Novel touchdown-PCR method for the detection of putrescine producing Gram-negative bacteria in food products

Formation of biogenic amines may occur in food due to metabolic activities of contaminating Gram-negative bacteria. Putrescine is assumed to be the major biogenic amine associated with microbial food spoilage. Gram-negative bacteria can form putrescine by three metabolic pathways that can include eight different enzymes. The objective of this study was to design new sets of primers able to detect all important enzymes involved in the production of putrescine by Gram-negative bacteria. Seven new sets of consensual primers based on gene sequences of different bacteria were designed and used for detection of the speA, adiA, adi, speB, aguA, speC, and speF genes. A newly developed touchdown polymerase chain reaction (PCR) method using these primers was successfully applied on several putrescine-producers. Selected PCR products were sequenced and high similarity of their sequences (99–91%) with known sequences of the corresponding genes confirmed high specificity of the developed sets of primers. Furthermore, all the investigated bacteria produced both putrescine and agmatine, an intermediate of putrescine production, which was confirmed by chemical analysis. The developed new touchdown PCR method could easily be used to detect potential foodborne Gram-negative producers of putrescine. The newly developed sets of primers could also be useful in further research on putrescine metabolism in contaminating microbiota.     

Antibacterial activity of the extracts from the fruit rinds of Garcinia cowa and Garcinia pedunculata against food borne pathogens and spoilage bacteria

The crude hexane and chloroform extracts from the fruit rinds of Garcinia cowa and Garcinia pedunculata were studied for their antibacterial activity against some foodborne pathogens and spoilage bacteria such as Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentrations (MICs) of the extracts determined by the agar dilution method were ranging from 15 to 500 μg/ml and 300 to 1250 μg/ml for G. cowa and G. pedunculata, respectively. However, the hexane and chloroform extracts from the fruit rinds of G. cowa exhibited marked inhibitory effect against all the test organisms and were more effective than that of G. pedunculata extracts. The antibacterial activity of all the extracts was more pronounced against the tested Gram-positive bacteria than the tested Gram-negative bacterium. Furthermore, this study is the first report on the in vitro antibacterial activity of extracts from the fruit rinds of G. cowa and G. pedunculata.     

Isolation and identification of pentagalloylglucose with broad-spectrum antibacterial activity from Rhus trichocarpa Miquel

Rhus trichocarpa Miquel has been utilised both as a food and for medicinal purposes. In this study, we determined that the methanol extracts of the stem and leaf portions of R. trichocarpa inhibited the growth of both Gram-negative and Gram-positive bacteria. The active constituent was isolated, and identified via mass spectrometry and NMR as 1,2,3,4,6-penta-O-galloyl-β-d-glucose. The compound also evidenced a broad spectrum of antibacterial activity against both Gram-negative and Gram-positive bacteria including Staphylococcus aureus (both methicillin-resistant S. aureus and quinolone-resistant S. aureus), Bacillus subtilis, Streptococcus mutans, Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa with MRC values of 16–32 μg/ml, whereas gallate failed to inhibit the growth of Gram-negative bacteria, even at a concentration of 128 μg/ml. The antibacterial activity of penta-O-galloylglucose was restored by the addition of Fe²⁺, whereas gallate was not, thereby indicating that its antibacterial activity could be attributable to the chelation of iron. The results of the time-kill study against S. aureus and E. coli revealed that penta-O-galloylglucose exhibited bacteriostatic activity. These findings indicate that the extracts of R. trichocarpa as well as its active component, penta-O-galloylglucose, may have profound potential for the control of both Gram-positive and negative pathogens.     

