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1.
Food Chem ; 368: 130833, 2022 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-34425342

RESUMO

To investigate the influences of different subcritical water conditions on apple pomace pectic polysaccharides (APP) extraction, 20 samples were successfully prepared and systematically analyzed. At low temperature region (100-120 °C), extraction effect was predominant and extracted APP was high molecular weight, esterification degree and galacturonic acid content as well as light color. At middle temperature region (140 °C), the balance of extraction and degradation effects was reached and led to the highest APP yield (14.89%). At high temperature region (160-180 °C), degradation effect was predominant and led to serious degradation of APP and more extraction of co-extracts, which endowed the APP with low viscosity and good antioxidant activities in vitro. Overall, the relationship between different subcritical water conditions and APP properties are preliminarily illuminated, which not only provides a promising way for directed extraction of specific APP, but also promotes the potential application of subcritical water to commercial pectin.


Assuntos
Malus , Pectinas , Polissacarídeos , Viscosidade , Água
2.
J Agric Food Chem ; 69(51): 15716-15727, 2021 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-34918923

RESUMO

T-2 toxin is a trichothecene mycotoxin commonly found in animal feed and agricultural products. Evidence indicates that T-2 toxin induces apoptosis and autophagy. This study investigated the role of ferroptosis in T-2 toxin cytotoxicity. RAS-selective lethal compound 3 (RSL3) and Erastin were applied to initiate ferroptosis. RSL3- and Erastin-initiated cell death were enhanced by T-2 toxin. Treatment with the ferroptosis inhibitor ferrostatin-1 markedly restored the sensitizing effect of T-2 toxin to RSL3- or Erastin-initiated apoptosis, suggesting that ferroptosis plays a vital role in T-2 toxin-induced cytotoxicity. Mechanistically, T-2 toxin promoted ferroptosis by inducing lipid reactive oxygen species (ROS), as N-acetyl-l-cysteine significantly blocked T-2 toxin-induced ferroptosis. Moreover, T-2 toxin decreased the expression of solute carrier family 7 member 11 (SLC7A11) and failed to further enhance ferroptosis in SLC7A11-deficient cells. SLC7A11 overexpression significantly rescued the enhanced ferroptosis caused by T-2 toxin. T-2 toxin induces ferroptosis by downregulating SLC7A11 expression. Ferroptosis mediates T-2 toxin-induced cytotoxicity by increasing ROS and downregulating SLC7A11 expression.


Assuntos
Ferroptose , Toxina T-2 , Animais , Apoptose , Lipídeos , Espécies Reativas de Oxigênio , Toxina T-2/toxicidade
3.
J Agric Food Chem ; 2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34747603

RESUMO

Companilactobacillus crustorum usually serves as a starter culture for the food industry. Recent studies revealed that this species also possesses probiotic properties. Genome engineering, including point mutation or gene deletion, is desired to understand the mechanisms of its probiotic and fermentation properties. To tackle the hurdle in genetic manipulation in C. crustorum, here, we established a fast and easy CRISPR/Cas9-based platform for precise genome editing in this species. The platform includes two CRISPR/Cas9 systems and a CRISPR/Cas9-based editing system. Using the developed methods, we were able to knockout 12 genes in C. crustorum by deleting a fragment located in the open reading frames. The editing efficiency ranged from 14.3 to 100%. Moreover, we developed a CRISPR-assisted cytidine base-editing system, enabling programmed C to T conversion in the chromosome for gene inactivation or point mutation. To further exploit this platform, we investigated the role of nine putative bacteriocin-encoding genes and found that bacteriocins BM173 and BM1157 mostly contributed to the antimicrobial activity of C. crustorum MN047 against Staphylococcus aureus and Escherichia coli. In addition, the regulation of bacteriocin expression was also revealed to be linked with the quorum-sensing modulator luxS. This work will dramatically accelerate the genetic engineering of C. crustorum and close-related species.

