The effect of adding antimicrobial peptides to milk inoculated with Staphylococcus aureus and processed by high-intensity pulsed-electric field

The use of high-intensity pulsed-electric field (HIPEF) and antimicrobial substances of natural origin, such as enterocin AS-48 (AS-48), nisin, and lysozyme, are among the most important nonthermal preservation methods. Thus, the purpose of this study was to evaluate the combined effect on milk inoculated with Staphylococcus aureus of the addition of AS-48 with nisin or lysozyme, or both, together with the use of HIPEF. Synergy was observed in the reduction of Staph. aureus counts with the following combination methods: i) addition of AS-48 and nisin, ii) addition of AS-48 plus use of HIPEF, and iii) addition of AS-48 and nisin plus use of HIPEF. Specifically, when 28 arbitrary units/mL of AS-48 and 20 IU/mL of nisin were added to the milk, and it was treated with HIPEF for 800 μs, over 6 log reductions were observed in the microorganism. In general, Staph. aureus inactivation was dependent on HIPEF treatment time, antimicrobial doses, and medium pH. During storage of the treated milk, survivor population was related to peptide concentration and temperature. Final cell viability was influenced by the sequence in which the treatments were applied: the addition of AS-48 or AS-48 and nisin was more effective before than after HIPEF treatment. The results obtained indicate that the combination of HIPEF and antimicrobials could be of great interest to the dairy industry, although it is necessary to study further the way in which the combined treatments act.     

Culture-independent study of the diversity of microbial populations in brines during fermentation of naturally-fermented Aloreña green table olives

Aloreña table olives are naturally fermented traditional green olives with a denomination of protection (DOP). The present study focused on Aloreña table olives manufactured by small and medium enterprises (SMEs) from Valle del Guadalhorce (Southern Spain) under three different conditions (cold storage, and ambient temperature fermentations in small vats and in large fermentation tanks). The microbial load of brines during fermentation was studied by plate counting, and the microbial diversity was determined by a culture-independent approach based on PCR-DGGE analysis. The viable microbial populations (total mesophilic counts, yeasts and molds, and lactic acid bacteria — LAB) changed in cell numbers during the course of fermentation. Great differences were also observed between cold, vat and tank fermentations and also from one SME to another. Yeasts seemed to be the predominant populations in cold-fermented olives, while LAB counts increased towards the end of vat and tank fermentations at ambient temperature. According to PCR-DGGE analysis, microbial populations in cold-fermented olives were composed mostly by Gordonia sp./Pseudomonas sp. and Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. together with halophilic archaea (mainly by haloarchaeon/Halosarcina pallida and uncultured archaeon/uncultured haloarchaeon/Halorubrum orientalis) and yeasts (Saccharomyces cerevisiae and Candida cf. apicola). Vat-fermented olives stored at ambient temperature included a more diverse bacterial population: Gordonia sp./Pseudomonas sp., Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. and Thalassomonas agarivorans together with halophilic archaea and yeasts (mainly S. cerevisiae and C. cf. apicola, but also Pichia sp., and Pichia manshurica/Pichia galeiformis). Some LAB were detected towards the end of vat fermentations, including Lactobacillus pentosus/Lactobacillus plantarum and Lactobacillus vaccinostercus/Lactobacillus suebicus. Only the tank fermentation showed a clear predominance of LAB populations (Lactobacillus sp., Lactobacillus paracollinoides, and Pediococcus sp.) together with some halophilic archaea and a more selected yeast population (P. manshurica/P. galeiformis). The present study illustrates the complexity of the microbial populations in naturally-fermented Aloreña table olives.     

