Response mechanisms to acid stress of acid-resistant bacteria and biotechnological applications in the food industry.

Acid-resistant bacteria are more and more widely used in industrial production due to their unique acid-resistant properties. In order to survive in various acidic environments, acid-resistant bacteria have developed diverse protective mechanisms such as sensing acid stress and signal transduction, maintaining intracellular pH homeostasis by controlling the flow of H+, protecting and repairing biological macromolecules, metabolic modification, and cross-protection. Acid-resistant bacteria have broad biotechnological application prospects in the food field. The production of fermented foods with high acidity and acidophilic enzymes are the main applications of this kind of bacteria in the food industry. Their acid resistance modules can also be used to construct acid-resistant recombinant engineering strains for special purposes. However, they can also cause negative effects on foods, such as spoilage and toxicity. Herein, the aim of this paper is to summarize the research progress of molecular mechanisms against acid stress of acid-resistant bacteria. Moreover, their effects on the food industry were also discussed. It is useful to lay a foundation for broadening our understanding of the physiological metabolism of acid-resistant bacteria and better serving the food industry.

Contribution of amino acids to Alicyclobacillus acidoterrestris DSM 3922T resistance towards acid stress.

Spoilage of juice and beverages by a thermo-acidophilic bacterium, Alicyclobacillus acidoterrestris, has been considered to be a major and widespread concern for juice industry. Acid-resistant property of A. acidoterrestris supports its survival and multiplication in acidic juice and challenges the development of corresponding control measures. In this study, intracellular amino acid differences caused by acid stress (pH 3.0, 1 h) were determined by targeted metabolomics. The effect of exogenous amino acids on acid resistance of A. acidoterrestris and the related mechanisms were also investigated. The results showed that acid stress affected the amino acid metabolism of A. acidoterrestris, and the selected glutamate, arginine, and lysine contributed to its survival under acid stress. Exogenous glutamate, arginine, and lysine significantly increased the intracellular pH and ATP level, alleviated cell membrane damage, reduced surface roughness, and suppressed deformation caused by acid stress. Additionally, the up-regulated gadA and speA genes and the enhanced enzymatic activity confirmed that glutamate and arginine decarboxylase systems played a crucial role in maintaining pH homeostasis of A. acidoterrestris under acid stress. Our research reveals an important factor contributing to acid resistance of A. acidoterrestris, which provides an alternative target for effectively controlling this contaminant in fruit juices.

New insights into thermo-acidophilic properties of Alicyclobacillus acidoterrestris after acid adaptation.

Alicyclobacillus acidoterrestris has unique thermo-acidophilic properties and is the main cause of fruit juice deterioration. Given the acidic environment and thermal treatment during juice processing, the effects of acid adaptation (pH 3.5, 3.2, and 3.0) on the resistance of A. acidoterrestris to heat (65 °C, 5 min) and acid (pH = 2.2, 1 h) stresses were investigated for the first time. The results showed that acid adaptation induced cross-protection against heat stress of A. acidoterrestris and acid tolerance response, and the extent of induced tolerance was increased with the decrease of adaptive pH values. Acid adaptation treatments did not disrupt the membrane potential stability and intracellular pH homeostasis, but reduced intracellular ATP concentration, increased cyclic fatty acids content, and changed the acquired Fourier transform infrared spectra. Transcription levels of stress-inducible (dnaK, grpE, clpP, ctsR) genes and genes related to spore formation (spo0A, ctoX) were up-regulated after acid adaptation, and spore formation was observed by scanning electron microscopy. This study revealed that the intracellular microenvironment homeostasis, expression of chaperones and proteases, and spore formation played a coordinated role in acid stress adaptive responses, with implications for applications in fruit juice processing.

Influences of acid and ethanol stresses on Oenococcus oeni SD-2a and its proteomic and transcriptional responses.