4.
Artigo em Inglês | MEDLINE | ID: mdl-34709598

RESUMO

Listeria monocytogenes is a well-known foodborne pathogen that usually lives as biofilm to cope with unfavorable surroundings. Bacteriocins have been reported as antimicrobial compounds, and their bactericidal actions have been extensively studied, but their antibiofilm actions have rarely been studied. Previous study indicated that bacteriocin BMP32r has a broad-spectrum antibacterial activity. In this study, the efficacy of BMP32r against the planktonic bacteria, inhibition of forming biofilm, destruction of mature biofilm, and kill persisters of L. monocytogenes ATCC 15,313 was determined. BMP32r exhibited the bactericidal effect on L. monocytogenes planktonic bacteria. Crystal violet staining showed that sub-minimum inhibitory concentrations (SICs) of BMP32r (1/32 × MIC and 1/16 × MIC) significantly (p < 0.001) inhibit the biofilm formation. In addition, the results of CCK-8, plate count, ruthenium red staining, scanning electron microscopy, and real-time quantitative PCR assay showed that SICs of BMP32r reduced cell adhesion, exopolysaccharide production, quorum sensing, and virulence genes expression in biofilm formation. Moreover, higher concentrations of BMP32r (2 × MIC and 4 × MIC) disrupt the mature biofilm by killing the bacteria in the biofilm and kill L. monocytogenes persisters bacteria effectively. Therefore, BMP32r has promising potential as an antibiofilm agent to combat L. monocytogenes.

5.
Eur J Nutr ; 2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34185157

RESUMO

PURPOSE: Gut microbiota has been reported to contribute to either prevent or promote colorectal cancer (CRC), and treatment with probiotics might be a promising intervention method. The present study aimed to evaluate the potential anti-CRC effects of Lactobacillus coryniformis MXJ32 on a colitis-associated (CA)-CRC mouse model. METHODS: The CA-CRC mouse model was induced by a single intraperitoneal injection of 10 mg/kg azoxymethane and followed by three 7-day cycles of 2% dextran sulfate sodium in drinking water with a 14-day recovery period. Mice were supplemented with L. coryniformis MXJ32 by oral gavage (1 × 109 CFU/day/mouse). The CA-CRC attenuating effects of this probiotic were assessed via intestinal barrier integrity, inflammation, and gut microenvironment. RESULTS: Treatment with L. coryniformis MXJ32 could significantly inhibit the total number of tumors and the average tumor diameter. This probiotic administration prevented the damage of intestinal barrier function by enhancing the expression of tight junction proteins (Occludin, Claudin-1, and ZO-1) and recovering the loss of goblet cells. Moreover, L. coryniformis MXJ32 alleviated intestinal inflammation via down-regulating the expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-γ, and IL-17a) and chemokines (Cxcl1, Cxcl2, Cxcl3, Cxcl5, and Ccl7). In addition, L. coryniformis MXJ32 supplementation increased the abundance of some beneficial bacteria (such as SCFAs-producing bacteria, Lactobacillus, Bifidobacterium, Akkermansia, and Faecalibaculum) and decreased the abundance of some harmful bacteria (such as pro-inflammatory bacteria, Desulfovibrio and Helicobacter), which in turn attenuated the overexpression of inflammation. CONCLUSION: Lactobacillus coryniformis MXJ32 could effectively ameliorate CA-CRC via regulating intestinal microenvironment, alleviating inflammation, and intestinal barrier damage, which further suggested that L. coryniformis MXJ32 could be considered as a functional food ingredient for the alleviation of CA-CRC.

6.
J Agric Food Chem ; 69(21): 5887-5896, 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34013725

RESUMO

Egg white ovomucin (OVM) is homologically related to MUC2, the key component of colonic mucous layer. This study investigated the effects of orally administered OVM from egg white on the colonic mucosal barrier and the development of colitis using a colitis C57BL/6J mice model. The results showed that daily supplementation of 125 and 250 mg/kg BW of OVM partially relieved the villous destruction and loss of intestinal barrier integrity, and hence decreased the epithelial barrier permeability. The supplementation also reduced the secretion of proinflammatory cytokines TNF-α and IL-6. Besides, OVM administration significantly increased the relative abundance of intestinal beneficial bacteria including Lactobacilli, Faecalibaculum, Ruminococcus, etc. and further upregulated the production of bacterial metabolites such as short-chain fatty acids (SCFAs), which is a direct source of energy for the proliferation of epithelia and goblet cells. In conclusion, OVM from egg white ameliorates colitis by enhancing the intestinal barrier function and abundance of intestinal bacteria, thereby increasing the number of SCFAs.