Metabolomic response of Brassica rapa submitted to pre-harvest bacterial contamination

Plants are continually challenged by natural pathogens. Among a number of environmental factors, pre-harvest contamination of plants with pathogens responsible for enteric diseases in humans is of major international concern. Despite the knowledge of how bacterial attack can affect the biological system of plants, little is known about the effect of the interaction of these bacteria on plant’s metabolome. In order to investigate the metabolic change of Brassica induced by its response to different typically food borne bacteria such as Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium and Shigella flexneri, ¹H NMR and two-dimensional NMR spectra, coupled with principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were applied to Brassica rapa which had been subjected to these pathogens during plant growth. The metabolic changes were found to vary according to bacterial species; for example, Gram-positive and Gram-negative bacteria had a different effect on the Brassica metabolome. While threonine and GABA were found to be the discriminating metabolites in Gram-positive bacteria treated plants, those treated with Gram-negative bacteria exhibited a significant increase in sinapoyl-malate, caffeoyl-malate and histidine. The detailed study of the effect of type of bacteria showed that amino acids, alcohols, carbohydrates and phenols were discriminating metabolites. These results prove the potential of NMR-based metabolomics as a tool to study the interaction of these food-borne bacteria with vegetables.     

PREPARATION OF CHITIN AND CHITOSAN OLIGOMERS AND THEIR APPLICATIONS IN PHYSIOLOGICAL FUNCTIONAL FOODS

Chitin and chitosan are known to possess multiple functional properties. Chitin is insoluble in any common solvent containing organic or mineral acid as well as water. Chitosan is water-insoluble and highly viscous in dilute acidic solutions. These solubility problems may restrict their use in physiological functional foods. However, chitin and chitosan oligomers are not only water-soluble and their solutions have low viscosity values, but they may also be absorbed in the human intestine. They may have much physiological functionality in the in vivo systems. This review demonstrates that chitin and chitosan oligomers can be prepared by chemical and enzymatic hydrolyses and that the oligomers with high degrees of polymerization, especially those with six residues or more, show strong physiological activities.     

Soybean whole-plant ensiled with chitosan and lactic acid bacteria: Microorganism counts,fermentative profile,and total losses

Chitosan is a biopolymer derived from chitin deacetylation, present in the exoskeleton of crustaceans and insects. Chitosan has been evaluated as rumen modulator and silage additive due to its antimicrobial properties. The objective of this study was to determine the effects of both chitosan and a bacterial additive on microbiological quality, chemical composition, nutrient in vitro degradation, fermentative profile, and total losses of whole-soybean plant silage (SS) harvested at R6 stage. Four treatments in a factorial arrangement were randomly assigned to 40 experimental minisilos as no additives (CON), 8 g/t fresh forage of microbial inoculant (INO; Kera SIL, Kera Nutrição Animal, Bento Gonçalves, Brazil); 5 g/kg of fresh forage chitosan (CHI); and CHI + INO. Microbial inoculant was composed of Lactobacillus plantarum (4.0 × 10¹⁰ cfu/g) and Propionibacterium acidipropionici (2.6 × 10¹⁰ cfu/g). The CHI and INO alone increased counts of lactic bacteria and anaerobic bacteria and decreased counts of mold and yeast in SS. The CHI or INO alone increased in vitro degradation of dry matter, crude protein, and neutral detergent fiber, and decreased nonfiber carbohydrate content of SS. Chitosan increased NH₃-N and lactate concentrations and decreased ethanol concentration in SS. The CHI increased dry matter recovery from SS; INO increased silage aerobic stability. The combination of CHI+INO showed the lowest value of gas losses. In general, the combination of CHI and INO had small positive effects on gas losses of SS; however, both CHI or INO alone improved nutrient in vitro degradation and decreased mold and yeast in SS. Chitosan or INO utilization improves SS quality.     