BACKGROUND: During winemaking, malolactic fermentation (MLF) is usually induced by Oenococcus oeni owing to its high resistance to wine stress factors. To ensure a controlled and efficient MLF process, starter cultures are inoculated in wine. In previous studies, O. oeni strains with sub-lethal acid or ethanol stresses showed higher freeze-drying vitality and better MLF performance. To explore the mechanisms involved, influences of acid and ethanol stresses on O. oeni SD-2a were investigated in this study to gain a better understanding of the cross-protection responses. RESULTS: The results showed that acid and ethanol stresses both caused damage to cell membranes and decreased cellular adenosine triphosphate concentration. At the same time, acid stress increased the uptake of glutathione, while ethanol stress led to cell depolarization. The results of comparative proteomic analysis highlighted that heat shock protein was induced with almost all acid and ethanol stresses. In addition, the expression of stress-relevant genes (hsp20, clpP, trxA, ctsR, recO, usp) increased greatly with ethanol and acid stress treatments. Finally, the viability of O. oeni was improved with acid and ethanol pretreatments after freeze-drying. CONCLUSIONS: This study demonstrated that acid and ethanol stresses had mixed influences on O. oeni SD-2a. Some physiological and molecular changes would contribute to a more stress-tolerant state of O. oeni, thereby improving the viability of lyophilized cells. © 2020 Society of Chemical Industry.

No more cleaning up - Efficient lactic acid bacteria cell catalysts as a cost-efficient alternative to purified lactase enzymes.

ß-galactosidases, commonly referred to as lactases, are used for producing lactose-free dairy products. Lactases are usually purified from microbial sources, which is a costly process. Here, we explored the potential that lies in using whole cells of a food-grade dairy lactic acid bacterium, Streptococcus thermophilus, as a substitute for purified lactase. We found that S. thermophilus cells, when treated with the antimicrobial peptide nisin, were able to hydrolyze lactose efficiently. The rate of hydrolysis increased with temperature; however, above 50 °C, stability was compromised. Different S. thermophilus strains were tested, and the best candidate was able to hydrolyze 80% of the lactose in a 50 g/L solution in 4 h at 50 °C, using only 0.1 g/L cells (dry weight basis). We demonstrated that it was possible to grow the cell catalyst on dairy waste, and furthermore, that a cell-free supernatant of a culture of a nisin-producing Lactococcus lactis strain could be used instead of purified nisin, which reduced cost of use significantly. Finally, we tested the cell catalysts in milk, where lactose also was efficiently hydrolyzed. The method presented is natural and low-cost, and allows for production of clean-label and lactose-free dairy products without using commercial enzymes from recombinant microorganisms. KEY POINTS: • Nisin-permeabilized Streptococcus thermophilus cells can hydrolyze lactose efficiently. • A low-cost and more sustainable alternative to purified lactase enzymes. • Reduction of overall sugar content. • Clean-label production of lactose-free dairy products.

First Insight into the Probiotic Properties of Ten Streptococcus thermophilus Strains Based on In Vitro Conditions.

The aim of this study was to evaluate probiotic properties of ten Streptococcus thermophilus strains (st1 to st10) isolated from pickles in China. These strains all had ß-galactosidase activity, which laid foundation for studying their probiotic properties. In this study, the bile salt hydrolase activity, lysozyme resistance, tolerance to simulated gastric juice, bile salt tolerance, and bacterial adhesion capacity to the Caco-2 cells of these selected strains were detected in vitro conditions. The results indicated that the bile salt hydrolase activities of st2, st6, and st9 were higher than that for other strains. St10 showed the greatest lysozyme resistance (> 80% survival), followed by st9, st8, st7, st5, and st6. As for the tolerance to simulated gastric juice, st5 possessed the highest survival rate (35%), followed by st6 (30%). St6 was the best performer in both bile salt tolerance and bacterial adhesion capacity to the Caco-2 cells. The results of fluorescence microscope and electron microscope further confirmed previous studies and more intuitively demonstrated the st6 strain's tolerance to harsh environments. Overall, these strains were expected to possess beneficial properties and have the potentiality to be probiotics.

Identification and Characterization of the Small Heat Shock Protein Hsp20 from Oenococcus oeni SD-2a.