Preservation of Manzanilla Aloreña cracked green table olives by high hydrostatic pressure treatments singly or in combination with natural antimicrobials

High hydrostatic pressure (HHP) treatments (singly or in combination with natural antimicrobials) were tested for stabilization of Manzanilla Aloreña seasoned olives stored at 25 °C. HHP (5 min) was highly effective on yeast populations at 300 MPa or higher. No viable yeasts were detected in samples treated at 400 MPa for up to three months. Low levels of endospore-forming bacteria were always detected after HHP treatments. Addition of nisin to the brines reduced bacterial counts by 1.4 log cycles but it had no effect on yeasts when tested singly or in combination with HHP treatment (400 MPa, 5 min). Thyme oil had almost no effect on yeast concentrations, but rosemary oil reduced yeast viable counts progressively during storage. Essential oils in combination with HHP (400 MPa, 5 min) significantly (p < 0.05) reduced the concentrations of aerobic mesophilic bacteria. Low-salt brined olives purged with N₂ or supplemented with ascorbic acid and then pressurized for 5 min at 450 or 550 MPa were preserved for up to 5 months without spoilage, suggesting that the NaCl content in brines of packed Manzanilla Aloreña table olives could be reduced considerably by application of HHP as a stabilization treatment.     

Differentiation and characterization by molecular techniques of Bacillus cereus group isolates from poto poto and dégué, two traditional cereal-based fermented foods of Burkina Faso and Republic of Congo

Poto poto (a maize sourdough) and dégué (a pearl millet-based food) are two traditional African fermented foods. The molecular biology of toxigenic and pathogenic bacteria found in those foods is largely unknown. The purpose of this study was to study the phylogenetic relatedness and toxigenic potential of 26 Bacillus cereus group isolates from these traditional fermented foods. The relatedness of the isolates was evaluated with repetitive element sequence-based PCR (REP-PCR) and 16S rDNA sequencing analysis. A multiplex real-time PCR assay targeting the lef and capC genes of B. anthracis pXO1 and pXO2 plasmids and the sspE chromosomal gene of B. cereus and B. anthracis also was carried out. Melting curve analysis of the sspE amplification product was used to differentiate B. cereus from B. anthracis, and the presence of the B. cereus enterotoxin genes was determined with PCR amplification. Isolates had 15 different REP-PCR profiles, according to which they could be clustered into four groups. 16S rDNA sequencing analysis identified 23 isolates as B. cereus or B. anthracis and three isolates as B. cereus or Bacillus sp. Multiplex real-time PCR amplification indicated the absence of the lef and capC genes of B. anthracis pXO 1 and pXO2 plasmids, and melting curve analysis revealed amplification of the 71-bp sspE product typical of B. cereus in all isolates instead of the 188-bp amplicon of B. anthracis, confirming the identity of these isolates as B. cereus. Four isolates had amylolytic activity. All isolates had lecithinase activity and beta-hemolytic activity. Enterotoxin production was detected in two isolates. The emetic toxin gene was not detected in any isolate. The nheB toxin gene was detected in 19 isolates by PCR amplification; one of these isolates also contained the hblD (L1) gene. The cytotoxin K cytK-1 gene was not detected, but the cytK-2 gene was clearly detected in six isolates.     

Bacteriocin-based strategies for food biopreservation

Bacteriocins are ribosomally-synthesized peptides or proteins with antimicrobial activity, produced by different groups of bacteria. Many lactic acid bacteria (LAB) produce bacteriocins with rather broad spectra of inhibition. Several LAB bacteriocins offer potential applications in food preservation, and the use of bacteriocins in the food industry can help to reduce the addition of chemical preservatives as well as the intensity of heat treatments, resulting in foods which are more naturally preserved and richer in organoleptic and nutritional properties. This can be an alternative to satisfy the increasing consumers demands for safe, fresh-tasting, ready-to-eat, minimally-processed foods and also to develop "novel" food products (e.g. less acidic, or with a lower salt content). In addition to the available commercial preparations of nisin and pediocin PA-1/AcH, other bacteriocins (like for example lacticin 3147, enterocin AS-48 or variacin) also offer promising perspectives. Broad-spectrum bacteriocins present potential wider uses, while narrow-spectrum bacteriocins can be used more specifically to selectively inhibit certain high-risk bacteria in foods like Listeria monocytogenes without affecting harmless microbiota. Bacteriocins can be added to foods in the form of concentrated preparations as food preservatives, shelf-life extenders, additives or ingredients, or they can be produced in situ by bacteriocinogenic starters, adjunct or protective cultures. Immobilized bacteriocins can also find application for development of bioactive food packaging. In recent years, application of bacteriocins as part of hurdle technology has gained great attention. Several bacteriocins show additive or synergistic effects when used in combination with other antimicrobial agents, including chemical preservatives, natural phenolic compounds, as well as other antimicrobial proteins. This, as well as the combined use of different bacteriocins may also be an attractive approach to avoid development of resistant strains. The combination of bacteriocins and physical treatments like high pressure processing or pulsed electric fields also offer good opportunities for more effective preservation of foods, providing an additional barrier to more refractile forms like bacterial endospores as well. The effectiveness of bacteriocins is often dictated by environmental factors like pH, temperature, food composition and structure, as well as the food microbiota. Foods must be considered as complex ecosystems in which microbial interactions may have a great influence on the microbial balance and proliferation of beneficial or harmful bacteria. Recent developments in molecular microbial ecology can help to better understand the global effects of bacteriocins in food ecosystems, and the study of bacterial genomes may reveal new sources of bacteriocins.     

