Assessment of Oligogalacturonide from Citrus Pectin as a Potential Antibacterial Agent against Foodborne Pathogens

Foodborne diseases are an important public health problem in the world. The bacterial resistance against presently used antibiotics is becoming a public health issue; hence, the discovery of new antimicrobial agents from natural sources attracts a lot of attention. Antibacterial activities of oligogalacturonide from commercial microbial pectic enzyme (CPE) treated citrus pectin, which exhibits antioxidant and antitumor activities, against 4 foodborne pathogens including Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Pseudomonas aeruginosa was assessed. Pectin hydrolysates from CPE hydrolysis exhibited antibacterial activities. However, no antibacterial activity of pectin was observed. Citrus oligogalacturonide from 24‐h hydrolysis exhibited bactericidal effect against all selected foodborne pathogens and displayed minimal inhibitory concentration at 37.5 μg/mL for P. aeruginosa, L. monocytogenes, and S. Typhimurium, and at 150.0 μg/mL for S. aureus.     

Partial Characterization of Nine Bacteriocins Produced by Lactic Acid Bacteria Isolated from Cold-Smoked Salmon with Activity against Listeria monocytogenes

Nine LAB bacteriocin-producers, isolated from vacuum-packaged cold-smoked salmon (CSS), were phenotypically and genotypically identified as Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus fermentum, Enterococcus faecium, and Pediococcus acidilactici. Their bacteriocins were partially characterized. The antimicrobial spectrum was determined against Listeria monocytogenes, E. faecalis, E. faecium, and Staphylococcus aureus. The molecular size of bacteriocins ranged from 2.8 to 4.5 kDa. They were inactivated by treatment with proteolytic enzymes but not by lipolytic or glycolytic enzymes. Maximal activity against L. monocytogenes ranged between 800 and 10000 AU/mL at pH 6.5. Most of the bacteriocins maintained full activity in a pH range of 2.0 to 8.0 but were partially or completely inactivated at pH 10.0. After heating at 60°C and 100°C, only two bacteriocins from Lb. curvatus strains partially lost activity. All bacteriocins showed a narrow spectrum of activity and a high anti-listerial activity, which is characteristic of the class IIa bacteriocins. Isolated bacteriocin-producing LAB could be used successfully in the bio-preservation of CSS and development of new potential bio-preservatives for CSS active against L. monocytogenes.     

Partial characterisation of enterocin KP produced by Enterococcus faecalis KP,a cheese isolate

Enterococcus faecalis KP, a bacteriocin‐producing strain, was identified using 16S rRNA gene sequence homology (99%). Enterocin KP from E. faecalis KP was sensitive to papain and ß‐mercaptoethanol, but resistant to trypsin, pepsin, lipase, catalase, α‐amylase, organic solvents, detergents, EDTA and heat treatment (90°C/30 min). It was active at a wide pH range (2.0–8.0) and produced at maximum level in de Mann Rogosa Sharpe broth at 32°C with an inoculum amount of 0.1–1.0% and an initial pH of 5.5–7.5. It was active against some Gram‐positive bacteria, including Listeria monocytogenes. It showed bactericidal activity against L. monocytogenes and its molecular weight was approximately 5.8 kDa.     

破壁方式对冠突散囊菌有性繁殖体提取物抑菌活性的影响

为对比3种破壁方法对冠突散囊菌有性繁殖体(子囊果和子囊孢子)提取物抑菌活性的影响,利用超声法、萌发法和玻璃珠振摇法处理冠突散囊菌有性繁殖体,运用抑菌圈法测定提取物对金黄色葡萄球菌和大肠杆菌的抑菌活性。结果表明,子囊果较易破碎,子囊孢子较难破碎;玻璃珠振摇法提取的子囊果提取物比子囊孢子提取物的抑菌活性更强,子囊果提取物对大肠杆菌和金黄色葡萄球菌的抑菌圈半径分别为5.8 mm和12.6 mm;玻璃珠振摇法破壁子囊孢子的破壁率最高,可达94.8%,其提取物具有抑菌活性,其余两种方法获得的提取物无明显的抑菌活性。3种方法中,玻璃珠振摇法是提取冠突散囊菌有性繁殖体中抑菌物质的最佳方法。     

Functional display of amylase on yeast surface from Rhizopus oryzae as a novel enzyme delivery method

