Low-intensity ultrasound-assisted adaptive laboratory evolution of Bacillus velezensis for enhanced production of peptides.

This work aimed to explore low-intensity ultrasound-assisted adaptive laboratory evolution (US-ALE) of Bacillus velezensis and fermentation performance of mutant strains were investigated by nitrogen transformation metabolism. Results showed ultrasound accelerated the process of adaptive evolution and enhanced cell dry weight, amylase and protease activity of mutant strains, accompanied with the improved transformation abilities of NO-3-N to NH4+-N. Compared with original strain, the total peptide-N, peptide-N (<3 kDa) and autolytic peptide-N of mutant strains increased by the maximum 23.17%, 66.07% and 30.30%, respectively, based on ideal fermentation medium. According to the actual liquid-state fermentation of soybean meal and corn gluten meal with mutant strains, the highest peptide yields of 50.63% and 23.67% were noticed in mutant strain US-ALE-BV3, accompanied with the improved amino acid composition by bacterial autolysis technology. Thus, this study showed that low-intensity ultrasound could accelerate the process of adaptive evolution and US-ALE will provide more possibilities for modifying fermentation strains.

Assessment of DNA mutagenicity induced by He-Ne laser using Salmonella typhimurium strains.

Helium-neon (He-Ne) laser mutagenesis is widely used in microbiology and plant breeding. In this study, two frameshift mutant representative strains of Salmonella typhimurium TA97a and TA98 and two base pair substitution types TA100 and TA102 were employed as model microorganisms to assess DNA mutagenicity induced by He-Ne laser (3 J·cm-2·s-1, 632.8 nm) for 10, 20, and 30 min. The results revealed that the optimal laser application was 6 h in the mid-logarithmic growth stage. Low-power He-Ne laser for short treatment inhibited cell growth, and continued treatment stimulated the metabolism. The effects of the laser on TA98 and TA100 were the most prominent. Sequencing results from 1500 TA98 revertants showed that there were 88 insertion and deletion (InDel) types in the hisD3052 gene, of which the InDels unique to laser were 21 more than that of the control. Sequencing results from 760 TA100 revertants indicated that laser treatment created Pro (CCC) in the product of the hisG46 gene more likely to be replaced by His (CAC) or Ser (TCC) than by Leu (CTC). Two unique non-classical base substitutions, CCC → TAC and CCC → CAA, also appeared in the laser group. These findings will provide a theoretical basis for further exploration of laser mutagenesis breeding. KEY POINTS: • Salmonella typhimurium served as model organism for laser mutagenesis study. • Laser promoted the occurrence of InDels in the hisD3052 gene of TA98. • Laser promoted the occurrence of base substitution in the hisG46 gene of TA100.

Studies on ultrasound-mediated insertion-deletion polymorphisms of DNA and underlying mechanisms based on Ames tester strains.

Low-lethality ultrasound technology has received more and more attention in regulating microorganisms of fermentation industry. Herein, two representative Ames tester strains TA97a and TA98 as model organisms were used to explore the effects of ultrasound on insertion-deletion (InDel) polymorphisms of microbial DNA and its underlying mechanisms. Results revealed that a promotion was observed in the reversion mutation of TA98 upon sonication. Sequencing results from 1752 TA98 revertants showed that there was a total of 127 InDels, of which the InDels unique to ultrasound were 36 more than that of the control. Compared with the control, ultrasound-mediated InDels of DNA displayed additional -29 bp deletion and +7 âˆ¼ +43 bp insertions of direct repeat sequences. Combined with the analysis of transcriptomics and prediction of secondary structure of single-stranded DNA from InDels core region (No. 832 âˆ¼ 915 bp) in hisD3052 gene of TA98 strain, ultrasound-mediated "thermal breathing" mechanism was proposed based on the formation of DNA hairpin structure with micro-homologous sequence. This finding implied that low-intensity ultrasound is expected to be developed a new low-lethal mutagenic technology for continuous mutagenesis.

Effects of ultrasound-assisted sodium bisulfite pretreatment on the preparation of cholesterol-lowering peptide precursors from soybean protein.

In order to take full advantage of the gastrointestinal digestive function, the effects of S-type ultrasound-assisted sodium bisulfite (UASB) pretreatment on the preparation of cholesterol-lowering peptide precursors derived from soybean protein were investigated and the structural characterizations of pretreated proteins were explored. UASB pretreatment with the operational mode of mono-frequency ultrasound at 28 kHz, ultrasonic power density of 200 W/L and ultrasonic time of 50 min exhibited the highest cholesterol-lowering activity (56.90%) of soybean protein hydrolysates (SPH) after simulated gastrointestinal digestion, which increased by 87.17% compared to the control. Under these conditions, the peptide content of SPH after simulated gastrointestinal digestion was not significantly different (p > 0.05) compared to the control. Further FTIR analysis showed that UASB pretreatment increased ß-turn and ß-sheet content and decreased α-helix and random coil content. The changes in the surface hydrophobicity and microstructures of soybean protein indicated that UASB pretreatment loosened soybean protein structure and exposed more hydrophobic groups. SDS-PAGE indicated that the restriction sites changed after UASB pretreatment. In conclusion, UASB pretreatment is an efficient method for the preparation of cholesterol-lowering peptide precursors.

