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Background Thermophiles can thrive at 50-80 °C and produce some enzymes with special promise for biocatalysis. A thermophilic protease-producing strain YYC4 was isolated from Yunyan cigarette and employed in solid-state fermentation (SSF) of unsterilized soybean meal (SBM). Results The isolate was identified as Bacillus licheniformis based on appearance of colonies, microscopic observation and 16S rDNA sequencing. After SSF, soluble and crude protein contents in SBM increased from 49.24 to 185.73 g kg-1 and from 404.18 to 479.46 g kg-1 , respectively, under the fermentation conditions of 107 cfu g-1 inoculation of strain YYC4, 1:1.8 (g mL-1 ) SBM to distilled water, 1.2 g kg-1 magnesium sulphate addition, 55 °C and 48 h. During fermentation, pH of the medium increased from 6.30 to 9.09 and protease activity especially neutral protease increased significantly from 13.5 to 181.31 U g-1 . Meanwhile, trypsin inhibitor (TI) activity was decreased from 8.19 to 3.19 mg g-1 . The safety of fermented SBM (FSBM) was verified by acute toxicity animal experiment. Analysis of microbial community in FSBM showed that Bacillus licheniformis YYC4 as a dominant strain inhibited most of the other microorganisms pre-existing in the materials during fermentation. Conclusion Increments of soluble and crude protein by 277.19% and 18.63% and decrement of harmful TI by 61.05% in SBM were achieved using thermophilic SSF by Bacillus licheniformis YYC4, providing a basis for the application of thermophiles in fermentation industry in an environmentally friendly and energy-saving way. © 2021 Society of Chemical Industry.

pH shift is an effective technique for modifying functional properties of food proteins. However, it can increase lysinoalanine (LAL) content under alkali conditions, thus limiting the use of proteins. This study investigated the inhibition effect of ultrasonic parameters on LAL formation in rapeseed protein isolates (RPI) during pH shift treatment (pH-ST). Results showed that the content of LAL decreased by 49.5% and 74.1%, following the use of ultrasound (28 kHz, 40 W/L, 40 °C, and 30 min) under alkali and acidic treatment, respectively. Structural analysis showed that after ultrasonic irradiation, increased sulfhydryl groups and amino acids reduced the dehydroalanine and, thus, decreased LAL content. Particle size, secondary structure, and microstructure (SEM, AFM) analyses showed relative dispersion in protein distribution, reducing intermolecular or intramolecular cross-linking, thereby lowering the LAL content. Thus, ultrasonic-aided pH-ST may be an operational technique toward minimizing LAL formation in RPI.

Background Fermentation efficiency of thermophiles of Bacillus licheniformis YYC4 and Geobacillus stearothermophilus A75, and mesophilic Bacillus subtilis 10 160 on soybean meal (SBM), was evaluated by examining the nutritional and protein structural changes. Results SBM fermentation by B. licheniformis YYC4, B. subtilis 10 160 and G. stearothemophilus A75 increased significantly the crude and soluble protein from 442.4 to 524.8, 516.1 and 499.9 g kg-1 , and from 53.9 to 203.3, 291.3 and 74.6 g kg-1 , and decreased trypsin inhibitor from 8.19 to 3.19, 2.14 and 5.10 mg g-1 , respectively. Bacillus licheniformis YYC4 and B. subtilis 10 160 significantly increased phenol and pyrazine content. Furthermore, B. licheniformis YYC4 fermentation could produce abundant alcohols, ketones, esters and acids. Surface hydrophobicity, sulfhydryl groups and disulfide bond contents of SBM protein were increased significantly from 98.27 to 166.13, 173.27 and 150.71, from 3.26 to 4.88, 5.03 and 4.21 μmol g-1 , and from 20.77 to 27.95, 29.53 and 25.5 μmol g-1 after their fermentation. Fermentation induced red shifts of the maximum absorption wavelength (λmax ) of fluorescence spectra from 353 to 362, 376 and 361 nm, while significantly reducing the fluorescence intensity of protein, especially when B. subtilis 10 160 was used. Moreover, fermentation markedly changed the secondary structure composition of SBM protein. Analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and atomic force microscopy showed that macromolecule protein was degraded into small-sized protein or peptide during fermentation of SBM. Conclusion Bacillus licheniformis YYC4 fermentation (without sterilization) improved nutrition and protein structure of SBM as B. subtilis 10 160, suggesting its potential application in the SBM fermentation industry. © 2021 Society of Chemical Industry.