Efficacy of enterocin AS-48 against bacilli in ready-to-eat vegetable soups and purees

The broad-spectrum bacteriocin enterocin AS-48 was tested for biopreservation of ready-to-eat vegetable foods (soups and purees) against aerobic mesophilic endospore-forming bacteria. By adding AS-48 (10 microg/ml), Bacillus cereus LWL1 was completely inhibited in all six vegetable products tested (natural vegetable cream, asparagus cream, traditional soup, homemade-style traditional soup, vegetable soup, and vichyssoise) for up to 30 days at 6, 15, and 22 degrees C. A collection of strains isolated from spoiled purees showed slightly higher resistance to AS-48 in the order Paenibacillus sp. > Bacillus macroides > B. cereus, although they were also completely inhibited in natural vegetable cream by AS-48 at 10 microg/ml. However, cocktails of five or eight strains composed of B. cereus (three strains), B. macroides (two strains), and Paenibacillus sp., Paenibacillus polymyxa, and Paenibacillus amylolyticus showed higher bacteriocin resistance with AS-48 of up to 50 microg/ml required for complete inactivation in natural vegetable cream stored at 22 degrees C. Repetitive extragenic palindromic sequence-based PCR (REP-PCR) analysis showed that paenibacilli (along with some B. cereus) was the predominant survivor in the cocktails after bacteriocin treatment. To increase the effectiveness of enterocin AS-48, the bacteriocin was tested (at 20 microg/ml) against the eight-strain cocktail in natural vegetable cream in combination with other antimicrobials. The combination of AS-48 and nisin had a slight but significant additive effect. Bactericidal activity was greatly enhanced by phenolic compounds (carvacrol, eugenol, geraniol, and hydrocinnamic acid), achieving a rapid and complete inactivation of bacilli in the tested puree at 22 degrees C.     

Treatment of vegetable sauces with enterocin AS-48 alone or in combination with phenolic compounds to inhibit proliferation of Staphylococcus aureus

The antimicrobial activity of enterocin AS-48 against Staphylococcus aureus was tested in vegetable sauces, alone and in combination with phenolic compounds. When added alone at 25 microg/ml, AS-48 inactivated all detectable staphylococci in napoletana and pesto sauces stored at 22 degrees C, but it only caused limited growth inhibition when these sauces were stored at 10 degrees C, as well as in other sauces such as carbonara and green sauce for fish. At 80 microg/ml, AS-48 eliminated all detectable staphylococci in napoletana, pesto, and green sauce for fish regardless of storage temperature, but it still had much more limited effect in carbonara sauce. Antistaphylococcal activity was potentiated significantly when AS-48 was used in combination with the phenolic compounds carvacrol, geraniol, eugenol, terpineol, caffeic acid, p-coumaric acid, citral, and hydrocinnamic acid. The efficacy of the combined treatments depended both on the phenolic compound and the type of sauce. In carbonara sauce stored at 22 degrees C, the combinations of 80 microg/ml AS-48 and 20 mM hydrocinnamic acid or 126 mM carvacrol reduced viable counts of staphylococci below detection limits for up to 30 days.     

