Inactivation mechanism of phenyllactic acid against Bacillus cereus spores and its application in milk beverage.

Phenyllactic acid (PLA) as a natural phenolic acid exhibits antibacterial activity against non-spore-forming bacteria, while the inhibitory effect against bacterial spore remained unknown. Herein, this study investigated the inactivation effect of PLA against Bacillus cereus spores. The results revealed that the minimum inhibitory concentration of PLA was 1.25 mg/mL. PLA inhibited the outgrowth of germinated spores into vegetative cells rather than germination of spores. PLA disrupted the spore coat, and damaged the permeability and integrity of inner membrane. Moreover, PLA disturbed the establishment of membrane potential due to the inhibition of oxidative metabolism. SEM observations further visualized the morphological changes and structural disruption caused by PLA. Besides, PLA caused the degradation of DNA of germinated spores. Finally, PLA was applied in milk beverage, and showed promising inhibitory effect against B. cereus spores. This finding could provide scientific basis for the application of PLA against spore-forming bacteria in food industry.

[Teaching reform and practice of "food enzymology and enzyme engineering" based on cutting-edge researches].

Food enzymology and enzyme engineering is an important professional course of food science. The course includes the basic theory of enzymology, enzyme engineering technology and the application of enzymes in food industry. Considering the knowledge gap between the teaching contents and the cutting-edge researches, the team constantly adjusted and optimized the course contents to enable students to keep up with state-of-the-art progress by carefully mining the cutting-edge researches. Taking cutting-edge researches as the breakthrough point, we explored the problem-based learning (PBL) teaching model under the guidance of outcome-based education (OBE) concept, and highlighted the importance of the teacher-student and student-student interactions to improve students' enthusiasm and participation. A diversified assessment system was established to evaluate the performance of students in the learning process. The teaching reform consolidated the basic knowledge and expanded the academic frontiers, and fostered students' ability in analyzing problems, designing solutions and achieving team communication. The course may give new insights into the teaching reform of food enzymology and enzyme engineering and other related courses.

Antibacterial mechanism of sucrose laurate against Bacillus cereus by attacking multiple targets and its application in milk beverage.

Sucrose laurate (SL) is a promising dual-functional additive due to its emulsification and antibacterial activity. However, the knowledge on the antibacterial action of SL against Bacillus cereus was lacking, and thus it was investigated from multiple targets. The antibacterial results demonstrated that the minimum inhibitory concentration of SL was 0.3125 mg/mL, and the time-killing curve confirmed the strong antibacterial activity of SL. The alkaline phosphatase assay suggested that SL disrupted the cell wall integrity. The flow cytometry and fluorescence spectroscopy analysis indicated that SL damaged the integrity of cell membrane and dissipated the transmembrane potential, resulting in the leakage of intracellular materials, which were further supported by scanning electron microscopy and transmission electron microscopy. iTRAQ-based proteomic analysis indicated that SL down-regulated cell wall-associated hydrolase, inhibited the synthesis of fatty acids, influenced nucleic acid synthesis, disturbed amino acid metabolism, and blocked HMP pathway and TCA cycle. Finally, the promising application of SL was evidenced in milk beverage. This investigation could provide scientific basis for the practical application of SL as a dual-functional food additive.

iTRAQ-based quantitative proteomic analysis of synergistic antibacterial mechanism of phenyllactic acid and lactic acid against Bacillus cereus.

Phenyllactic acid (PLA) as a phenolic acid by lactic acid (LA) bacteria shows enhanced antibacterial activity when coexisting with LA, while the antibacterial mechanism of PLA combined with LA was unknown. Hence, the antibacterial mechanism of PLA and LA was investigated against Bacillus cereus. Flow cytometry and TEM analysis demonstrated that single PLA and LA disrupted the membrane integrity and the morphology, while combined PLA and LA synergistically enhanced the damage. iTRAQ-based proteomic analysis suggested that PLA down-regulated kdpB and inhibited K+ transport, disturbed the function of ribosome and expression of competence genes; LA down-regulated periplasmic phosphorus-binding proteins and inhibited phosphorus transport, disturbed the function of ribosome, TCA cycle, as well as purine and pyrimidine metabolism; and combined PLA and LA inhibited K+ and phosphorus transport, and influenced the synthesis of purine and pyrimidine metabolism. The investigation could provide some insights into the application of PLA in food preservation.

Biosynthesis of Neokestose Laurate Catalyzed by Candida antarctica Lipase B and Its Antimicrobial Activity against Food Pathogenic and Spoilage Bacteria.

To increase the functionality and broaden the potential application of neokestose, neokestose laurate was biosynthesized using Candida antarctica lipase B as biocatalyst, for which a mixture of 20% DMSO in 2-methyl-2-butanol (v/v) was chosen as the reaction medium. The optimum conditions for biosynthesis were as follows: a molar ratio of vinyl laurate to neokestose of 12, a temperature of 50 °C, molecular sieves of 100 g/L, and enzyme loading of 10 g/L. Under the optimal conditions, the conversion rate was achieved over 80%. The synthesized chemical 6'-O-lauroylneokestose confirmed by nuclear magnetic resonance (NMR) exhibited good emulsification with critical micelle concentration (CMC) of 352 µM and broad antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus, Listeria monocytogenes, Streptococcus mutans, Bacillus subtilis, and Bacillus cereus. Conclusively, 6'-O-lauroylneokestose was evidenced to be a dual-functional agent with emulsification and antibacterial activity, showing promising application potential in the food industry.

Antibacterial activity of phenyllactic acid against Listeria monocytogenes and Escherichia coli by dual mechanisms.

Phenyllactic acid (PLA), a phenolic acid phytochemical, is considered to be a promising candidate for use as a chemical preservative due to its broad antimicrobial activity. The antibacterial target of PLA has rarely been reported, thus investigations were performed to elucidate the antibacterial mechanism of PLA against Listeria monocytogenes and Escherichia coli. Flow cytometry analysis stained with propidium iodide (PI) demonstrated that PLA could damage the membrane integrity of L. monocytogenes, while it could not disrupt that of E. coli. The uptake of 1-N-phenylnaphthylamine (NPN) indicated that PLA interrupted the outer membrane permeability of E. coli. Scanning electron microscopy (SEM) observation visualized the damage caused by PLA as morphological changes in L. monocytogenes and E. coli. Fluorescence assays demonstrated that PLA could interact with bacterial genomic DNA in the manner of intercalation. This finding suggested dual antibacterial targets of PLA, namely membrane and genomic DNA.

Eichhornia crassipes: Agro-waster for a novel thermostable laccase production by Pycnoporus sanguineus SYBC-L1.

The aim of this study was to explore the utilization of an intractable waster of Eichhornia crassipes in laccase production by Pycnoporus sanguineus SYBC-L1. E. crassipes as the sole carbon and nitrogen source was confirmed to produce laccase (7.26 U/g dry substrate). The fermentation medium for the maximum enzyme production was optimized and the laccase was then purified and characterized. The optimized culture medium was 25.1% E. crassipes, 13.9% sawdust, 1.5 mM CuSO4, 40 µM gallic acid, 65% moisture content and initial pH 6.0. A maximum laccase activity of 32.02 U/g dry substrate was detected at 9th day, which was 4.5-fold compared to the initial medium. The molecular mass of the purified Lac-S was 58.4 kDa. The optimum activity of Lac-S for DMP was at pH 3.0 and 70°C. Lac-S showed not only high catalytic activities at low temperature, but also good stabilities toward pH and temperature. The residual catalytic activities of Lac-S were 30%, 40% and 50% at 0°C, 10°C and 20°C, and the half-lives at 50°C, 60°C and 70°C were 21.7, 9.7 and 1.5 h, respectively. The results provide a significant basis for E. crassipes further utilization and Lac-S specific application in harsh industry.

Effect of the treatment by slightly acidic electrolyzed water on the accumulation of γ-aminobutyric acid in germinated brown millet.

The accumulation of γ-aminobutyric acid and the microbial decontamination are concerned increasingly in the production of sprouts. In this work, the effect of the treatment by slightly acidic electrolyzed water on the accumulation of γ-aminobutyric acid in the germinated brown millet was evaluated by high performance liquid chromatography during germination. The results showed that slightly acidic electrolyzed water with appropriate available chlorine (15 or 30 mg/L) could promote the accumulation of γ-aminobutyric acid by up to 21% (P < 0.05). However, the treatment with slightly acidic electrolyzed water could not enhance the sprouts growth of the germinated brown millet. The catalase and peroxidase activities of the germinated brown millet during germination were in agreement with the sprouts growth. Our results suggested that the accumulation of γ-aminobutyric acid was independent of the length of sprouts in germinated grains. Moreover, the treatment with slightly acidic electrolyzed water significantly reduced the microbial counts in the germinated millet (P < 0.05) and the treatment with high available chlorine concentration (15 and 30 mg/L) showed stronger anti-infection potential in the germinated brown millet than that of lower available chlorine concentration (5 mg/L). In conclusion, the treatment with slightly acidic electrolyzed water is an available approach to improve the accumulation of γ-aminobutyric acid and anti-infection potential in the germinated brown millet, and it can avoid too long millet sprouts.

Metabolic mechanism of phenyllactic acid naturally occurring in Chinese pickles.

Phenyllactic acid, a phenolic acid phytochemical with the antimicrobial activity, was rarely reported in food besides honey and sourdough. This study evidenced a new food source of phenyllactic acid and elucidated its metabolic mechanism. Phenyllactic acid naturally occurred in Chinese pickles with concentrations ranged from 0.02 to 0.30 mM in 23 pickle samples including homemade and commercial ones. Then, lactic acid bacteria capable of metabolizing phenyllactic acid were screened from each homemade pickle and a promising strain was characterized as Lactobacillus plantarum. Moreover, the investigation of the metabolic mechanism of phenyllactic acid in pickles suggested that the yield of phenyllactic acid was positively related to the content of phenylalanine in food, and the addition of phenylalanine as precursor substance could significantly promote the production of phenyllactic acid. This investigation could provide some insights into the accumulation of phenyllactic acid in pickle for long storage life.

Improved stability and controlled release of ω3/ω6 polyunsaturated fatty acids by spring dextrin encapsulation.

Food grade biopolymers, such as dextrin, have been suggested as a technological solution for the controlled delivery of health promoting substances. The main focus of this work is to improve the stability of poly-unsaturated fatty acids (PUFAs) and controlled release by encapsulating with helical spring dextrin (SD). The encapsulation was formed between SD with a DP¯ of 62 and α-linolenic acid (ALA) or linoleic acid (LA) at 60 °C and characterized by WXRD, DSC, TGA and SEM. Under conditions which simulated the human environment of the gastrointestinal system, 21.7% and 18.5% of SD-ALA and SD-LA were released, respectively. A molecular dynamics simulation indicated that the space of helix cavity for ALA-SD complex was larger than that for LA-SD complex. This research work supports the idea that these complexes not only can improve the stability of ALA and LA, but also can achieve the targeted delivery of functional lipids or other bioactive components to the small intestine.

Comparative study of spring dextrin impact on amylose retrogradation.

The effects of spring dextrin on amylose recrystallization were investigated by wide-angle X-ray diffraction (WXRD) and differential scanning calorimetry (DSC). Recrystallinity of amylose was reduced in terms of adding SD(7), SD(9), or SD(11). Alternatively, SD(3) or SD(5) accelerated the degree of crystallinity. DSC data were analyzed using the Avrami equation and confirmed the results of WXRD. Finally, molecular dynamic simulation was adapted to predict the behavior of polymers in water, and the results showed that the small spring dextrins disturbed amylose retrogradation by inhibiting or altering amylose-amylose interaction.

Low-cost production of 6G-fructofuranosidase with high value-added astaxanthin by Xanthophyllomyces dendrorhous.

The effects of medium composition and culture conditions on the production of (6)G-fructofuranosidase with value-added astaxanthin were investigated to reduce the capital cost of neo-fructooligosaccharides (neo-FOS) production by Xanthophyllomyces dendrorhous. The sucrose and corn steep liquor (CSL) were found to be the optimal carbon source and nitrogen source, respectively. CSL and initial pH were selected as the critical factors using Plackett-Burman design. Maximum (6)G-fructofuranosidase 242.57 U/mL with 5.23 mg/L value-added astaxanthin was obtained at CSL 52.5 mL/L and pH 7.89 by central composite design. Neo-FOS yield could reach 238.12 g/L under the optimized medium conditions. Cost analysis suggested 66.3% of substrate cost was reduced compared with that before optimization. These results demonstrated that the optimized medium and culture conditions could significantly enhance the production of (6)G-fructofuranosidase with value-added astaxanthin and remarkably decrease the substrate cost, which opened up possibilities to produce neo-FOS industrially.

Biochemical characterization of an intracellular 6G-fructofuranosidase from Xanthophyllomyces dendrorhous and its use in production of neo-fructooligosaccharides (neo-FOSs).

An intracellular 6G-fructofuranosidase (endo-type enzyme) extracted from Xanthophyllomyces dendrorhous 269 efficiently hydrolyzes fructosyl-ß-(2→1)-linked sucrose to produce neo-kestose as a main transglycosylation product. The enzyme with a molecular weight of 33 kDa was purified by DEAE-52 cellulose chromatography. Thirty-fivefold purification and a 13.4% enzyme activity recovery were achieved. Optimum enzyme activity occurred at pH 6.4 and 45 °C and the enzyme was stable at pH 4-7 and at 45 °C. Using sucrose as a substrate, the Km and Vmax values were, respectively, 511 mmol/l and 233 µmol/(min mg) for transfer activity and 62 mmol/l and 164 µmol/(min mg) for hydrolytic activity. Under optimum conditions, a maximum concentration (73.9 g/l) of neo-fructooligosaccharides catalyzed by the endo-enzyme was obtained. These findings suggest that the purified endo-enzyme exhibits a high transfructosylation activity and it has potential for the industrial production of neo-FOSs.

Production of neo-fructooligosaccharides using free-whole-cell biotransformation by Xanthophyllomyces dendrorhous.

The effects of production parameters on the biotransformation of sucrose were investigated to enhance the yield of neo-FOS by Xanthophyllomyce dendrorhous cells. Cells showed optimal beta-fructofuranosidase activity at neutral pH condition and the yield of neo-FOS showed no significant differences between buffer and buffer-free systems. Cell concentration negatively affected the maximum neo-FOS yield. Sucrose concentration positively increased the maximum yield of neo-FOS. Elevating the reaction temperature to 30 degrees C, the neo-FOS productivity increased 1.85-fold compared with that at 20 degrees C. Meanwhile, cell age of 32 h enabled the biotransformation of sucrose more efficiently. In addition, free cells exhibited a higher productivity over immobilized cells. The maximum neo-FOS concentration finally reached 227.72 g/l from 400 g/l sucrose under the optimal conditions.