The activity of ACE inhibitory peptides is closely related to their amino acid species, hydrophobic amino acid content, sequence structure, and C-terminal amino acids. In this study, the analysis o...

Ultrasonic-assisted enzymolysis is the application of ultrasound technology to enhance the process of enzymolysis. Traditional enzymolysis has been met with certain limitations which includes uneven stirring, decreased enzyme activity and protein accumulation as a result of minimal contact between the enzyme and substrate. Ultrasound assisted enzymolysis is based on the potential for cell molecule modification by ultrasound to improve bioavailability of nutrients while retaining the original quality, and the potential use of radical sonochemistry to achieve targeted hydroxylation of polyphenols and carotenoids. The cavitation effects and mechanical action of ultrasound can be advantageous during enzymolysis when applied appropriately. Ultrasound pretreatment of substrates can boost biological activity, alter protein structure, enhance protein conversion rate and accelerate enzyme hydrolysis. This review therefore, explored the principle of ultrasonic-assisted enzymolysis and its effects on protein extraction, biological activity and enzyme hydrolysis as reported by different researchers. Ultrasonic-assisted enzymolysis shows great potentials in enzyme hydrolysis and can be employed as a processing tool by the food and pharmaceutical industries.

This study aimed to investigate the effect of ultrasound curing with various working modes and frequency combinations, including mono-, dual- and tri-frequency, on the content of NaCl and tenderness of pork loins (Longissimus dorsi). The physical qualities, myoglobin, moisture migration, distribution, and microstructure of pork were also evaluated. The results displayed that the NaCl content of samples cured by simultaneous ultrasound (100 W/L) working mode with a frequency combination of 20, 40, and 60 kHz was higher than that of other ultrasound working modes. The effect of ultrasonic brining was significantly better than the static curing when the saline solution was35 mL. In addition, the samples cured by simultaneous ultrasound had better physical qualities, including more pickling absorptivity, less cooking loss, and lower hardness, tenderness, and chewiness value. The intensity of lightness was reduced, although redness and yellowness remained unaltered compared to static curing. The myoglobin content decreased drastically without changing the oxygenation level, and the relaxation time of T

Proteolysis kinetics and structure attributes of sunflower meal protein (SMP) pretreated using sonication of frequency type single (20, 40 kHz) and dual (20/40 kHz) were examined. A simplified model with impeded enzyme reaction was developed and the proteolysis of SMP in a heterogeneous system was successfully described. Initial observed rate (kin) increased after sonication, demonstrating the impacts of sonication on SMP by enhancing proteolysis (enzymolysis) and altering the structure of SMP, which was validated by circular dichroism (CD) spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier transform infrared (FTIR) spectroscopy and sodium dodecyl sulfide polyacrylamide gel electrophoresis (SDS-PAGE) analyses. From SMP characterization, there was a reduction in α-helix, enhancement in absorption intensity and alterations in functional groups of SMP following sonication. SEM and AFM observations indicated that sonicated SMP unfolded and exhibited more heterogenous structures, and irregular small-sized particles than control, especially at 20/40 kHz. SDS-PAGE profile displayed notable changes in molecular weight after sonication, which evidenced limited unfolding in SMP conformation. The kinetic model has possibility of being used to control enzymolysis; and structural attributes could guide in the development of ultrasonic equipment for use of pretreating protein for enzymolysis.

