (HAs) in Meat Patties, under Different Smoking Temperatures and Durations.

In this study, UPLC-MS/MS was used to study the effects of smoking duration and temperature on the formation of heterocyclic amines (HAs) in smoke-processed meat patties. Four kinds of free HAs­including F-7,8-DiMeIQx; F-MeAαC; F-Harman and F-Norharman­and six kinds of protein-bound HAs­including B-AαC; B-7,8-DiMeIQx; B-Glu-p-1; B-MeAαC; B-Harman and B-Norharman­were detected and quantified. Among the free HAs, we observed a 23-fold content increase (p < 0.05), from 0−4 h (at 0 h and 4 h they were 4.24 ng·g−1 and 98.33 ng·g−1, respectively), and the content of the free HAs decreased to 78.80 ng·g−1, at 5 h. At the same time, the free HAs content increased from 53.52 ng·g−1, at 50 °C, to 127.16 ng·g−1, at 60 °C, and then decreased continuously. The content of the free HAs was the highest at 60 °C. For the protein-bound HAs, their content was found to generally decrease with the increase in smoking duration and temperature. However, at 5 h, the content of protein-bound HAs slightly increased to 984.2 ng·g−1. Meanwhile, at 90 °C, it increased to 1643.53 ng·g−1. Additionally, a total of 16 volatile organic compounds (VOCs) were found in all of the meat samples, of which 10 VOCs (one acid, three aldehydes and seven phenols) were significantly related to the formation of free HAs. These findings showed that all the different types of HAs were produced under low-temperature processing, which provided scientific insights into the potential generation of HAs during meat smoking processes and could be used as a reference to minimize the risks of cancer related to the consumption of smoked meat products.

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.