Oenococcus oeni can exert its function in hostile wine conditions during the malolactic fermentation process. Therefore, it is an important microbial resource for exploring resistance genes. Hsp20 is an important small heat shock protein from O. oeni. The conserved consensus motif "A-x-x-x-x-G-x-L" of Hsp20 announced its role as a member of the small heat shock protein family. The hsp20 gene from O. oeni SD-2a was cloned to create the recombinant plasmid pTriEx-Hsp20. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and the Hsp20 protein was induced by isopropyl-ß-D-thiogalactoside (IPTG). The hsp20 gene from O. oeni SD-2a was successfully expressed, and a 20-kDa fusion protein was identified by SDS-PAGE. The purified Hsp20 protein was obtained using Ni-affinity chromatography. Additionally, BL21(DE3)/Hsp20 and BL21(DE3)/Ctrl were treated at high temperatures of 42 and 52 °C, at pH values of 2.0-12.0, under oxidative shock with 0.1% (v/v) and 0.2% (v/v) H2O2, and under an osmotic shock of 430 and 860 mM NaCl to compare the effects of heterologous expression of the Hsp20 protein from O. oeni SD-2a for stress resistance. Notably, Hsp20 overexpression showed enhanced resistance than the control strain did when confronted with different elevated stress conditions. The results demonstrated heterologous expression of the hsp20 gene from O. oeni SD-2a significantly improved the resistance of the host E. coli bacteria against stress conditions.

Antibacterial activity of selenium-enriched lactic acid bacteria against common food-borne pathogens in vitro.

Selenium (Se) is an essential trace element for human health and animal nutrition. The aim of this study was to evaluate the inhibitory activities of Se-enriched lactic acid bacteria (LAB), Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus, against pathogenic Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes in vitro. The results indicated that the accumulation amount of Se by Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus reached 12.05 ± 0.43 µg/mL and 11.56 ± 0.25 µg/mL, respectively, accompanied by the relative maximum living cells when sodium selenite was 80 µg/mL. Oxford cup double plate assay showed that bacterial culture solution and cell-free culture supernatant (CFCS) from Se-enriched LAB exerted stronger antibacterial activity than those from the non-Se strains. The growth of pathogenic bacterial culture with CFCS at any growth stages was worse than that without CFCS; moreover, the inhibiting effect of CFCS of Se-enriched LAB was more significant than that of non-Se strains. Results from a scanning electron microscope equipped with energy dispersion X-ray spectrometry showed that elemental Se nanoparticles, which characteristically energy peak around 1.42 keV, were deposited on the cell surfaces of Lactobacillus delbrueckii ssp. bulgaricus. In addition, CFCS of Se-enriched LAB induced more serious cell structure damage of pathogenic bacteria than did non-Se LAB.

Free and Bound Phenolic Compound Content and Antioxidant Activity of Different Cultivated Blue Highland Barley Varieties from the Qinghai-Tibet Plateau.