Fungal amylase has great importance in fermentation industry such as brewing, food fermentation, starch hydrolysis and for improving microbial populations in chicken intestine through feed applications. In the present investigation, alpha amylase cDNA from Rhizopus oryzae was cloned, sequenced, and successfully surface anchored in functional form using Saccharomyces cerevisiae EBY100 as host, yielding enzyme activity of 4.35 (±0.5) U/ml. The surface displayed yeast expressed amylase activity using plate assay, produced glucose and maltose as hydrolysis products using starch as substrate. The targeted and armed yeast with displayed enzyme was evaluated for its characterization at various pH and temperatures. The engineered yeast showed optimal activity at neutral pH and at 50°C incubation temperature. Reducing sugars produced by displayed enzyme were visualized by paper chromatography. The data suggested successful heterologous expression and display of amylase enzyme on yeast cell surface. The displayed system could further be utilized in fermentation industry for improving cost-effectiveness of the process.     

Synergistic Reduction of Salmonella in a Model Raw Chicken Media using a Combined Thermal and Acidified Organic Acid Salt Intervention Treatment

ABSTRACT: Salmonella-contaminated poultry products are considered major contributors to foodborne illness. The anti-Salmonella activity of organic acid salts has been studied in food products and poultry feed but rarely in combination with nonchemical treatments. Here, we investigated the combination of acidified organic acid salt solutions with thermal treatment as an effective Salmonella intervention applicable in poultry carcass processing. A model raw chicken media was used to propagate Salmonella prior to the intervention treatment. Salmonella Typhimurium strains LT2 and ATCC nr 14028 grew similarly in the model raw chicken media at 37 and 42 °C, reaching stationary phase 24 h after inoculation. Four log₁₀CFU of either Salmonella Typhimurium strain at stationary phase was exposed to 2.5% organic acid salt solutions (at pH 4) for 1 min at 55 °C. All organic acid salt treatments yielded significant Salmonella Typhimurium reductions, ranging from 1 log (sodium acetate) to almost 4 logs (sodium butyrate). Exposure to pH 4 water at 55 °C or the organic acid salt solutions at room temperature had no effect. The combined thermal and acidified organic acid salt intervention produced a significant, synergistic reduction of Salmonella Typhimurium and may represent an effective method for decontamination of poultry carcasses during processing.     

Effects of Polyphosphates on Cell Numbers, Enterotoxin A, and Extracellular Protein in Staphylococcus aureus 196E

Effects of sodium salts of pyro- (SAPP), tripoly- (STPP), and hexametaphosphate (SHMP) on cell numbers, enterotoxin A (SEA) and extracellular protein production (ECP) of Staphylococcus aureus strain 196A were studied in 4% N-Z amine broth plus 1% yeast extract after 24 hr at 30°C. At pH 7.0, concentrations lower than 56 mM (1.2%) SAPP, 27 mM (1%) STPP or 3 mM (0.4%) SHMP had no antibacterial effects and a bacteriostatic effect showed at slightly higher concentrations. SAPP was the least effective inhibitor at neutral pH, but displayed enhanced inhibitory effects at pH 5.5. Reduction in SEA and ECP paralleled cell growth suppression; their concentrations were 1400 ng/mL and 1.5 mg/mL, respectively, in phosphate free broth (9 × 10⁹ CFU/mL), and nondetectable when cell numbers were ≤10⁶ per mL.     

Inhibition of Three Pathogens on Bologna and Summer Sausage Using Antimicrobial Edible Films

Whey protein isolate (WPI) films (pH 5.2) containing 0.5 to 1.0% p‐aminobenzoic acid (PABA) and/or sorbic acid (SA) were assessed for antimicrobial and mechanical properties while in contact with sliced bologna and summer sausage that were inoculated with Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica subsp. enterica serovar Typhimurium DT104. WPI films containing SA or PABA decreased L. monocytogenes, E. coli, and S. Typhimurium populations by 3.4 to 4.1,3.1 to 3.6, and 3.1 to 4.1 logs, respectively, on both products after 21 d at 4 °C. Background flora was inhibited compared with controls. Film tensile strength decreased while % elongation remained unchanged following 72 h of product contact.     

Effects of Heat-Stable Alakaline Protease Activity of Atlantic Menhaden (Brevootii tyrannus) on Surimi Gels

Heat stable protease isolated from the sarcoplasmic fraction of menhaden (Brevoorti tyrannus) muscle tissue was characterized as to optimum temperature and pH against casein substrate and its degradative action on actomyosin and surimi during heating. The optimum conditions for activity were 60°C at a pH of 7.5 to 8.0. Activity dropped off remarkably at temperatures below 45°C or above 70°C and when pH was below 7.0 or above 8.0. The enzyme(s) was capable of degrading actomyosin as observed by sodium dodecyl sulfate electrophoresis. This implicated a causative role for this protease system in the texture degradation observed during thermal processing of menhaden surimi at temperatures of 50-70°C.     