Assuntos
Colite , Ovomucina , Animais , Bactérias/genética , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colo , Sulfato de Dextrana , Modelos Animais de Doenças , Mucosa Intestinal , Camundongos , Camundongos Endogâmicos C57BL
7.
Food Chem Toxicol ; 153: 112242, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33930482

RESUMO

The nonsteroidal estrogenic compound bisphenol A (BPA) is widely present in several industrial and medical products including plastic food containers and sealants in dentistry. There are growing concerns on the toxic effects of this compounds since BPA is known to have reproductive toxicity. This study evaluated the effects of low-dose BPA exposure on decidual stromal cells (DSCs) of mice. The results showed that although 10 nM of BPA have no significant effect on the cell viability, it alters the expression of decidualization-related genes including Prl8a2, Prl3c1, Ptgs2, and Mmp2. Moreover, we found that low-dose BPA exposure induces UPR response in DSCs. However, the expression of the three major UPR receptors (Perk, Ire 1, and Xbp1) did not change significantly. Interestingly, the expression of Luman, a novel receptor of UPR, was significantly upregulated in a dose-dependent manner. Lentivirus containing shLuman sequence was used to generate stable Luman silencing DSCs. It's showed that Luman knockdown could affect the expression of decidualization-related genes in decidual cells after BPA treatment. In summary, these results suggest that Luman plays a key role in low dose BPA-induced decidual toxicity of DSCs in mouse.


Assuntos
Compostos Benzidrílicos/toxicidade , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Decídua/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fenóis/toxicidade , Células Estromais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Sobrevivência Celular , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Feminino , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Probiotics Antimicrob Proteins ; 13(6): 1632-1643, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33851347

RESUMO

The beneficial effects of probiotics on ameliorating ulcerative colitis (UC) have attracted much attention in recent years. Nevertheless, the number of these identified probiotics is still limited. In addition, the adhesion abilities of probiotics are considered to be a key determinant for probiotic efficacy. However, the relationship between the adhesion abilities of probiotics and their role in ameliorating UC has been poorly studied to date. This study measured the adhesion abilities of four Lactobacillus strains to Caco-2 cells and their anti-adhesion effects on Caco-2 cells against pathogenic bacteria, as well as their application in ameliorating the symptoms of dextran sulfate sodium-induced UC, and further illustrated the relationship between these two potential probiotic properties of probiotics and their beneficial effects on UC. Results suggested that the adhesion abilities of the four tested Lactobacillus strains exists highly strain-specific and the mechanisms of their anti-adhesion effect on Caco-2 cells against Escherichia coli may be different. Moreover, all these strains had promising effects on ameliorating UC by reducing inflammatory response and improving the intestinal mucosal barrier function, as well as promoting the production of SCFAs. In conclusion, the four tested Lactobacillus strains can be considered as alternative dietary supplements in alleviating UC. In addition, it could be concluded that there is no significant correlation between the adhesion abilities of probiotics and their role in ameliorating UC, which further illustrated that the adhesion properties of probiotics in vitro may not be suitable as the key criterion for screening potential strains with UC-alleviating effects.

9.
Compr Rev Food Sci Food Saf ; 20(1): 863-899, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33443793

RESUMO

Bacteriocins are generally considered as low-molecular-weight ribosomal peptides or proteins synthesized by G+ and G- bacteria that inhibit or kill other related or unrelated microorganisms. However, low yield is an important factor restricting the application of bacteriocins. This paper reviews mining methods, heterologous expression in different systems, the purification technologies applied to bacteriocins, and identification methods, as well as the antibacterial mechanism and applications in three different food systems. Bioinformatics improves the efficiency of bacteriocins mining. Bacteriocins can be heterologously expressed in different expression systems (e.g., Escherichia coli, Lactobacillus, and yeast). Ammonium sulfate precipitation, dialysis membrane, pH-mediated cell adsorption/desorption, solvent extraction, macroporous resin column, and chromatography are always used as purification methods for bacteriocins. The bacteriocins are identified through electrophoresis and mass spectrum. Cell envelope (e.g., cell permeabilization and pore formation) and inhibition of gene expression are common antibacterial mechanisms of bacteriocins. Bacteriocins can be added to protect meat products (e.g., beef and sausages), dairy products (e.g., cheese, milk, and yogurt), and vegetables and fruits (e.g., salad, apple juice, and soybean sprouts). The future research directions are also prospected.