The influence of freeze and convection (at 40 and 50 °C) drying on the physical, functional, and rheological attributes of sunflower protein (SP) and its hydrolysate (SPH) was investigated. Compared with convectively-dried samples, the lightness, turbidity, bulk density, and particle size values of the freeze-dried SP and SPH were substantially higher, but the browning index was lower (p < 0.05). Additionally, freeze-dried samples exhibited good solubility and foaming characteristics, whereas lower emulsion properties with the most pH values were observed. Furthermore, SPHs possessed higher solubility as well as foamability over SPs under varying pH values (2.0–10.0), whilst reduction in the emulsion activity index was clearly observed (p < 0.05). Convectively-dried powders exhibited greater viscosity and consistency coefficient; and significantly lower flow behavior index of dispersions, relative to the respective freeze-dried preparations, indicating that dehydration methods influenced the flow behavior of the investigated samples. From a molecular weight analysis, convectively-dried samples at various temperatures were characterized with high proportion of small-sized particles at ≤1 kDa fractions over the respective powders obtained by freeze drying. The observations made, thus, would benefit food processors and manufacturers in electing better dehydration technique based on the desired traits of SP and SPH powders for successful application in food product formulations.

This study investigated the effect of allicin binding on the structure, antioxidant and antibacterial properties of soy protein isolate (SPI). Results showed that allicin bound to 82.6 % free thiol groups of SPI at a molar ratio of 0.5. The combination of allicin and SPI significantly affected the structure of protein. Result of circular dichroism showed that the content of α-helix decreased by 26.9 % and the content of β-sheet increased by 12.2 % over control when the molar ratio was 0.5. The result of surface hydrophobicity signified the unfolding of SPI with the action of allicin. These results implied that allicin binding might be a suitable method for the modification of SPI. Furthermore, the antibacterialand antioxidant experiments indicated that allicin-SPI conjugates not only had the capacity to inhibit the growth of Escherichia coli and Staphyloccocus aureus, but also had DPPH and ABTS radicals scavenging activities.

Highlights • The ethanol yield increased by 34.87% after optimal ultrasonic irradiation. • Transcriptomics and metabolomics together revealed high yield mechanisms of ethanol. • Sonication treatment changed the expression of coding gene of ethanol synthetase. • Ultrasonic irradiation decreased the expression of organic acid in yeast., This study reports an enhancement of ethanol yield in Saccharomyces cerevisiae with low-intensity ultrasonic irradiation using fixed mode frequency generated by a self-developed six-frequencies (20, 23, 25, 28, 33, 40 kHz) ultrasonic device in our group. After sonication treatment, the ethanol production potential was determined. Under the optimal conditions of ultrasonic treatment (ultrasonic frequency 28 kHz, power density 180 W/L, and treatment time 24 h), the maximum ethanol yield increased by 34.87% compared to the control. Transcriptome sequencing showed that the ultrasonic treatment had expressional regulations on genes involved in pyruvate metabolism, glycolysis, pentose phosphate pathway, glucose transport, and reducing power production. The quantitative real-time polymerase chain reaction (qRT-PCR) further confirmed the changes in gene expression (up- or down-regulation). Metabolomics revealed that ultrasonic treatments increased intracellular glucose and nicotinamide adenine dinucleotide (NADH) contents, which are key metabolites for ethanol synthesis. Besides, ultrasonic treatments decreased the acetate and its derivatives resulting in lowered reverse consumption of pyruvate and thus promoted ethanol synthesis. These changes in gene expression and metabolites content might be the main reason why the ethanol yield in Saccharomyces cerevisiae increased after ultrasonic irradiation.

BACKGROUND Surfactin, a good biological surfactant, is derived from the metabolites of microorganisms. However, the ability of natural strains to produce surfactin is low, and so the presented study aimed to use a novel mutagenesis technology to increase their yields. RESULTS Atmospheric and room temperature plasma (ARTP) was used to conduct mutation breeding of Bacillus subtilis CICC 10721, and a mutant strain M45 with a higher surfactin yield of 34.2% and a stable subculture was screened out. From the fermentation kinetics study, it was found that the maximum cell dry weight, maximum growth rate and surfactin synthesis parameters of the mutant strain M45 were all greater than that of the original strain. Scanning electron microscope and laser scanning confocal microscope observations showed that the spore morphology changed after ARTP treating, and the intracellular Ca2+ concentration of the mutant increased. Genome resequencing analysis showed that 66 single nucleotide poymorphism non-synonymous mutation sites occurred in M45, and the identification results of the fermentation broth extract from M45 showed that it is composed of C12 -C16 surfactin. CONCLUSION ARTP mutagenesis was found to change the morphology of bacteria, membrane permeability and genes related to the synthesis and secretion of surfactin. The present study provides a basis for industrial production of surfactin and an understanding of the mutagenesis mechanism. © 2021 Society of Chemical Industry.

