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      Exploring Coagulase-Negative Staphylococci Diversity from Artisanal Llama Sausages: Assessment of Technological and Safety Traits

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          Abstract

          Llama sausage is still produced following artisanal procedures, with the autochthonous microbiota being mainly responsible for the fermentation process. In this work, the taxonomical identification and technological-safety criteria of coagulase-negative staphylococci (CNS) isolated from two different productions of llama sausages (P: pilot and A: artisanal) were investigated. Staphylococcus ( S) equorum and S. saprophyticus were the species most frequently found in P production, followed by S. succinis and S. warneri; a wider species variability was observed in A factory being S. equorum, S. capitis, S. xylosus, S. pasteuri, S. epidermidis and S. saprophyticus as the main identified species. The technological characterization of 28 CNS strains showed their ability to hydrolyze gelatin and tributyrin together with a relevant nitrate reductase activity. Phenotypic and genotypic approaches were conducted to investigate the main safety traits. Llama’s CNS strains exhibited weak decarboxylase and hemolytic activity and low biofilm production; additionally, no enterotoxin genes were detected. Correlation analysis between phenotypic and genotypic data showed low values for the biofilm parameters, while high correlation was observed for oxacillin, ampicillin, tetracycline and aminoglycosides resistance and their genetic determinants. Data obtained may contribute to broaden knowledge about the autochthonous strains of this poorly studied fermented product, thus helping to select an appropriate combination of potential starter cultures to improve llama sausage safety and quality.

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          Improved screening procedure for biogenic amine production by lactic acid bacteria.

          An improved screening plate method for the detection of amino acid decarboxylase-positive microorganisms (especially lactic acid bacteria) was developed. The suitability and detection level of the designed medium were quantitatively evaluated by confirmation of amine-forming capacity using an HPLC procedure. The potential to produce the biogenic amines (BA) tyramine, histamine, putrescine, and cadaverine, was investigated in a wide number of lactic acid bacteria (LAB) of different origin, including starter cultures, protective cultures, type strains and strains isolated from different food products. Also, several strains of Enterobacteriaceae were examined. Modifications to previously described methods included lowering glucose and sodium chloride concentrations, and increasing the buffer effect with calcium carbonate and potassium phosphate. In addition, pyridoxal-5-phosphate was included as a codecarboxylase factor for its enhancing effect on the amino acid decarboxylase activity. The screening plate method showed a good correlation with the chemical analysis and due to its simplicity it is presented as a suitable and sensitive method to investigate the capacity of biogenic amine production by LAB. Tyramine was the main amine formed by the LAB strains investigated. Enterococci, carnobacteria and some strains of lactobacilli, particularly of Lb. curvatus. Lb. brevis and Lb. buchneri, were the most intensive tyramine formers. Several strains of lactobacilli, Leuconostoc spp., Weissella spp. and pediococci did not show any potential to produce amines. Enterobacteriaceae were associated with cadaverine and putrescine formation. No significant histamine production could be detected for any of the strains tested.
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            Bacterial diversity in typical Italian salami at different ripening stages as revealed by high-throughput sequencing of 16S rRNA amplicons.

            The bacterial diversity involved in food fermentations is one of the most important factors shaping the final characteristics of traditional foods. Knowledge about this diversity can be greatly improved by the application of high-throughput sequencing technologies (HTS) coupled to the PCR amplification of the 16S rRNA subunit. Here we investigated the bacterial diversity in batches of Salame Piacentino PDO (Protected Designation of Origin), a dry fermented sausage that is typical of a regional area of Northern Italy. Salami samples from 6 different local factories were analysed at 0, 21, 49 and 63 days of ripening; raw meat at time 0 and casing samples at 21 days of ripening where also analysed, and the effect of starter addition was included in the experimental set-up. Culture-based microbiological analyses and PCR-DGGE were carried out in order to be compared with HTS results. A total of 722,196 high quality sequences were obtained after trimming, paired-reads assembly and quality screening of raw reads obtained by Illumina MiSeq sequencing of the two bacterial 16S hypervariable regions V3 and V4; manual curation of 16S database allowed a correct taxonomical classification at the species for 99.5% of these reads. Results confirmed the presence of main bacterial species involved in the fermentation of salami as assessed by PCR-DGGE, but with a greater extent of resolution and quantitative assessments that are not possible by the mere analyses of gel banding patterns. Thirty-two different Staphylococcus and 33 Lactobacillus species where identified in the salami from different producers, while the whole data set obtained accounted for 13 main families and 98 rare ones, 23 of which were present in at least 10% of the investigated samples, with casings being the major sources of the observed diversity. Multivariate analyses also showed that batches from 6 local producers tend to cluster altogether after 21 days of ripening, thus indicating that HTS has the potential for fine scale differentiation of local fermented foods.
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              Biofilms in Food Processing Environments: Challenges and Opportunities

              This review examines the impact of microbial communities colonizing food processing environments in the form of biofilms on food safety and food quality. The focus is both on biofilms formed by pathogenic and spoilage microorganisms and on those formed by harmless or beneficial microbes, which are of particular relevance in the processing of fermented foods. Information is presented on intraspecies variability in biofilm formation, interspecies relationships of cooperativism or competition within biofilms, the factors influencing biofilm ecology and architecture, and how these factors may influence removal. The effect on the biofilm formation ability of particular food components and different environmental conditions that commonly prevail during food processing is discussed. Available tools for the in situ monitoring and characterization of wild microbial biofilms in food processing facilities are explored. Finally, research on novel agents or strategies for the control of biofilm formation or removal is summarized.
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                Author and article information

                Journal
                Microorganisms
                Microorganisms
                microorganisms
                Microorganisms
                MDPI
                2076-2607
                27 April 2020
                May 2020
                : 8
                : 5
                : 629
                Affiliations
                [1 ]DISTAS, Università Cattolica del Sacro Cuore, via Emilia Parmense 84, 29122 Piacenza, Italy; annalisa.rebecchi@ 123456unicatt.it (A.R.); daniela.bassi@ 123456unicatt.it (D.B.)
                [2 ]Biotechnology Research Centre (CRB), via Milano 24, 26100 Cremona, Italy; francesco.miragoli@ 123456unicatt.it (F.M.); constanzamaria.lopez@ 123456unicatt.it (C.L.)
                [3 ]Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Famaillá, Famaillá 4172, Tucumán, Argentina
                Author notes
                Author information
                https://orcid.org/0000-0001-9020-3853
                https://orcid.org/0000-0002-5503-7631
                Article
                microorganisms-08-00629
                10.3390/microorganisms8050629
                7284484
                32349211
                afd0e089-5553-46c1-ad49-002b54b8ebe9
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 13 March 2020
                : 02 April 2020
                Categories
                Article

                llama meat,fermented sausages,coagulase-negative staphylococci,starter cultures,safety traits

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