Assuntos
Bacteriocinas , Animais , Antibacterianos/farmacologia , Bacteriocinas/genética , Bovinos , Escherichia coli/genética , Lactobacillus , Leite
10.
Sci Total Environ ; 770: 145321, 2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-33515886

RESUMO

The conversion of lignocellulosic biomass to bioethanol is a potential approach to alleviate the energy crisis and environmental deterioration. To improve the conversion efficiency of bioethanol from wheat straw (WS), the optimization of subcritical water pretreatment and high solid hydrolysis were investigated in this study. Response surface methodology (RSM) accompanied with glucose concentration after enzymatic hydrolysis as a more reasonable response value was applied for the pretreatment optimization, and the optimum conditions were obtained as 220.51 °C of extraction temperature, 22.01 min of extraction time and 2.50% (w/v) of substrate loading. After pretreatment, the hemicellulose decreased by 18.37%, and the cellulose and lignin increased by 25.92% and 8.81%, respectively, which were consistent with the destroyed microstructure and raised crystallinity. The high efficiency of separate hydrolysis and fermentation (SHF) was verified by five commercial cellulases, and yields of hydrolysis and fermentation were 77.85-89.59% and 93.34-96.18%, respectively. Based on the high solid (15%) hydrolysis and fermentation, the ethanol concentration was significantly improved to 37.00 g/L. Interestingly, 64.47% of lignin was accumulated in the solid residue after enzymatic hydrolysis and it did not affect the efficiency of SHF, which further suggested that subcritical water mainly affected the structure of WS rather than the removal of lignin. Therefore, subcritical water pretreatment combined with high solid hydrolysis is a more effective solution for bioethanol conversion, which is also a promising strategy to utilize all components of lignocellulosic biomass.


Assuntos
Triticum , Água , Biomassa , Fermentação , Hidrólise , Lignina/metabolismo , Triticum/metabolismo
11.
J Dairy Sci ; 104(2): 1474-1483, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33246623

RESUMO

There is an increasing demand for dairy products, but the presence of food-spoilage bacteria seriously affects the development of the dairy industry. Bacteriocins are considered to be a potential antibacterial or antibiofilm agent that can be applied as a preservative. In this study, bacteriocin BM173 was successfully expressed in the Escherichia coli expression system and purified by a 2-step method. Furthermore, it exhibited a broad-spectrum antibacterial activity, high thermal stability (121°C, 20 min), and broad pH stability (pH 3-11). Moreover, the minimum inhibitory concentration values of BM173 against E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were 14.8 µg/mL and 29.6 µg/mL, respectively. Growth and time-kill curves showed that BM173 exhibited antibacterial and bactericidal activity. The results of scanning electron microscopy and transmission electron microscopy demonstrated that BM173 increased membrane permeability, facilitated pore formation, and even promoted cell lysis. The disruption of cell membrane integrity was further verified by propidium iodide uptake and lactic dehydrogenase release. In addition, BM173 exhibited high efficiency in inhibiting biofilm formation. Therefore, BM173 has promising potential as a preservative used in the dairy industry.