Highlights • The ethanol yield of S. cerevisiae could be increased by using ultrasound. • The mode of ultrasound at 28 kHz had the best ethanol yield. • The activity changes of HK, PFK and PK were closely related with the ethanol yield., In this study, the effect of sonication on the fermentation process of a single-celled fungus was examined. During the experiment, Saccharomyces cerevisiae (S. cerevisiae) was used as the starting strain for ethanol fermentation (batch fermentation) in a 7.5 L automated fermentation tank. The fermentation tank connected with a six-frequency ultrasonic equipment. Non-sonication treatment was set up as the control. Sonication treatment with power density of 280 W/L and 48 h of treatment time were set up as trial groups for investigating the influence of different ultrasound frequency including 20, 23, 25, 28, 33 and 40 kHz on the changes in dry cell-weight, glucose consumption rate, and ethanol yield. The results showed that the dry cell-weight, glucose consumption rate, and ethanol content reached the best results under the ultrasonic condition of 28 kHz ultrasound frequency in comparison with other ultrasound frequency. The dry cell-weight and ethanol content of the 28 kHz ultrasonic treatment group increased by 17.30% and 30.79%, respectively in comparison with the control group The residual sugar content dropped to a lower level within 24 h, which was consistent with the change in ethanol production. Besides, the results found that the glucose consumption rate increased compared to the control. It indicated that ultrasound accelerated glucose consumption contributed to increase the rate of ethanol output. In order to explore the mechanism of sonication enhanced the content of ethanol output by S. cerevisiae, the morphology, permeability of S. cerevisiae and key enzyme activities of ethanol synthesis were investigated before and after sonication treatment. The results showed that after sonication treatment, the extracellular nucleic acid protein content and intracellular Ca2+ concentration increased significantly. The morphology of S. cerevisiae was observed by SEM and found that the surface of the strain had wrinkles and depressions after ultrasonic treatment. furthermore after sonication treatment, the activities of three key enzymes which catalyze three irreversible reactions in glycolysis metabolism, namely, hexokinase, phosphofructokinase and pyruvate kinase increased by 59.02%, 109.05% and 87.27%, respectively. In a word, low-intensity ultrasound enhance the rate of ethanol output by S. cerevisiae might due to enhancing the growth and cell permeability of strains, and increasing the activities of three key enzymes of ethanol biosynthesis.

Recently, insects have aroused the interest of researchers as potential therapeutic resources against malignant diseases such as cancer. In this study, the effects of aqueous extracts from mysore thorn borer (MTB) (Anoplophora chinensis) and mealworm larvae (MWL) (Tenebrio molitor) against cancer cells were investigated. MWL aqueous extract showed higher antiproliferative effects against Caco-2 and HepG2 cells compared to MTB. The IC50 (48 hr) of MWL aqueous extract were 11.44 and 20 mg/ml for Caco-2 and HepG2 respectively. Flow cytometry analysis showed that MWL aqueous extract induced apoptosis in Caco-2 and HepG2 increasing from 2.06% to 74.34% and from 0.04% to 42.14% after 24 hr respectively. Caspase activity assay showed that apoptosis was mediated via death receptor pathway mediated by caspase-8 and -9 followed by the activation of caspase-3; caspase-3 may have induced DNA damage and cell death. These effects may be correlated to its free amino acids. The results of this study demonstrate the potentials of MWL in the development of natural anticancer therapeutics in the future. PRACTICAL APPLICATIONS: Natural nutraceuticals from insects might be useful for the treatment and prevention of cancers such as colorectal and liver cancer. In recent years, edible insects have caught the attention of researchers, because of their potential as an alternative source of food and nutraceuticals. The results of our study showed that MWL extract might provide important anticancer compounds against colon and liver cancer.