Antibacterial activity and action target of phenyllactic acid against Staphylococcus aureus and its application in skim milk and cheese

Phenyllactic acid (PLA) has been demonstrated to possess antibacterial activity and capacity to prolong food shelf life. However, studies on the performance of PLA in inhibiting Staphylococcus aureus and its effectiveness when applied to dairy products are largely lacking. Here, antibacterial activity (planktonic and biofilm states) of PLA against S. aureus CICC10145 (S. aureus_45) were investigated. The results showed that PLA inhibited growth of S. aureus_45 and formation of S. aureus_45 biofilm. Next, the antibacterial action target of PLA was uncovered from both physiological and phenotypic perspectives. The results showed that PLA decreased cell metabolic activity and cell viability, damaged cell membrane integrity, triggered leakage of intracellular contents (DNA, proteins, and ATP), and caused oxidative stress damage and morphological deformation of S. aureus_45. In practical application, the antibacterial activity of PLA against S. aureus_45 cells was further confirmed in skim milk and cheese as dairy food models, and the antibacterial effects can be adequately maintained during storage for 21 d, at least at 4°C. These findings suggested that PLA could be a potential candidate for controlling S. aureus outgrowth in dairy foods.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF PEACH PECTINESTERASE

Pectinesterase (EC 3.1.1.11) was extracted from peaches (Prunus persica) and partially purified by preparative free solution isoelectric focusing. On SDS-PAGE gels, protein bands at 36.3 and 33.9 kilodaltons represented the major bands; minor bands were observed at 108.4, 40.7, and 17.0 kilodaltons. The pH optimum for pectinesterase activity in the partially purified extract was 8.0. The enzyme was stable at 30°C for 30 min between pH values of 5 and 8. Peach pectinesterase is stable when heated at 55°C for 5 min in 0.1 M NaCl, 50 mM sodium phosphate, pH 7, buffer. However, residual activity decreased to 23% of 65°C for 5 min and was inactivated at 70°C for 5 min. The energy of activation of peach pectinesterase was determined to be 34, 600 J/mol °K. The Q₁₀ between 30°C and 60°C was estimated to be 1.5–1.6.     

Incorporation of Essential Oils and Nanoparticles in Pullulan Films to Control Foodborne Pathogens on Meat and Poultry Products

The incorporation of essential oils and nanotechnology into edible films has the potential to improve the microbiological safety of foods. The aim of this study was to evaluate the effectiveness of pullulan films containing essential oils and nanoparticles against 4 foodborne pathogens. Initial experiments using plate overlay assays demonstrated that 2% oregano essential oil was active against Staphylococcus aureus and Salmonella Typhimurium, whereas Listeria monocytogenes and Escherichia coli O157:H7 were not inhibited. Two percent rosemary essential oil was active against S. aureus, L. monocytogenes, E. coli O157:H7, and S. Typhimurium, when compared with 1%. Zinc oxide nanoparticles at 110 nm were active against S. aureus, L. monocytogenes, E. coli O157:H7, and S. Typhimurium, when compared with 100 or 130 nm. Conversely, 100 nm silver (Ag) nanoparticles were more active against S. aureus than L. monocytogenes. Using the results from these experiments, the compounds exhibiting the greatest activity were incorporated into pullulan films and found to inhibit all or some of the 4 pathogens in plate overlay assays. In challenge studies, pullulan films containing the compounds effectively inhibited the pathogens associated with vacuum packaged meat and poultry products stored at 4 °C for up to 3 wk, as compared to control films. Additionally, the structure and cross‐section of the films were evaluated using electron microscopy. The results from this study demonstrate that edible films made from pullulan and incorporated with essential oils or nanoparticles may improve the safety of refrigerated, fresh or further processed meat and poultry products.     