Assuntos
Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Biofilmes/efeitos dos fármacos , Escherichia coli/fisiologia , Lactobacillus/química , Staphylococcus aureus/fisiologia , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Permeabilidade da Membrana Celular/efeitos dos fármacos , Conservantes de Alimentos , Testes de Sensibilidade Microbiana
12.
Int J Biol Macromol ; 164: 2162-2176, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32791267

RESUMO

Bacteriocins are a subclass of antibacterial peptides considered to be the most promising alternative to antibiotics. A large number of unknown bacteriocins are hidden in lactic acid bacteria. In this study, by combining the genome with LC-MS/MS, 14 novel bacteriocins produced by Lactobacillus rhamnosus LS-8 were detected. Moreover, these bacteriocins were successfully cloned via plasmid pET-28a(+) and pET-30a(+) and heterologously expressed in Escherichia coli BL21. Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 were used to confirm their antibacterial activity. Subsequently, the four bacteriocins (pH 25, S68, S81, and S137) with the strongest antibacterial ability were selected, and their expression conditions were optimized. Purification was performed by cation exchange chromatography and high performance liquid chromatography, and the active parts were collected and analyzed by mass spectrometry. The mass spectrometry analysis revealed that peptide coverage was >71.39%. The MICs of the four bacteriocins against four pathogenic bacteria ranged from 5.38 to 19.84 µg/mL. In addition, these bacteriocins significantly inhibited the growth of four standard pathogenic bacteria. They also exhibited broad-spectrum bacteriostasis on Gram-positive and Gram-negative bacteria. Therefore, these new bacteriocins have great potential in the study of alternative antibiotics.


Assuntos
Bacteriocinas/metabolismo , Lactobacillus rhamnosus/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Cromatografia Líquida/métodos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Plasmídeos/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Espectrometria de Massas em Tandem/métodos
13.
Int J Biol Macromol ; 164: 845-854, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32702420

RESUMO

Multidrug-resistant (MDR) bacterial infection still poses a serious threat to public health, therefore, effective and safe antimicrobial agents are urgently needed. In this study, recombinant bacteriocin BMP32 (BMP32r) prepared by the Escherichia coli expression system had a broad-spectrum antibacterial activity even against some MDR bacteria and its minimum inhibitory concentration ranged from 9.2 to 36.8 mg/L. Furthermore, BMP32r showed good stable performance in heat, pH and storage. Moreover, the scanning electron microscope and transmission electron microscope revealed that BMP32r killed indicator strains through cell wall destruction, pore formation, and the membrane permeability increasing which was proved by propidium iodide uptake investigation. The wound healing of an animal MDR S. aureus infected model was promoted by BMP32r, and the safety was verified by the cytotoxicity assay that the viability of HFF cells remained 87.3% in even when the concentration of BMP32r was as high as 147.2 mg/L. In addition, no abnormalities or damages to major organs was found in vivo assessments after treatment with BMP32r. In conclusion, BMP32r has great potential to be developed as a safe antimicrobial agent to treat MDR bacterial infections.


Assuntos
Anti-Infecciosos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Bacteriocinas/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Anti-Infecciosos/química , Infecções Bacterianas/microbiologia , Bacteriocinas/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Humanos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Cicatrização/efeitos dos fármacos
14.
Braz J Microbiol ; 51(4): 2033-2048, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32537676

RESUMO

Bacteriocins are ribosomally synthesized peptides with antibacterial activity against food-borne pathogenic bacteria that cause spoilage, possessing important potential for use as a natural preservative in the food industry. The novel bacteriocin BM1300 produced by Lactobacillus crustorum MN047 was identified after purification in this study. It displayed broad-spectrum antibacterial activity against some selected Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration (MIC) values of BM1300 against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were 13.4 µg/mL and 6.7 µg/mL, respectively. Moreover, BM1300 showed excellent thermal (between 60 and 120 °C), pH (2-11), and chemical (Tween-40, Tween-80, Triton X-100, and EDTA) stabilities. Time-kill curves revealed that BM1300 exhibited bactericidal activity against S. aureus and E. coli. The scanning and transmission electron microscopy indicated that BM1300 acted by disrupting the cell membrane integrity and increasing cell membrane permeabilization of indicator bacteria. The disruption of cell membrane integrity caused by BM1300 was further demonstrated by the uptake of propidium iodide (PI) and the release of intracellular lactate dehydrogenase (LDH) and nucleic acid and proteins. Moreover, BM1300 affected cell cycle distribution to exert antibacterial activity collaboratively. Meanwhile, BM1300 inhibited the growth of S. aureus and E. coli of beef meat and improved the microbiological quality of beef meat. These findings place BM1300 as a potential biopreservative in the food industry.


Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Lactobacillus/química , Animais , Antibacterianos/classificação , Eritrócitos/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise , Camundongos , Testes de Sensibilidade Microbiana
15.
J Food Sci ; 85(5): 1523-1535, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32282078

RESUMO

Bacteriocins are defined as ribosomally synthesized antibacterial peptides/proteins that either kill or inhibit the growth of other bacteria. In the present study, the physicochemical properties, mode of action, and potential use in food preservation of a novel bacteriocin BM1122 from Lactobacillus crustorum MN047 were studied. It exhibited a broad inhibitory spectrum against selected Gram-positive and Gram-negative bacteria. Kinetic curves revealed efficient time-dependent bactericidal activity. Moreover, BM1122 possessed low hemolytic activity and good thermal stability between 60 and 120 °C. It was resistant to a wide range of pH (2 to 11) and proteinases. The scanning and transmission electron microscopy showed that BM1122 led to plasmolysis of Staphylococcus aureus and pore formation in Escherichia coli. Flow cytometric analysis demonstrated that BM1122 destroyed cell membrane integrity. Additionally, BM1122 could also inhibit biofilm formation and disturb the normal cell cycles of S. aureus and E. coli. Finally, BM1122 may enhance the inhibition of S. aureus and E. coli on beef meat stored at 4 °C for a duration of 10 days. These findings indicated that BM1122 had the potential for use as a natural preservative in the food industry. PRACTICAL APPLICATION: Fresh raw meats are highly perishable products. Bacteriocin BM1122 with a broad antibacterial spectrum can inhibit the growth of microorganisms in beef meat during refrigerated storage.


Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Lactobacillus/química , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Bacteriocinas/química , Bacteriocinas/metabolismo , Bovinos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Conservação de Alimentos , Lactobacillus/metabolismo , Carne Vermelha/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
16.
Front Microbiol ; 11: 281, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32174898

RESUMO

The composting ecosystem provides a potential resource for finding new microorganisms with the capability for cellulose degradation. In the present study, Congo red method was used for the isolating of thermostable lignocellulose-degrading bacteria from chicken manure compost. A thermophilic strain named as Geobacillus thermodenitrificans Y7 with acid-resident property was successfully isolated and employed to degrade raw switchgrass at 60°C for 5 days, which resulted in the final degradation rates of cellulose, xylan, and acid-insoluble lignin as 18.64, 12.96, and 17.21%, respectively. In addition, GC-MS analysis about aromatic degradation affirm the degradation of lignin by G. thermodenitrificans Y7. Moreover, an endocellulase gene belong to M42 family was successfully cloned from G. thermodenitrificans Y7 and expressed in Escherichia coli BL21. Recombinant enzyme Cel-9 was purified by Ni-NTA column based the His-tag, and the molecular weight determined as 40.4 kDa by SDA-PAGE. The characterization of the enzyme Cel-9 indicated that the maximum enzyme activity was realized at 50°C and pH 8.6 and, Mn2+ could greatly improve the CMCase enzyme activity of Cel-9 at 10 mM, which was followed by Fe2+ and Co2+. Besides, it also found that the ß-1,3-1,4, ß-1,3, ß-1,4, and ß-1,6 glucan linkages all could be hydrolyzed by enzyme Cel-9. Finally, during the application of enzyme Cel-9 to switchgrass, the saccharification rates achieved to 1.81 ± 0.04% and 2.65 ± 0.03% for 50 and 100% crude enzyme, respectively. All these results indicated that both the strain G. thermodenitrificans Y7 and the recombinant endocellulase Cel-9 have the potential to be applied to the biomass industry.