Inactivation of Gram-negative pathogens in refrigerated milk by reuterin in combination with nisin or the lactoperoxidase system

Inhibitory activity of reuterin (β-hydroxypropionaldehyde) combined with the antimicrobial peptide nisin or the lactoperoxidase system (LPOS) against food-borne Gram-negative pathogens in milk refrigerated at 4 and 8 °C was investigated. At 4 °C, reuterin (8 AU/ml) and LPOS were bactericidal against Salmonella enterica, Campylobacter jejuni, Aeromonas hydrophila and Yersinia enterocolitica, whereas the only effect recorded for nisin was a slight inhibition of Escherichia coli O157:H7. At 8 °C, reuterin was bactericidal against all the Gram-negative pathogens studied. Same results were attained with LPOS, except for its effect on Y. enterocolitica which was only bacteriostatic. The combination of reuterin with nisin did not enhance the antimicrobial effect of reuterin. A strong synergistic bactericidal activity of reuterin in combination with LPOS on E. coli O157:H7 and S. enterica was observed in milk at 4 °C, and against all the Gram-negative bacteria assayed in milk refrigerated at 8 °C. The application of both antimicrobials would be a useful means to inhibit pathogenic microorganisms, which may be present in milk due to postpasteurization contamination.     

壳聚糖在果汁澄清中的应用

壳聚糖可由海洋生物虾、蟹的外壳中提取甲壳素经过脱乙酰基得到,是迄今为止发现的唯一的阳离子动物纤维和碱性多糖。作为天然澄清剂,壳聚糖属于阳性,能与果汁中属阴性的胶体物质产生静电作用,对胶体物质有很强的凝集力,因此可以使混浊的果汁变的澄清。     

Isolation and characterisation of a novel antibacterial peptide from bovine αS1-casein

Bovine casein was hydrolysed with a range of proteolytic enzymes including pepsin, trypsin, α-chymotrypsin and β-chymotrypsin, and assessed for antibacterial activity. The pepsin digest of bovine casein, which showed antibacterial activity, was fractionated using reverse phase high performance liquid chromatography and the antibacterial peptides isolated were characterised using electrospray ionisation mass spectrometry. Two antibacterial peptides were identified, a novel peptide (Cp1) which corresponded to residues 99–109 of bovine α_S1-casein and a previously reported peptide (Cp2) which corresponded to residues 183–207 of bovine α_S2-casein. The minimum inhibitory concentration (MIC) of Cp1 and Cp2 were determined against a range of bacterial cultures. Cp1 exhibited an MIC of 125 μg mL⁻¹ against all Gram-positive bacteria tested, and MIC ranging between 125 and >1000 μg mL⁻¹ against the Gram-negative bacteria tested. Cp2 was generally far more potent against the Gram-positive bacteria, exhibiting an MIC of 21 μg mL⁻¹, compared to MICs ranging from 332 to >664 μg mL⁻¹ against most of the Gram-negative bacteria tested.     

Antibacterial activity of Thai edible plants against gastrointestinal pathogenic bacteria and isolation of a new broad spectrum antibacterial polyisoprenylated benzophenone,chamuangone

Twenty-two edible plant extracts were subjected to evaluation of their antibacterial activity against some gastrointestinal pathogenic bacteria, including Escherichiacoli, Salmonella typhimurium, Salmonella typhi, Shigella sonnei and Helicobacter pylori using the disc diffusion and broth microdilution methods. Sixteen of the plant extracts exhibited antibacterial activity against one or more tested bacteria. Only Garcinia cowa leaf extracts exhibited antibacterial activity against all tested bacteria. Purification of the ethyl acetate extract of G. cowa leaves using an antimicrobial assay-guided isolation afforded a new polyprenylated benzophenone, chamuangone, that exhibited satisfactory antibacterial activity against Streptococcus pyogenes (minimum inhibitory concentration (MIC) 7.8 μg/ml), Streptococcus viridans and H. pylori (MICs 15.6 μg/ml), and Staphylococcus aureus, Bacillus subtilis and Enterococcus sp. (MICs 31.2 μg/ml).     