In this study, the polyphenols composition and antioxidant properties of 12 blue highland barley varieties planted on the Qinghai-Tibet Plateau area were measured. The contents of the free, bound and total phenolic acids varied between 166.20-237.60, 170.10-240.75 and 336.29-453.94 mg of gallic acid equivalents per 100 g of dry weight (DW) blue highland barley grains, while the free and bound phenolic acids accounted for 50.09% and 49.91% of the total phenolic acids, respectively. The contents of the free, bound and total flavones varied among 20.61-25.59, 14.91-22.38 and 37.91-47.98 mg of catechin equivalents per 100 g of dry weight (DW) of blue highland barley grains, while the free and bound flavones accounted for 55.90% and 44.10% of the total flavones, respectively. The prominent phenolic compounds in the blue hulless barley grains were gallic acid, benzoic acid, syringic acid, 4-coumaric acid, naringenin, hesperidin, rutin, (+)-catechin and quercetin. Among these, protocatechuic acid, chlorogenic acid and (+)-catechin were the major phenolic compounds in the free phenolics extract. The most abundant bound phenolics were gallic acid, benzoic acid, syringic acid, 4-coumaric acid, benzoic acid, dimethoxybenzoic acid, naringenin, hesperidin, quercetin and rutin. The average contribution of the bound phenolic extract to the DPPH• free radical scavenging capacity was higher than 86%, that of free phenolic extract to the ABTS•+ free radical scavenging capacity was higher than 79%, and that of free phenolic (53%) to the FRAP antioxidant activity was equivalent to that of the bound phenol extract (47%). In addition, the planting environment exerts a very important influence on the polyphenol composition, content and antioxidant activity of blue highland barley. The correlation analysis showed that 2,4-hydroxybenzoic acid and protocatechuic acid were the main contributors to the DPPH• and ABTS•+ free radical scavenging capacity in the free phenolic extract, while chlorogenic acid, vanillic acid, ferulic acid and quercetin were the main contributors to the free radical scavenging capacity in the bound phenol extract. The study results show that the blue highland barley grains have rich phenolic compounds and high antioxidant activity, as well as significant varietal differences. The free and bound phenolic extracts in the blue hulless barley grains have an equivalent proportion in the total phenol, and co-exist in two forms. They can be used as a potential valuable source of natural antioxidants, and can aid in enhancing the development and daily consumption of foods relating to blue highland barley.

Rapid concentration detection and differentiation of bacteria in skimmed milk using surface enhanced Raman scattering mapping on 4-mercaptophenylboronic acid functionalized silver dendrites.

A novel method was developed to rapidly concentrate, detect, and differentiate bacteria in skimmed milk using surface enhanced Raman scattering (SERS) mapping on 4-mercaptophenylboronic acid (4-MPBA) functionalized silver (Ag) dendrites. The 4-MPBA functionalized Ag dendritic SERS substrate was used to capture the bacterial cells and enhance the bacterial signal. Salmonella, a significantly important food pathogen, was used as the representative strain to optimize and evaluate the developed method. The capture efficiency for Salmonella enterica subsp enterica BAA1045 (SE1045) was 84.92 ± 3.25% at 106 CFU/mL and as high as 99.65 ± 3.58% at 103 CFU/mL. Four different strains, two gram-negative and two gram-positive, can be clearly distinguished by their SERS spectra using principle component analysis. A mapping technique was utilized to automatically collect 400 spectra over an area of 60 µm × 60 µm to construct a visual image for a sensitive and statistically reliable detection within 30 min. Using this method, we were able to detect as low as 103 CFU/mL bacterial cells in 50 mM NH4HCO3 solution and 102 CFU/mL cells in both 1% casein and skimmed milk. Our results demonstrate the feasibility of using SERS mapping method coupled with 4-MPBA functionalized Ag dendrites for rapid and sensitive bacteria detection in complex liquid samples. Graphical Abstract A novel SERS mapping method based on 4-mercaptophenylboronic acid functionalized silver (Ag) dendrites was developed to rapidly concentrate, detect, and differentiate bacteria.

Label-free mapping of single bacterial cells using surface-enhanced Raman spectroscopy.

Here we presented a simple, rapid and label-free surface-enhanced Raman spectroscopy (SERS) based mapping method for the detection and discrimination of Salmonella enterica and Escherichia coli on silver dendrites. The sample preparation was first optimized to maximize sensitivity. The mapping method was then used to scan through the bacterial cells adsorbed on the surface of silver dendrites. The intrinsic and distinct SERS signals of bacterial cells were used as the basis for label-free detection and discrimination. The results show the developed method is able to detect single bacterial cells adsorbed on the silver dendrites with a limit of detection as low as 10(4) CFU mL(-1), which is two orders of magnitude lower than the traditional SERS method under the same experimental condition. The time needed for collecting a 225 points map was approximately 24 minutes. Moreover, the developed SERS mapping method can realize simultaneous detection and identification of Salmonella enterica subsp. enterica BAA1045 and Escherichia coli BL21 from a mixture sample using principle component analysis. Our results demonstrate the great potential of the label-free SERS mapping method to detect, identify and quantify bacteria and bacterial mixtures simultaneously.