This study used a He-Ne laser with pulsed light irradiation to produce mutant strains of Phellinus igniarius strain JQ9 with enhanced characteristics for fermentation (17.685 ± 3.092 g/L) compared with the parent strain (12.062 ± 1.119 g/L). The combined treatment conditions were as follows: He-Ne laser irradiation for 30 min using a spot diameter of 10 mm, pulsed light treatment power set at 100 J, a treatment distance of 14.5 cm, and a flash frequency of 0.5 s. The production of bioactive polysaccharides and small biocompounds such as polyphenols, flavonoids, and triterpenes increased together with mycelium production. The results showed that polyphenol content was significantly correlated with L*, a*, and b* values (R = -0.594, P < 0.01; R = 0.571, P < 0.01; and R = 0.500, P < 0.05; respectively). Antagonistic and random amplified polymorphic DNA analyses indicated that the genetic material of the screened mutants was altered. The mutant screening using a He-Ne laser with pulsed light irradiation could be an effective method for the development of Phellinus strains and could thus improve mycelium production.

In this study, the influences of ultrasonic treatments with different working modes (fixed frequency and sweeping frequency) and various frequencies on the bacterial growth and metabolism yield of Saccharomyces cerevisiae (S. cerevisiae) were investigated by employing an in situ ultrasonic irradiation slot coupled with a flask fermentation equipment. The results revealed that the in situ ultrasonic treatment could promote the bacterial growth and metabolism yield, and the effect of fixed frequency ultrasound (FF) was higher than sweeping frequency ultrasound (SF). The content of metabolite in the fermentation broth increased with FF and decreased after SF treatments. On the other hand, when the frequency of FF was more than 33 kHz, the growth of S. cerevisiae became weaken, and the mortality rate of S. cerevisiae increased in fermentation broth. At FF of 23 kHz and 48 h fermentation-time, ethanol content increased by 19.33%, and the content of β-phenylethanol and other volatile metabolites such as esters also increased. In conclusion, FF could significantly improve the growth of S. cerevisiae, and the FF of 23 kHz showed the optimum impact on fermentation process of S. cerevisiae.

Herein the effect of low intensity ultrasound on the fermentation of skim milk medium by Lactobacillus paracasei were investigated to obtain optimum ultrasonic conditions for the highest yield of yoghurt peptides. The results showed that the fermented skim milk medium treated with ultrasound with its seed culture without ultrasonic treatment was an optimum scheme. In this scheme with the ultrasonic conditions of 28 kHz, ultrasonic pulsed model of on-time 100 s and off-time 10 s, 100 W/L for the treatment time of 30 min after the fermentation time of 9 h, the peptide content in the fermented skim milk media increased by 49.5% and the viable cells in the same media increased by 43.5% compared with those in the untreated samples. By response surface methodology (RSM) analysis and its verification experiments, a reasonably accurate empirical model was established for investigating and predicting the relationship between skim milk concentration, ultrasonic treatment time, power and the yield of yoghurt peptides. The former two parameters 12.6% w/v and 35 min were taken in the verification experiments in which the peptide content of the fermented media reached 5.9 mg/mL with an increase by 64.23% and the peptide yield was 14.2%, similar to its theoretical value of 14.6% according to the empirical model. The comparison of extracellular enzyme activities in the fermented skim milk media between with and without ultrasonic treatment under the conditions in the optimum scheme indicated that the mechanism of the ultrasound-activated peptide content increment might be the extracellular enzyme activities immediately activated by the ultrasound, effect of which would disappear in the progress of fermentation after the ultrasound was removed.