Isolation of antibacterial components from infusion of Caesalpinia paraguariensis bark. A bio-guided phytochemical study

The antimicrobial activities and toxicity of infusion, decoction and tincture of Caesalpinia paraguariensis Burk. bark (CPBEs) were investigated to validate its traditional use as drink additive and to identify microbicidal component(s). The minimum inhibitory concentrations (MIC) of CPBEs against aerobic bacteria (Gram-negative and Gram-positive species) were determined using standardised dilution methods. The LC₅₀ were determined by Brine Shrimp Test. CPBEs showed bacteriostatic and bactericidal activity against tested strains. The highest activity was observed for infusion (MIC:200 μg/mL) against Morganella morganii, Erwinia carotovora, Bacillus spp., Staphylococcus spp. and Enterococcus spp. The bacterial species were susceptible to CPBEs (MIC:200–1993 μg/mL) at lower concentration than sodium benzoate, a known food preservative. Two bioactive components were isolated from liophylised infusion by bio-guided chromatographic procedures; these were identified by spectrometric techniques as ellagic and 3-O-methylellagic acids. This study demonstrated that C. paraguariensis bark infusion it is safe for human consumption and a possible source of food natural preservatives.     

Chemical composition and antibacterial activity of Indian seagrasses against urinary tract pathogens

Seagrasses have a long history of being used for a variety of remedial purposes, such as the fever, skin diseases, muscle pains, wounds and stomach problems. Hence it is essential to study their bioactive metabolites and medicinal properties when considering their food applications. The chemical composition of six seagrasses were determined and evaluated for their potential to urinary tract infection bacteria (UTI). The chemical composition determined by GC–MS yielded 24 compounds. For the first time 4H-Pyran-4-one-2,3-dihydro-3,5-dihydroxy-6-methyl- (6.68%), d-allose (3.67%) and 5-Caranol, trans,trans-(+)- (2.14%) were identified from Halodule pinifolia. d-Allose is a aldo-hexose (sugar) used as a potential inhibitor of glycosidases and low-calorie carbohydrate sweeteners. Among the six seagrasses tested, H. pinifolia and Cymodocea rotundata exhibited predominant growth inhibitory activity against all the UTI bacteria. This study shows the presence of various biological metabolites in tested seagrasses that can be used effectively in food and pharmacological industries.     

壳聚糖的抑菌机制及其在肉及肉制品中的应用研究进展

肉及肉制品因微生物浸染易腐败变质,常通过添加抗氧化剂和防腐剂对肉及肉制品进行防腐保鲜。壳聚糖是一种无抗原性、无毒性、具有良好生物相容性的天然防腐剂,其在肉及肉制品的防腐保鲜、改善品质等方面有重要的作用。壳聚糖对革兰氏阴性菌和革兰氏阳性菌均有较好的抑菌作用,在肉及肉制品中具有广阔的应用前景,复合型壳聚糖更是未来肉制品抑菌发展的总趋势。本文从菌种的类型概括壳聚糖的抑菌机制,综述了壳聚糖在肉及肉制品中的防腐保鲜应用,最后对目前我国肉类工业中的壳聚糖应用瓶颈进行剖析及展望未来的研究重点,为壳聚糖在肉类工业的应用提供参考价值。     

Chitosan effects on quality properties of Greek style fresh pork sausages

The effect of chitosan (0.5% and 1%) added individually or in combination with nitrites (150 ppm) on microbiological (Total Viable Counts, Lactic acid bacteria, Pseudomonas spp., Brochothrix thermosphacta, Enterobacteriaceae, yeasts and moulds), physicochemical-chemical (pH, chemical composition, lipid oxidation) and sensory properties of fresh pork sausages stored at 4 °C for 28 days was investigated. Chitosan addition resulted in significant (p < 0.05) inhibition of microbial growth, while nitrites did not seem to protect sausages from microbial spoilage. A gradual reduction of nitrites was observed till the end of storage, when nitrites were almost depleted in all nitrite containing samples. The rate of lipid oxidation in fresh pork sausages was significantly decreased (p < 0.05) by addition of increasing levels of chitosan, while samples containing both chitosan and nitrites showed the lowest malondialdehyde (MDA) values, indicating a synergistic antioxidative effect. Consequently, the samples containing the combination of nitrites and chitosan at any level deteriorated less rapidly and were judged as more acceptable than all the other samples.     