Characterization of Lactococcus lactis response to ampicillin and ciprofloxacin using surface-enhanced Raman spectroscopy.

Decades of antibiotic use or misuse has resulted in antibiotic resistance in lactic acid bacteria, a group of common culture starters and probiotic microorganisms. This has urged researchers to study how lactic acid bacteria respond to antibiotics, so as to have a better strategy to identify and predict the antibiotic-resistant bacteria. This study aimed to characterize the biochemical profiles of Lactococcus lactis responding to antibiotics using surface-enhanced Raman spectroscopy (SERS). Lactococcus lactis exposed to antibiotics was mixed with 50-nm gold nanoparticles for subsequent SERS measurements. The SERS spectra analyzed by principal component analysis showed no significant change after 30 min of antibiotic treatment, whereas distinct changes were clearly observed after 60 and 90 min of antibiotic treatment. Different antibiotics induced different spectral changes, and these changes revealed the detailed biochemical information of cellular responses. This study demonstrates that the SERS method developed not only senses the changes in the bacterial cell wall, but also reveals details of the biochemical profiles, which help us to understand how lactic acid bacteria respond to antibiotics, as well as to set a base for the detection of antibiotic susceptibility of bacteria by SERS.

Exploring the growth characteristics of Alicyclobacillus acidoterrestris for controlling juice spoilage with zero additives.

Fruit juice spoilage that caused by contaminated Alicyclobacillus has brought huge losses to beverage industry worldwide. Thus, it is very essential to understand the growth and metabolism processing of Alicyclobacillus acidoterrestris (A. acidoterrestris) in controlling juice spoilage caused by Alicyclobacillus. In this work, simulative models for the growth and metabolism of A. acidoterrestris were systematically conducted in the medium and fruit juice. The results showed that low temperature (4 ℃) and strong acidic environment (pH 3.0-2.0) of medium inhibited the growth and reproduction of A. acidoterrestris. In addition, with decreasing temperature, the color, smell and turbidity of commercially available juice supplemented with A. acidoterrestris significantly improved. This work provided a clear exploration of growth characteristics of A. acidoterrestris by applying theory (medium) to reality (fruit juices), and pave fundamental for exploring the zero additives of controlling juice spoilage.

Transcriptomic and Metabolomic Profiling Uncovers Response Mechanisms of Alicyclobacillus acidoterrestris DSM 3922T to Acid Stress.

Alicyclobacillus acidoterrestris, which has strong acidophilic and heat-resistant properties, can cause spoilage of pasteurized acidic juice. The current study determined the physiological performance of A. acidoterrestris under acidic stress (pH 3.0) for 1 h. Metabolomic analysis was carried out to investigate the metabolic responses of A. acidoterrestris to acid stress, and integrative analysis with transcriptome data was also performed. Acid stress inhibited the growth of A. acidoterrestris and altered its metabolic profiles. In total, 63 differential metabolites, mainly enriched in amino acid metabolism, nucleotide metabolism, and energy metabolism, were identified between acid-stressed cells and the control. Integrated transcriptomic and metabolomic analysis revealed that A. acidoterrestris maintains intracellular pH (pHi) homeostasis by enhancing amino acids decarboxylation, urea hydrolysis, and energy supply, which was verified using real-time quantitative PCR and pHi measurement. Additionally, two-component systems, ABC transporters, and unsaturated fatty acid synthesis also play crucial roles in resisting acid stress. Finally, a model of the responses of A. acidoterrestris to acid stress was proposed. IMPORTANCE Fruit juice spoilage caused by A. acidoterrestris contamination has become a major concern and challenge in the food industry, and this bacterium has been suggested as a target microbe in the design of the pasteurization process. However, the response mechanisms of A. acidoterrestris to acid stress still remain unknown. In this study, integrative transcriptomic, metabolomic, and physiological approaches were used to uncover the global responses of A. acidoterrestris to acid stress for the first time. The obtained results can provide new insights into the acid stress responses of A. acidoterrestris, which will point out future possible directions for the effective control and application of A. acidoterrestris.