Staple food and health: a comparative study of physiology and gut microbiota of mice fed with potato and traditional staple foods (corn, wheat and rice).

The effects of potato and traditional staple foods (corn, wheat and rice) on physiology and gut microbiota were investigated by feeding ICR mice for 12 months. Compared with traditional staple foods, potato significantly improved the food and water intake and survival rate, and inhibited the swelling of viscera of mice, accompanied by a decreased white blood cell count and urine bilirubin content. Furthermore, potato significantly increased the relative abundance of Bacteroides and Faecalibacterium, which are short-chain fatty acid producing bacteria and play very important roles in the maintenance of human health. Meanwhile, potato significantly decreased the relative abundance of spoilage bacteria Pseudomonas and Thiobacillus. Analysis of putative metagenomes indicated that the potato diet upregulated the gene abundance of glycan biosynthesis and metabolism, digestive system and immune system. These findings indicated that potato has the potential to be an excellent substitute for traditional staple foods owing to its good physiological function and favorable gut microbiota modulation.

Antihypertensive effect of rapeseed peptides and their potential in improving the effectiveness of captopril.

BACKGROUND: The main objective of this study was to evaluate the safety and antihypertensive activity of rapeseed peptides and to investigate their potential synergy with captopril. RESULTS: The peptides were nontoxic with the maximum tolerated dose exceeding 25 g kg-1 BW d-1 for mice and they had angiotensin converting enzyme (ACE) inhibitory activity with IC50 value of 1.27 mg mL-1 . Rapeseed peptides did not have a synergistic effect with captopril on inhibiting ACE activity in simulated digestion tests in vitro. But in vivo they could synergistically augment the amplitude range of lowering blood pressure with captopril by approximately 9% and prolong the antihypertensive effect duration time by over 20% in antihypertension tests of spontaneously hypertensive rats. In addition, the inhibiting effect of rapeseed peptides on ACE activity was noticeable in some rat organs in vivo. Nevertheless, when compared to captopril group, the potential synergy of rapeseed peptides with captopril did not cause a further decrease in ACE activity in the organs but their synergy further improved levels of NO (12.7%) and endothelial nitric oxide synthase (74.1%) in rat serum. Further studies of some peptides identified from rapeseed peptides showed that some of the rapeseed peptides (Cys-Leu, Val-Ala-Pro) could markedly increase contents of NO and endothelial nitric oxide synthase. CONCLUSIONS: Rapeseed peptides have antihypertensive activity and they showed potential synergy with captopril in antihypertensive performance in vivo. The synergy was not from ACE inhibition but from other pathways, like improvement in endogenous vasodilator contents. © 2020 Society of Chemical Industry.

Analysis in protein profile, antioxidant activity and structure-activity relationship based on ultrasound-assisted liquid-state fermentation of soybean meal with Bacillus subtilis.

This study investigated effects of ultrasound on the contents of peptide and soluble protein, antioxidant activity, functionalities and structural characteristics of fermented soybean meal (FSBM) with Bacillus subtilis systematically. The results showed that there were significant effects of ultrasound treatments (frequency, treatment time and power density) on the contents of peptide and soluble protein (p < 0.05). Under the optimum ultrasound conditions (power density of 0.08 W/mL, frequency of 33 kHz and treatment time of 1 h) by single factor experiment, the contents of peptide and soluble protein increased by 31.27% and 18.79% compared to those of the control, respectively. Additionally, the in vitro antioxidant activity (•OH scavenging rate, Fe2+ chelating capacity and DPPH radical scavenging rate) and functional properties (emulsifying activity and emulsifying stability) of FSBM were found to be noticeably improved by ultrasound (p < 0.05). The fourier transform infrared (FTIR) spectroscopy revealed that ultrasound caused protein molecules to unfold with a decrease content of α-helix and ß-turn and an increase in the proportion of ß-sheet and random coil. Besides, atomic force microscope (AFM) results indicated that ultrasonication generally increased the surface roughness of protein and the protein sonicated with higher frequency (≥33 kHz) exhibited a greater height compared to lower frequency ultrasonication. Structure-activity relationship analysis illustrated that there was a good linear relationship between •OH scavenging rate and ß-sheet/ß-turn with Pearson's correlation coefficient r of -0.86/0.90. Collectively, the selection of ultrasonic parameters is essential for the preparation of functional protein and bioactive peptide by enhancing fermentation of agroindustrial by-products.