A polysaccharide of Irpex lacteus, a white-rot fungus with lignocellulose-degrading activities, has been used as a commercial medicine for nephritis treatment. Previously, a low-intensity electromagnetic field (LI-EMF) was found to increase the biomass and polysaccharide content of Irpex lacteus and induce twists on the cell surface. In this study, RNA-sequencing (RNA-seq) technology was used to analyze the underlying mechanism of LI-EMF's influence on Irpex lacteus. We identified 3268, 1377, and 941 differentially expressed genes (DEGs) in the LI-EMF-treated samples at recovery times of 0 h, 3 h, and 6 h, respectively, indicating a significant decline in the influence of the LI-EMF treatment on Irpex lacteus with the passage of recovery time. Moreover, 30 upregulated and 14 downregulated DEGs overlapped in the LI-EMF-treated samples at the recovery times of 0 h, 3 h, and 6 h, implying the important lasting effects of LI-EMF. The reliability of the RNA-seq data were validated by quantitative real-time PCR (qRT-PCR). The DEGs related to transcription factors, cell proliferation, cell wall, membrane components, amino acid biosynthesis and metabolism, and polysaccharide biosynthesis and metabolism were significantly enriched in the LI-EMF-treated samples. The experiments confirmed that the LI-EMF treatment significantly increased the content of amino acids with a considerable increase in the content of essential amino acids. Therefore, the global gene expression changes explained the pleiotropic effects of Irpex lacteus induced by the LI-EMF treatment. These findings provide the requisite data for the appropriate design and application of LI-EMF in the fermentation of microorganisms to increase production. Bioelectromagnetics. 40:104-117, 2019. © 2019 Bioelectromagnetics Society.

Compared with traditional thermal sterilization process, pulsed magnetic field (PMF) is a non-thermal sterilization technology with superior advantages of maintaining the nutrition and flavor of food. But little is known about its antimicrobial mechanism. To explore its antimicrobial mechanism, the variation of cell membrane, intracellular biological macromolecules and respiratory metabolism of E. coli O157: H7 after PMF treatment, were investigated. The results showed that PMF could cause cell membrane damage and increase cell membrane permeability, reducing the content of three intracellular macromolecules (protein, ATP and DNA), as well as exhibiting inhibition for ATPase activity. The inhibition rate and superposition rate test confirmed the pathway that affecting the respiratory metabolism of E. coli O157:H7 treated with PMF was hexose monophophate pathway (HMP), via reducing the activity of key enzyme glucose-6-phosphate dehydrogenase in HMP. Besides, PMF also exhibited superior antibacterial effect on E. coli O157:H7 when it was applied in vegetable juices.

Raman spectroscopy was used to monitor the enzymatic hydrolysis process of grape seed protein. The degree of hydrolysis (DH), IC50 of the ACE inhibitory activity, and peptide content of the digestive products of grape seed protein were analyzed offline. The partial least squares (PLS), interval partial least squares (IPLS), and joint interval partial least squares (Si-PLS) models of DH, IC50 , and peptide content were established and the optimal pretreatment method was selected. In the optimal model, the corrected model r of the grape seed protein hydrolysis degree is 0.997, the Root Mean Square Error of Cross Validation (RMSECV) is 0.507%. The predicted model r value is 0.9932, the Root Mean Square Error of Prediction (RMSEP) is 1.15%. The corrected model r value of the IC50 is 0.9965, the RMSECV is 11.9%. The r value and RMSEP of predicted model are 0.9978 and 9.64%. The corrected model r value of the peptide content is 0.9955, the RMSECV is 12.7%, the predicted model r value is 0.9953, and the RMSEP is 15.4%. These results showed that in situ real-time monitoring of grape seed protein hydrolysis process can be achieved by Raman spectroscopy. PRACTICAL APPLICATIONS: This study uses Raman spectroscopy method to establish the quantification of proteolysis, IC50, and peptide content of the simulated digestive products during grape seed proteolysis. Analyze the model to monitor and evaluate the target parameters during the entire grape seed proteolysis process. In situ real-time monitoring of grape seed proteolysis is of great significance to the entire grape seed active peptide industry.

This study sought to establish an optimized procedure for extraction of protein from microwave‐dried larvae of an edible insect, Hermetia illucens (HIL), and to determine and compare the amino acid (AA) component, intrinsic fluorescence (IF), and physicochemical/functional properties (PFPs) of protein isolates. Box–Behnken's design with the Box–Wilson methodology was applied in the optimization process. Following single‐factor investigations, the relational analysis indicated that the polynomial model could be used to illustrate the influence of extraction parameters on protein yield. Response–surface plots showed that extraction variables influenced (p