Factors affecting the susceptibility of Staphylococcus aureus CCRC 12657 to water soluble lactose chitosan derivative

In this study, the susceptibility of Staphylococcus aureus CCRC 12657 to water-soluble lactose chitosan derivative as influenced by pH, temperature and cell age were investigated. Besides, the effect of this chitosan derivative on enterotoxin production and cell leakage of S. aureus CCRC 12657 was also examined.It was found that the susceptibility of S. aureus CCRC 12657 to lactose chitosan derivative varied with incubation temperature, pH and cell age. The lactose chitosan derivative exerted a higher antibacterial activity against S. aureus CCRC 12657 at 37°C than at 22 and 5°C. At pH 6.5–7.0, S. aureus CCRC 12657 was more susceptible to the chitosan derivative than at pH 6.0 or 7.5. Besides, cells of S. aureus CCRC 12657 in the late-exponential phase (12 h) were most susceptible to the chitosan derivative followed by cells in the stationary phase (24 h) and mid-exponential phase (6 h). Supernatant of cell suspension showed a marked increase in absorbance at 280 nm after the cells of the test organism were exposed to deionized water-containing lactose chitosan derivative. Furthermore, it was also noted that the lactose chitosan derivative not only caused the death of S. aureus CCRC 12657 but also inhibited enterotoxin production in Brain–Heart infusion broth.     

The linamarase of Leuconostoc mesenteroides: Production,isolation and some properties

Leuconostoc mesenteroides was found to produce highly active linamarase when linamarin was incorporated in its growth medium. The enzyme was isolated from the bacterium and partially purified using diethylaminoethyl (DEAE) cellulose. Its activity was measured spectrophotometrically using linamarin extract. This yielded 62.2 mg CN^? g^?1 of linamarin. A study of some of its properties showed it was active in the temperature range of -10 to +45°C, with an optimum at 29°2°C. Activity was observed over a wide pH range, 4.0–8.0, with optimum at 6.0–6.5. Its pH of stability was 5.5–7.5, while above pH 8.0 there was a rapid loss of activity. Incubating the enzyme at 50°C led to loss of over 90% of its activity within 18 min. The optimal substrate concentration was 0.15–0.20 ml^?1. Whereas above 0.25 mg ml^?1 there was no observable increase in activity, loss of activity became more pronounced below 0.10 mg ml^?1 of substrate.     

Isolation and identification of a plant lysozyme from <Emphasis Type="Italic">Momordica charantia L</Emphasis>

A plant lysozyme exhibiting antifungal activity and with a molecular mass of 20.1 kDa in SDS–polyacrylamide gel electrophoresis was isolated from Momordica charantia L using a procedure that involved aqueous extraction, vacuum freeze-drying of extraction, and ion exchange chromatography on high-performance liquid chromatography on Source Q and POROS 50 HS. Its N-terminal sequence was determined to be Q-L-C-E-L-A-A-C-M-K-R-H-G-L-D, showing relatively high homology (about 80%) with that of hen egg white lysozyme. Its pI was estimated to be 9.9. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 50 °C. Moreover, the lysozyme exerted an antifungal action toward Mucorracem osus and Rhizoctonia solani, in addition to an antibacterial action against Escherichia coli and Staphylococcus aureus.     

Control of Two Major Pathogens on Fresh Poultry Using a Combination Potassum Sorbate/Karbon Dioxide Packaging Treatment

Salmonella enteriditis and Staphylococcus aureus were separately inoculated onto fresh chicken thighs prior to dipping in 0, 1.0, 2.5, or 5.0% potassium sorbate solutions adjusted to pH 6.0. Treated samples were packaged in Nylon/Plexar/Surlyn bags under air, vacuum, 20%, 60%, or 100% CO₂ atmospheres and stored at 10°C ± 1.0°C for 10 days. Changes in gaseous headspace composition, sorbate concentrations, surface pH, and microbial numbers were monitored during the storage period. S. enteriditis was more sensitive to potassium sorbate than S. aureus; growth on poultry of the latter organism was more effectively inhibited by exposure to high levels of CO₂. Increased concentrations of sorbate dip solutions in combination with higher concentrations of CO₂ in the package environment provided a more effective inhibitory system against growth of both pathogens on fresh poultry.     