Isolation and characterization of a nisin-like bacteriocin produced by a Lactococcus lactis strain isolated from charqui,a Brazilian fermented,salted and dried meat product

A Lactococcus lactis subsp. lactis strain (L. lactis 69) capable to produce a heat-stable bacteriocin was isolated from charqui, a Brazilian fermented, salted and sun-dried meat product. The bacteriocin inhibited, in vitro, Listeria monocytogenes, Staphylococcus aureus, several lactic acid bacteria isolated from foods and spoilage halotolerant bacteria isolated from charqui. The activity of the bacteriocin was not affected by pH (2.0–10.0), heating (100 °C), and chemical agents (1% w/v). Treatment of growing cells of L. monocytogenes ScottA with the cell-free supernatant of L. lactis 69 resulted in complete cell inactivation. L. lactis 69 harbored the gene for the production of a nisin-like bacteriocin, and the amino acid sequence of the active peptide was identical to sequences previously described for nisin Z. However, differences were observed regarding the leader peptide. Besides, the isolate was able to survive and produce bacteriocins in culture medium with NaCl content up to 20%, evidencing a potential application as an additional hurdle in the preservation of charqui.     

Effect of enterocin AS-48 in combination with biocides on planktonic and sessile Listeria monocytogenes

Enterocin AS-48 was tested on a cocktail of Listeria monocytogenes strains in planktonic and sessile states, singly or in combination with biocides benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide, triclosan, poly-(hexamethylen guanidinium) hydrochloride, chlorhexidine, hexachlorophene, and the commercial sanitizers P3 oxonia and P3 topax 66. Combinations of sub-inhibitory bacteriocin concentrations and biocide concentrations 4 to 10-fold lower than their minimum inhibitory concentrations (MIC) completely inhibited growth of the planktonic listeriae. Inactivation of Listeria in biofilms formed on polystyrene microtiter plates required concentrations of enterocin AS-48 greater than 50 μg/ml, and biocide concentrations ten to 100-fold higher. In combination with enterocin AS-48 (25 or 50 μg/ml), microbial inactivation increased remarkably for all biocides except P3 oxonia and P3 topax 66 solutions. Polystyrene microtiter plates conditioned with enterocin solutions (0.5-25 μg/ml) decreased the adherence and biofilm formation of the L. monocytogenes cell cocktail, avoiding biofilm formation for at least 24 h at a bacteriocin concentration of 25 μg/ml.     

Biodiversity of the microbial community in a Spanish farmhouse cheese as revealed by culture-dependent and culture-independent methods

The microbial diversity within Alberquilla cheese, made from a spontaneously fermented mixture of raw goats' and sheep's milk in the Alpujarra mountains (Granada, south-east Spain), has been studied by the classical culturing method and also by molecular analysis of community DNA. A collection of 206 isolates was obtained from the cheese on different selective/differential media, which were then re-grouped to 52 after randomly amplified polymorphic DNA (RAPD)-PCR analyses. Isolates on Man-Rogosa and Sharpe-agar (MRS), M17-glucose agar and Kenner Fecal (KF)-agar medium were identified by specific PCR or 16S rRNA gene sequencing and belonged mainly to the lactic-acid bacteria group. The predominant genus was Lactobacillus, which accounted for more than 50% of the isolates, the most abundant species being Lactobacillus paracasei, followed by considerably less quantities of Lb. plantarum and Lb. brevis. Other lactic-acid bacteria identified were Pediococcus urinaequi, Leuconostoc pseudomesenteroides, Leuc. mesenteroides, Lactococcus lactis and even the enterococci Enterococcus faecium and E. devriesei. Cluster analyses of RAPD-PCR patterns revealed a high degree of diversity among the lactobacilli. The Gram-negative bacterial strains belonged mainly to Hafnia alvei species. The microbes occurring in Alberquilla cheese were also studied by PCR temporal temperature-gradient gel electrophoresis (TTGE) of the 16S rRNA V3 region and partial 16S rRNA sequencing of the TTGE bands. The results showed a major presence of lactic-acid bacteria closely related to Lc. lactis, Lb. paracasei, Lb. plantarum, Lb. brevis, Lb. acidophilus and Enterococcus sp. The non-lactic-acid bacterium detected was identified as Escherichia coli. All the Enterococcus strains showed great susceptibility to the most clinically relevant antibiotics, harbouring only the virulence gene efaAfm. On the basis of their antimicrobial activity against Listeria monocytogenes we chose two strains of Ln. mesenteroides that produced mesenterocin B105 and mesenterocin Y105, as revealed by PCR techniques.