17.
AMB Express ; 10(1): 42, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32140794

RESUMO

Endocellulase is a key cellulase for cellulosic material pretreatment in the industry by hydrolyzing long cellulose chains into short chains. To investigate the endocellulase characteristics from Bacillus subtilis 1AJ3, and increase its production yield, this paper cloned an endocellulase gene denoted CEL-5A from strain 1AJ3 and expressed in E. coli BL21 (DE3). The CEL-5A gene was sequenced with a full-length of 1500 bp, encoding a totally of 500 amino acids, and containing two domains: the GH5 family catalytic domain (CD) and the CBM3 family cellulose-binding domain (CBD). Recombinant endocellulase Cel-5A with a His-tag was purified of the Ni-NTA column, and SDS-PAGE results demonstrated that Cel-5A exhibited a molecular weight of 56.4 kDa. The maximum enzyme activity of Cel-5A was observed at pH 4.5 and 50 °C. Moreover, it was active over the broad temperature region of 30-60 °C, and stable within the pH range of 4.5-10.0. In addition, Co2+ was able to increase enzyme activity, while the majority of metal ions demonstrated stable enzyme activity under low- concentration. The substrate specificity of Cel-5A exhibited a high specific activity on the ß-1,3-1,4 glucan linkage from barley. The Michaelis-Menten constant and the maximum velocity of the recombinant Cel-5A for CMC-Na were determined as 14.87 mg/mL and 19.19 µmol/min/mg, respectively. When Cel-5A was applied to the switchgrass and coffee grounds, its color became lighter and the biomass was observed to loosen following hydrolyzation. The saccharification rate reached 12% of the total weight of switchgrass in 20 h. These properties highlight the potential application of Cel-5A as an endocellulase in the pretreatment of biomass, for example, in the coffee grounds/waste, and related industries.

18.
BMC Genomics ; 21(1): 103, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32000663

RESUMO

BACKGROUND: Luman is a member of CREB3 (cAMP responsive element-binding) subfamily of the basic leucine-zipper (bZIP) transcription factors. It may play an important regulatory role during the decidualization process since Luman was highly expressed in the decidual cells. However, the exact molecular mechanisms of how Luman regulating decidualization is unknown. RESULTS: Using an in vitro model, we prove that Luman knockdown significantly affects the decidualization process of mice endometrial stromal cells (ESCs) as the expression of two decidual markers PRL8a2 and PRL3c1 were repressed. We employed massively parallel RNA sequencing (RNA-Seq) to understand the changes in the transcriptional landscape associated with knockdown of Luman in ESCs during in vitro decidualization. We found significant dysregulation of genes related to protein processing in the endoplasmic reticulum (ER). Several genes involved in decidualization including bone morphogenetic proteins (e.g. BMP1, BMP4, BMP8A, BMP2, and BMP8B), growth factor-related genes (e.g. VEGFB, FGF10, and FGFR2), and transcription factors (IF4E, IF4A2, WNT4, WNT9A, ETS1, NOTCH1, IRX1, IDB1, IDB2, and IDB3), show altered expression. We also found that the knockdown of Luman is associated with increased expression of cell cycle-related genes including cycA1, cycB1, cycB2, CDK1, CDK2, and PLPK1, which resulted in an increased proportion of ESCs in the G1 phase. Differentially expressed genes (DEGs) were highly enriched on ECM-receptor interaction signaling, endoplasmic reticulum protein processing, focal adhesion, and PI3K-Akt signaling pathways. CONCLUSIONS: Luman knockdown results in widespread gene dysregulation during decidualization of ESCs. Genes involved in protein processing in ER, bone morphogenetic protein, growth factor, and cell cycle progression were identified as particularly important for explaining the decidual deficiency observed in this in vitro model. Therefore, this study provides clues as to the underlying mechanisms that may expand our understanding of gene regulation during decidualization.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Decídua/metabolismo , Perfilação da Expressão Gênica/veterinária , Redes Reguladoras de Genes , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Endométrio/metabolismo , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Camundongos , Análise de Sequência de RNA/veterinária
19.
BMC Genomics ; 21(1): 157, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-32050906