Changes of glycinin conformation due to pH, heat and salt determined by differential scanning calorimetry and circular dichroism

Conformational changes of glycinin caused by either pH, heat or salt were investigated using differential scanning calorimetry (DSC) and circular dichroism (CD). Glycinin of more than 97% purity was used for the experiments. Without the presence of salt (NaCl), glycinin (protein concentration of 16%) was most stable at pH 4.5; the temperature for midpoint of transition (T_m) and enthalpy change (ΔH) were 93.5 °C and 18.1 J g^?1 protein, respectively. Both parameters decreased gradually as the pH of the protein solution was increased to 9.5. The endothermic peak of glycinin disappeared when the pH was >11.5 or <3.0. DSC results showed that with increasing of salt concentration, glycinin was substantially stabilized even in acidic (pH 3.0) and alkaline (pH 11.5) conditions; the degree of stabilization was more significantly affected at the acidic pH. The CD profiles of soy glycinin were, however, not significantly altered even in acidic and alkaline conditions. Thermal denaturation was suppressed by NaCl even in 90 °C, pH 8.0, when the salt concentration was 0.45 m . The stabilizing effects of NaCl coincided with an increase in the percentage of α‐helix in glycinin. Therefore, soy glycinin appeared to have a well‐ordered structure under the pH conditions studied, especially in the presence of NaCl. The λ_min signal at 222 nm remained unchanged up to 80 °C, but the negative intensity increased substantially when the temperature was >80 °C.     

Candida and Pseudomonas Lipase-Catalyzed Hydrolysis of Butteroil in the Absence of Organic Solvents

Candida rugosa lipase (CRL) and Pseudomonas fluorescein lipase (PFL) were used to hydrolyze a low-melting point butteroil fraction in the absence of orgaic solvents. CRL and PFL displayed pH activity optima of 6.7 and 7.8, and temperature-activity optima of 40–50°C and 35°C, respectively. Maximum activity for PFL was observed at 5% (v/v) water content. CRL showed higher specificity towards butyric acid relative to PFL after 3 hours of hydrolysis at 40°C (22.3 mol% vs 7.8 mol%, respectively). Both enzymes displayed, however, high specificities towards palmitic and oleic acids. CRL's inherent specificity towards butyric acid makes it an ideal choice for a biological catalyst to generate useful dairy flavor profiles.     

Antibacterial mechanism of adzuki bean seed coat polyphenols and their potential application in preservation of fresh raw beef

The monomeric phenolic components of adzuki bean seed coat polyphenols (ABSCPs) were analysed, and their antibacterial activity and mechanism against two tested bacteria (Escherichia coli ATCC8797 and Staphylococcus aureus ATCC12600) were evaluated for their potential application in preservation of fresh raw beef. ABSCP contained 13 kinds of monomeric phenols, and the minimum inhibition concentration (MIC) of ABSCP against E. coli and S. aureus was 1250 and 625 μg ml⁻¹, respectively. The mechanism of ABSCP against E. coli and S. aureus was associated with increased cell protein and nucleic acid leakage, increased cell membrane potential, decreased intracellular ATP concentration and morphological changes in bacterial cell. In addition, ABSCP with whey protein isolate (WPI) was applied to fresh raw beef and this remarkably retarded microbial growth by maintaining the total microbial quality below the acceptable limit of 6 lg CFU g⁻¹ for 14 days. Lower pH and total volatile base nitrogen values were observed in the coated samples during refrigerated storage (4 °C). As a result, we found that ABSCP, as a natural food bacteriostatic agent, can effectively inhibit the growth of E. coli and S. aureus and combined with WPI to apply to fresh raw beef to extend its shelf life.     

Identification and Partial Characterization of a Bacteriocin‐Like Inhibitory Substance (BLIS) from Lb. Bulgaricus K41 Isolated from Indigenous Yogurts

Forty‐two strains of Lactobacillus bulgaricus isolated from locally made yogurts were examined and compared for bacteriocin producing ability using spot on lawn assay which improved by taking photo and image processing. Lb. bulgaricus K41 exhibited the highest inhibition level against indicators. K41 Bacteriocin‐like inhibitory substance is sensitive to proteolytic enzymes (proteinase K, pepsin, and trypsin) but α‐amylase makes slight reduction in its activity and it is resistant to lipase. This antibacterial peptide is extremely heat‐stable (121 °C for 15 min) and remains active over a wide pH range (pH = 2 to 10); also nonionic detergents (Tween‐20, Tween‐80, and Triton X100) showed no effect on its activity. The inhibitory spectrum is against Gram‐positive bacteria (except Staphylococcus aureus) with extremely antilisterial activity and it is almost ineffective against Gram‐negative bacteria. The mode of its action was identified as bactericidal against Listeria monocytogenes. The properties of K41 bacteriocin‐like inhibitory substance add to its safety as a biopreservative produced by a generally recognized as safe (GRAS) bacterium suggesting it can be used in hurdle technology for ready‐to‐eat foods as one of the main sources of Listeria contaminations.