Identification and decarboxylase activities of bacteria isolated from decomposed mahimahi (Coryphaena hippurus) after incubation at 0 and 32°C

Identification and amine-forming ability of bacterial isolates from decomposed mahimahi (Coryphaena hippurus) were studied in order to identify spoilage microflora and determine the potential for a chemical index of spoilage based on the metabolites produced. Mesophilic bacteria isolated from a fish incubated at 32°C for 24 h were essentially all Gram-negative rods; 89% of these were Vibrio alginolyticus. Strong histamine-forming (> 100 mg/100 ml) mesophiles consisted of eight cultures of Morganella morganii and one of Proteus mirabilis. Weak histamine-forming (< 10 mg/100 ml) mesophiles were all V. alginolyticus, and these comprised 15% of the isolates assigned to that species. Decarboxylation of ornithine and lysine occurred in 38 and 92%, respectively, of the mesophilic isolates. Psychrotrophic isolates obtained from a fish incubated 14 days at 0°C were predominantly Gram-negative genera. Of these, 9% were histamine-forming cultures of Alteromonas putrefaciens, a weak histamine former that produced < 1 mg/100 ml at 5 and 20°C. Decarboxylation of ornithine and lysine occurred in 13 and 15%, respectively, of the psychrotrophic isolates.     

Effects of gassericins A and T, bacteriocins produced by Lactobacillus gasseri, with glycine on custard cream preservation

Lactobacillus gasseri LA39 and LA158 isolated from human-infant feces produce bacteriocins named gassericins A and T, respectively. Both gassericins have high heat stability (121°C, 10 min), good pH tolerance (pH 2-11), and strong bactericidality against many gram-positive bacteria, especially lactic acid bacteria, and thus are expected to be effective food preservatives. A microwell plate assay against 12 strains of custard cream spoilage bacteria showed that the gassericins had broader antibacterial spectra than nisin A. Although the gassericins allowed gram-negative isolates to grow, they successfully inhibited the growth of all tested bacterial strains in microwells with the addition of glycine. Glycine was bacteriostatic against many strains except lactic acid bacteria. For practical use, gassericin A was efficiently produced by cultivation in a food-grade medium improved using cheese whey, nourishing proteose peptone, and surfactant yolk lecithin. The practical preservative effect of gassericin A and glycine was verified from the viability of 4 isolated strains, Bacillus cereus, Lactococcus lactis ssp. lactis, Achromobacter denitrificans, and Pseudomonas fluorescens, in custard creams. Custard cream containing 123 arbitrary units of gassericin A per milliliter entirely growth-inhibited the 2 gram-positive strains. In custard cream containing an insufficient amount of gassericin A (49 arbitrary units/mL), the gram-positive strains gradually grew but were completely inhibited by the addition of 0.5% (wt/wt) glycine. The 2 gram-negative strains did not multiply even in the additive-free custard cream, probably because of the unsuitable growth environment. This is the first report showing the combined effect of bacteriocin and glycine and their application for food preservation, which may be helpful for future use in the food industry.     

Diversity and assessment of potential risk factors of Gram-negative isolates associated with French cheeses

The goal of this study was to identify at the species level a large collection of Gram-negative dairy bacteria isolated from milks or semi-hard and soft, smear-ripened cheeses (cheese core or surface samples) from different regions of France. The isolates were then assessed for two risk factors, antibiotic resistance and volatile and non-volatile biogenic amine production in vitro. In total, 173 Gram-negative isolates were identified by rrs and/or rpoB gene sequencing. A large biodiversity was observed with nearly half of all Gram-negative isolates belonging to the Enterobacteriaceae family. Overall, 26 different genera represented by 68 species including potential new species were identified among the studied Gram-negative isolates for both surface and milk or cheese core samples. The most frequently isolated genera corresponded to Pseudomonas, Proteus, Psychrobacter, Halomonas and Serratia and represented almost 54% of the dairy collection. After Pseudomonas, Chryseobacterium, Enterobacter and Stenotrophomonas were the most frequently isolated genera found in cheese core and milk samples while Proteus, Psychrobacter, Halomonas and Serratia were the most frequently isolated genera among surface samples. Antibiotic resistance profiles indicated that resistances to the aminosid, imipemen and quinolon were relatively low while more than half of all tested isolates were resistant to antibiotics belonging to the monobactam, cephem, fosfomycin, colistin, phenicol, sulfamid and some from the penam families. Thirty-six% of isolates were negative for in vitro biogenic amine production. Among biogenic amine-producers, cadaverine was the most frequently produced followed by isoamylamine, histamine and putrescine. Only low levels (<75 mg/l) of tyramine were detected in vitro.     