RESUMO

BACKGROUND: Plant growth-promoting rhizobacteria (PGPR) are good alternatives for chemical fertilizers and pesticides, which cause severe environmental problems worldwide. Even though many studies focus on PGPR, most of them are limited in plant-microbe interaction studies and neglect the pathogens affecting ruminants that consume plants. In this study, we expand the view to the food chain of grass-ruminant-human. We aimed to find biocontrol strains that can antagonize grass pathogens and mammalian pathogens originated from grass, thus protecting this food chain. Furthermore, we deeply mined into bacterial genomes for novel biosynthetic gene clusters (BGCs) that can contribute to biocontrol. RESULTS: We screened 90 bacterial strains from the rhizosphere of healthy Dutch perennial ryegrass and characterized seven strains (B. subtilis subsp. subtilis MG27, B. velezensis MG33 and MG43, B. pumilus MG52 and MG84, B. altitudinis MG75, and B. laterosporus MG64) that showed a stimulatory effect on grass growth and pathogen antagonism on both phytopathogens and mammalian pathogens. Genome-mining of the seven strains discovered abundant BGCs, with some known, but also several potential novel ones. Further analysis revealed potential intact and novel BGCs, including two NRPSs, four NRPS-PKS hybrids, and five bacteriocins. CONCLUSION: Abundant potential novel BGCs were discovered in functional protective isolates, especially in B. pumilus, B. altitudinis and Brevibacillus strains, indicating their great potential for the production of novel secondary metabolites. Our report serves as a basis to further identify and characterize these compounds and study their antagonistic effects against plant and mammalian pathogens.


Assuntos
Burkholderiales/genética , Genoma Bacteriano , Genômica , Lolium/fisiologia , Desenvolvimento Vegetal , Mineração de Dados , Genômica/métodos , Humanos , Filogenia , Desenvolvimento Vegetal/genética , Simbiose
20.
BMC Biotechnol ; 20(1): 2, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31910834

RESUMO

BACKGROUND: Cellulosic biomass degradation still needs to be paid more attentions as bioenergy is the most likely to replace fossil energy in the future, and more evaluable cellulolytic bacteria isolation will lay a foundation for this filed. Qinling Mountains have unique biodiversity, acting as promising source of cellulose-degrading bacteria exhibiting noteworthy properties. Therefore, the aim of this work was to find potential cellulolytic bacteria and verify the possibility of the cloning of cellulases from the selected powerful bacteria. RESULTS: In present study, 55 potential cellulolytic bacteria were screened and identified from the rotten wood of Qinling Mountains. Based on the investigation of cellulase activities and degradation effect on different cellulose substrates, Bacillus methylotrophicus 1EJ7, Bacillus subtilis 1AJ3 and Bacillus subtilis 3BJ4 were further applied to hydrolyze wheat straw, corn stover and switchgrass, and the results suggested that B. methylotrophicus 1EJ7 was the most preponderant bacterium, and which also indicated that Bacillus was the main cellulolytic bacteria in rotten wood. Furthermore, scanning electron microscopy (SEM) and X-ray diffraction analysis of micromorphology and crystallinity of wheat straw also verified the significant hydrolyzation. With ascertaining the target sequence of cellulase ß-glucosidase (243 aa) and endoglucanase (499 aa) were successfully heterogeneously cloned and expressed from B. methylotrophicus 1EJ7, and which performed a good effect on cellulose degradation with enzyme activity of 1670.15 ± 18.94 U/mL and 0.130 ± 0.002 U/mL, respectively. In addition, based on analysis of amino acid sequence, it found that ß-glucosidase were belonged to GH16 family, and endoglucanase was composed of GH5 family catalytic domain and a carbohydrate-binding module of CBM3 family. CONCLUSIONS: Based on the screening, identification and cellulose degradation effect evaluation of cellulolytic bacteria from rotten wood of Qinling Mountains, it found that Bacillus were the predominant species among the isolated strains, and B. methylotrophicus 1EJ7 performed best on cellulose degradation. Meanwhile, the ß-glucosidase and endoglucanase were successfully cloned and expressed from B. methylotrophicus for the first time, which provided new materials of both strain and the recombinant enzymes for the study of cellulose degradation and its application in industry.


Assuntos
Bacillus/classificação , Celulases/genética , Triticum/química , Madeira/microbiologia , Bacillus/enzimologia , Bacillus/genética , Bacillus/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Celulases/metabolismo , Clonagem Molecular , Hidrólise , Microscopia Eletrônica de Varredura , Difração de Raios X
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