Effects of ultrasound pretreatment with different frequencies and working modes on the enzymolysis and the structure characterization of rice protein.

The effects of ultrasound pretreatment with different frequencies and working modes, including mono-frequency ultrasound (MFU), dual-frequency ultrasound (DFU) and tri-frequency ultrasound (TFU), on the degree of hydrolysis (DH) of rice protein (RP) and angiotensin-I-converting enzyme (ACE) inhibitory activity of RP hydrolysate were investigated. Ultraviolet-visible (UV) spectroscopy, fourier transform infrared (FTIR) spectroscopy, surface hydrophobicity and scanning electron microscopy (SEM) of RP pretreated with ultrasound were measured. The results showed that ultrasound pretreatment did not increase DH of RP significantly (p>0.05). However, all the ultrasound pretreatment increased the ACE inhibitory activity of RP hydrolysate significantly (p<0.05). The MFU of 20kHz showed higher ACE inhibitory activity compared to that of other MFU. The ACE inhibitory activity of sequential DFU was higher than that of simultaneous with the same frequency combination. Sequential TFU of 20/35/50kHz produced the highest increase in ACE inhibitory activity in contrast with other ultrasound frequencies and working modes. All the results under ultrasound pretreatment showed that ultrasound frequencies and working modes were of great effect on the ACE inhibitory activity of RP. The changes in UV-Vis spectra and surface hydrophobicity indicated the unfolding of protein and exposure of hydrophobic groups by ultrasound. The FTIR analysis showed that all the ultrasound pretreatment with different frequencies and working modes decreased α-helix, ß-turn content and increased ß-sheet, random coil content of RP. The SEM results indicated that ultrasound pretreatment resulted in the deformation of RP. In conclusion, the frequency selection of ultrasound pretreatment of RP is essential for the preparation of ACE inhibitory peptide.

Quantitative studies on structure-ORAC relationships of anthocyanins from eggplant and radish using 3D-QSAR.

The 3-dimensional quantitative structure activity relationship (3D-QSAR) models were established from 21 anthocyanins based on their oxygen radical absorbing capacity (ORAC) and were applied to predict anthocyanins in eggplant and radish for their ORAC values. The cross-validated q(2)=0.857/0.729, non-cross-validated r(2) = 0.958/0.856, standard error of estimate = 0.153/0.134, and F = 73.267/19.247 were for the best QSAR (CoMFA/CoMSIA) models, where the correlation coefficient r(2)pred = 0.998/0.997 (>0.6) indicated a high predictive ability for each. Additionally, the contour map results suggested that structural characteristics of anthocyanins favourable for the high ORAC. Four anthocyanins from eggplant and radish have been screened based on the QSAR models. Pelargonidin-3-[(6''-p-coumaroyl)-glucosyl(2 → 1)glucoside]-5-(6''-malonyl)-glucoside, delphinidin-3-rutinoside-5-glucoside, and delphinidin-3-[(4''-p-coumaroyl)-rhamnosyl(1 → 6)glucoside]-5-glucoside potential with high ORAC based the QSAR models were isolated and also confirmed for their relative high antioxidant ability, which might attribute to the bulky and/or electron-donating substituent at the 3-position in the C ring or/and hydrogen bond donor group/electron donating group on the R1 position in the B ring.

Removal of off-flavours from radish (Raphanus sativus L.) anthocyanin-rich pigments using chitosan and its mechanism(s).

In this paper, we examined the role of chitosan in the removal of off-flavours from radish anthocyanin-rich pigments and studied the mechanisms of the process. Four radish glucosinolates (glucoraphenin, dehydroerucin, glucobrassicin, and glucoerucin) were identified by LC-MSn from root extracts and dehydroerucin was found to be the major glucosinolate in red radish roots. Application of chitosan with 76%, 83% or 89% deacetylation in radish extracts attributed to 26%, 35% or 43% adsorption rate for glucosinolates, and 28%, 26% or 22% for anthocyanins, respectively. HS-SPME/GC-MS analysis demonstrated that the concentration of volatile compounds decreased by 70%, resulting in the loss of odorous compounds. The changes in chitosan spectra before/after adsorption and after desorption at 1590 and 3360cm(-1) and at broad bands from 2600 to 2000cm(-1) suggest that the dominant adsorption mechanisms of glucosinolates on chitosan may be electrostatic attractions, including hydrogen bonds and charge neutralisation.