Sensitivity of four pathogenic bacteria to Turkish thyme and oregano hydrosols

The two thyme (Thymus vulgaris L. and Thymus serpyllum L.) and three oregano (Origanum vulgare L., Origanum onites L. and Origanum majorana L.) hydrosols, widely used in food products and as drinks, were tested for their inhibitory effects against four pathogenic bacteria (Escherichia coli ATCC 25922, E. coli O157:H7 ATCC 33150, Staphylococcus aureus ATCC 2392 and Yersinia enterocolitica ATCC 1501). Two principles were used in determining the antibacterial effects of spice hydrosols: inhibition zones were determined by paper disc diffusion method, and the bacteriostatic effects, bactericidal effects and the growth inhibition levels were studied as colony forming units (cfu) in broth cultures. All tested bacteria were inhibited by all of the spice hydrosols using paper disc diffusion method. According to serial dilution method, while all spice hydrosols at 10 and 25 mL/100 mL concentrations had generally bacteriostatic activities, the same hydrosols appeared to have bactericidal effects at concentrations above 50 mL/100 mL. The most sensitive of the bacteria against the spice hydrosols was S. aureus. In addition, the most inhibitive of the spice hydrosols on the four pathogenic bacteria were O. onites L. and O. majorana L.     

Assessment of antimicrobial activity of coffee brewed in three different ways from different origins

The antimicrobial effect against pathogens such as Staphylococcus aureus, Enterecoccus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa, Escherichia coli and Salmonella choleraesius was determined in four types of coffee (Coffea arabica L. cv. Colombia, decaffeinated cv. Colombia, cv. Ethiopia and cv. Kenya). Coffee was seen to have significant activity against the growth of food spoilage bacteria. Among the Gram-positive bacteria, coffee was strongly active against S. aureus, moderately active against L. monocytogenes and had a slightly inhibitory effect against E. faecalis. However, coffee samples were found to be less active against Gram-negative bacteria. The results show that espresso Colombia coffee has better antimicrobial activity than filter and Italian coffee with significant differences (p?<?0.05). Taking into account the origin, there were significant differences (p?<?0.05) between Kenya and decaffeinated Colombia, on the one hand, and Ethiopia and Colombia coffee, on the other, the two last showing the highest antimicrobial activity. The antimicrobial activity of the coffee from different origins studied in this paper increased with concentration. Typical coffee compounds were also analysed, and only caffeic and chlorogenic acids showed any inhibitory effect against the growth of all the analysed bacteria. The antibacterial properties of coffee means that it has a promising potential as natural food ingredient to extend the shelf life of foods such as cake, cookies or biscuits, coffee flavoured with shakes, yoghurt.     

Antimicrobial Activity of Thyme (Tymus vulgaris) and Oregano (Origanum vulgare) Essential Oils against Some Food-borne Microorganisms

The aim of this study was to investigate antibacterial effects of oregano and thyme essential oils (EOs) on some food-borne bacteria. GC-MS analysis of EOs was performed in order to determine their composition and phenols were predominant constituents. The investigation of the antibacterial effects of EOs was performed on Salmonella Enteritidis, Salmonella Thyphimurium, Staphylococcus aureus, methicillin resistant Staphylococcus aureus, Escherichia coli and Bacillus cereus, and MICs were determined by broth microdilution method. EOs exhibited antibacterial activity against all tested microorganisms.