Integrated transcriptomic and proteomic analysis reveals the response mechanisms of Alicyclobacillus acidoterrestris to heat stress.

Alicyclobacillus acidoterrestris can survive pasteurization and is implicated in pasteurized fruit juice spoilage. However, the mechanisms underlying heat responses remain largely unknown. Herein, gene transcription changes of A. acidoterrestris under heat stress were detected by transcriptome, and an integrated analysis with proteomic and physiological data was conducted. A total of 911 differentially expressed genes (DEGs) was observed. The majority of DEGs and differentially expressed proteins (DEPs) were exclusively regulated at the mRNA and protein level, respectively, whereas only 59 genes were regulated at both levels and had the same change trends. Comparative analysis of the functions of the specifically or commonly regulated DEGs and DEPs revealed that the heat resistance of A. acidoterrestris was primarily based on modulating peptidoglycan and fatty acid composition to maintain cell envelope integrity. Low energy consumption strategies were established with attenuated glycolysis, decreased ribosome de novo synthesis, and activated ribosome hibernation. Terminal oxidases, cytochrome bd and aa3, in aerobic respiratory chain were upregulated. Meanwhile, the MarR family transcriptional regulator was upregulated, reactive oxygen species (ROS) was discovered, and the concentration of superoxide dismutase (SOD) increased, indicating that the accompanied oxidative stress was induced by high temperature. Additionally, DNA and protein damage repair systems were activated. This study provided a global perspective on the response mechanisms of A. acidoterrestris to heat stress, with implications for better detection and control of its contamination in fruit juice.

Effect of inorganic and organic nitrogen supplementation on volatile components and aroma profile of cider.

To investigate the impact of inorganic and organic nitrogen on volatile compounds of cider, various levels (60, 150 and 240 mg N/L) of diammonium hydrogen phosphate (DAP) and amino acids mixture were added to apple juice before fermentation, respectively. The qualitative and quantitative of volatile substances were carried out by GC-MS. Further, sensory and electronic nose analysis was performed to analyze the aroma profile of cider. Both DAP and amino acids mixture promoted fermentation, significantly affected volatile compositions and increased perceived aromas of cider. Adding 60 mg N/L DAP significantly increased volatile compounds content, especially medium-chain fatty acid ethyl esters (MCFAEEs) and fatty acids (FAs), whereas higher alcohols and acetate esters concentrations were remarkably increased with 150 mg N/L amino acids supplementation. Partial least-squares (PLS) regression analysis suggested that MCFAEEs contributed the most to tropical fruity aroma. The study provides a theoretical foundation for high-quality cider brewing.

Acid adaptive response of Alicyclobacillus acidoterrestris: A strategy to survive lethal heat and acid stresses.

Alicyclobacillus acidoterrestris causes the spoilage of pasteurized acidic fruit juice, seriouslydecreasing quality and posing a significant safety concern. We previously discovered that acid adaptation could induce stress adaptive responses of A. acidoterrestris, however, the underlying mechanisms of this induction have not been fully elucidated. In this work, the effects of acid adaptation (pH = 3.0, 1 h) on intracellular pH (pHi) and the morphophysiological properties of A. acidoterrestris under lethal heat and acid stresses were investigated, and gene expression profiles after acid adaptation were measured by transcriptomic analysis. The results showed that acid adaptation increased the pHi of A. acidoterrestris cells in response to lethal stresses, enhanced membrane integrity, decreased surface shrinkage and roughness, and altered the Fourier transform infrared spectra profiles. After acid adaptation of A. acidoterrestris, 517 differentially expressed genes (DEGs) were detected. Consistent with resistance phenotypes, DEGs included genes related to cell surface modification and pHi homeostasis. Specifically, the barrier function of cell membrane was strengthened during acid adaptation by increasing fatty acid (FA) chain length, promoting unsaturated FA biosynthesis, and maintaining balanced synthesis of zwitterionic and acidic phospholipids. To reduce excessive intracellular protons, cells upregulated glutamate decarboxylation, urease system, and branched-chain amino acid synthesis. Additionally, the nucleotide salvage pathway was activated, and homologous recombination, UvrD-mediated transcription-coupled, and ribonucleotide excision repair pathways were applied to repair DNA lesions. Sporulation metabolism was also induced. The findings of this study provide insight into the multiple layers of acid adaptive response strategies of A. acidoterrestris, with implications for the formulation of improved control measures in the fruit juice industry.

Deciphering the antibacterial activity and mechanism of p-coumaric acid against Alicyclobacillus acidoterrestris and its application in apple juice.

Alicyclobacillus acidoterrestris has been regarded as the main hazardous factor causing the spoilage of commercially pasteurized fruit juice due to its unique thermo-acidophilic properties. p-Coumaric acid, primarily isolated from plants and having high biological activity, is supposed to be a viable food additive. Herein, the antibacterial activity and mechanism of p-coumaric acid against A. acidoterrestris and its application in apple juice were investigated. The results showed that p-coumaric acid had an active inhibition against A. acidoterrestris vegetative cells, and both minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were 0.2 mg/mL. MIC of p-coumaric acid against spores was also 0.2 mg/mL, while MBC was >1.6 mg/mL. Treatment of p-coumaric acid caused significant damage of cell integrity, decrease of intracellular ATP, hyperpolarization of cell membrane, degradation of whole cell protein, and malformation of cell morphology. Agarose gel retardation and fluorescence spectroscopy assays revealed that p-coumaric acid could interact with DNA and intercalate into the groove of DNA double helix to disturb normal cellular functions. Furthermore, the addition of p-coumaric acid at MIC caused 2.07 log drop in A. acidoterrestris vegetative cells and effectively inhibited the germination of spores in apple juice storage at 20 °C for 7 days. The low-temperature storage (4 °C) could inactivate A. acidoterrestris cells in apple juice, but the addition of p-coumaric acid further accelerated degradation of vegetative cells and the inactivation time was shortened from 5 days to 3 days compared with control. Importantly, the addition of p-coumaric acid could increase total phenolic content and ABTS radical scavenging activity and had no remarkable influence on pH, total soluble solids, color and sensory qualities of apple juice, regardless of storage temperature. These results revealed that p-coumaric acid could be expected to serve as an alternative or supplement agent for inhibiting the development of A. acidoterrestris in fruit juice.

[Application of near infrared spectral fingerprint technique in lamb meat origin traceability].

Near infrared spectra of 99 lamb meat samples from three pasturing areas and two farming areas of China were scanned and analyzed to seek a cheap, rapid and effective method for lamb meat origin traceability. Two chemometric methods including linear discriminant analysis based on principal component analysis (PCA+LDA) and partial least squares discriminant analysis (PLS-DA) were used to develop the discriminate models. It was showed that there were significantly differences among the lamb meat samples from five regions based on NIR spectra after second derivative (Savitzky-Golay, 9 point) and multiplicative scattering correction (MSC) transformation in the whole wavelength. The discrimination of two models was best for classification of pasturing area and farming area, with both correctly classified by 100%. The correct classification rate of samples from five different regions using PCA+LDA model was 91.2%, higher than using PLS-DA model (76.7%). These results demonstrate that near infrared reflectance spectroscopy (NIRS) combined with chemometric analysis can be used as an effective method to classify lamb meat according to its geographical origin.

Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis of Alicyclobacillus acidoterrestris Under Acid Stress.

Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of A. acidoterrestris under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene ctsR showed that dnaG and dnaN were the optimal multiple RGs for normalization at pH 4.0; ytvI, dnaG, and 16s rRNA at pH 3.5; icd and dnaG at pH 3.0; and ytvI, dnaG, and spoVE at pH 2.5. This study revealed for the first time that A. acidoterrestris had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium.