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Lactococcus (Lc.) paracarnosus and the phylogenetically closely related Lc. carnosus species are common members of the microbiota in meat stored under modified atmosphere and at low temperature. The effect of these strains on meat spoilage is controversially discussed. While some strains are known to cause spoilage, others are being studied for their potential to suppress the growth of spoilage and pathogenic bacteria. In this study, Lc. paracarnosus DSM 111017T was selected based on a previous study for its ability to suppress the growth of meat spoilers, including Brochothrix thermosphacta. The mechanism by which this bioprotective strain inhibits competing bacteria and how it contributes to spoilage are not yet known. To answer these two questions, we investigated the effect of four different headspace gas mixtures (simulated air (21 % O2/79 % N2); HiOx-MAP (70 % O2/30 % CO2); nonOx-MAP (70 % N2/ 30 % CO2); simulated vacuum (100 % N2) and the presence of Brochothrix (B.) thermosphacta TMW 2.2101 on the growth and transcriptional response of Lc. paracarnosus DSM 111017T when cultured on a meat simulation agar surface at 4 °C. Analysis of genes specifically upregulated by the gas mixtures used revealed metabolic pathways that may lead to different levels of spoilage metabolites production. We propose that under elevated oxygen levels, Lc. paracarnosus preferentially converts pyruvate from glucose and glycerol to uncharged acetoin/diacetyl instead of lactate to counteract acid stress. Due to the potential production of a buttery off-flavour, the strain may not be suitable as a protective culture in meat packaged under high­oxygen conditions. 70 % N2/ 30 % CO2, simulated vacuum- and the presence of Lc. paracarnosus inhibited the growth of B. thermosphacta TMW 2.2101. However, B. thermosphacta did not affect gene regulation of metabolic pathways in Lc. paracarnosus, and genes previously predicted to be involved in B. thermosphacta growth suppression were not regulated at the transcriptional level. In conclusion, the study indicates that the gas mixture used in packaging significantly affects the metabolism and spoilage potential of Lc. paracarnosus and its ability to inhibit B. thermosphacta growth.

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In this work, two autochthonous LAB strains (Lactiplantibacillus paraplantarum BPF2 and Pediococcus acidilactici ST6), isolated from spontaneously fermented sausages produced in Spain, were tested to produce Spanish fermented sausages (salchichón) in pilot plants, due to their promising technological and anti-listerial activity. These products were compared with a sample obtained with a commercial starter (RAP) and a spontaneously fermented control sample. Physico-chemical parameters, microbial counts, metagenomic analysis, biogenic amines content and organoleptic profile of the obtained samples were studied to assess the performances of the native starters. In fact, traditional and artisanal products obtained through spontaneous fermentations can represent an important biodiversity reservoir of strains to be exploited as new potential starter cultures, to improve the safety, quality and local differentiation of traditional products. The data underlined that ST6 strain resulted in a final lower percentage if compared with the other LAB used as starter cultures. The use of starters reduced the BA concentration observed in the sausages obtained with spontaneous fermentation and the BPF2 and ST6 strains were able to decrease the level of products rancidity. Moreover, a challenge test against L. monocytogenes were performed. The data confirmed the effectiveness in the inhibition of L. monocytogenes by the two bacteriocinogenic strains tested, with respect to RAP and control samples, highlighting their ability to produce bacteriocins in real food systems. This work demonstrated the promising application in meat industry of these autochthonous strains as starter cultures to improve sensory differentiation and recognizability of typical fermented sausages.

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The main objective of this study was to innovate soft and semi-cooked sheep milk cheese production processes with the use of a commercial protective culture able to control Listeria monocytogenes growth. A freeze-dried commercial culture of Lactobacillus plantarum was tested in DS cheese and PS cheese, two types of pasteurized sheep milk, raw-paste cheeses aged for no less than 20 and 30 days respectively. In the first step, in vitro tests were conducted to identify the most suitable matrix for the growth of L. plantarum in order to create a subculture that could be used at industrial cheese-making plants. During the second phase of the study, L. plantarum culture was introduced in the manufacturing process of the cheeses in a production plant. Finally, a challenge test was conducted on portioned DS and PS cheeses in order to evaluate the activity of the protective culture against L. monocytogenes: the cheeses were portioned, experimentally contaminated with L. monocytogenes strains, vacuum packed and stored at +4°C (correct storage conditions) and at +10°C (thermal abuse). Cheeses were analysed at the end of the shelf-life to evaluate the presence and growth of L. monocytogenes, to enumerate lactic acid bacteria and determine chemicalphysical features. The results confirmed that protective cultures are a useful technological innovation to control L. monocytogenes growth during cheese storage without altering composition, microflora and chemical- physical characteristics of the product. However, the use of protective cultures should be applied as an integration of risk control measures and not as a substitute for preventive actions.

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Listeria monocytogenes contamination that occurs during and post-processing of dairy products is a serious concern for consumers, and bioprotective cultures can be applied to control the growth of the pathogen in sheep milk cheeses. However, to respect specifications provided for protected designation of origin (PDO) cheeses, only autochthonous microorganisms can be used as bioprotective cultures in these products. This in vitro study aimed to evaluate thermophilic lactic acid bacteria (LAB) isolated from sheep milk as bio-preservative agents to control L. monocytogenes growth in PDO cheese. Results were compared with those obtained with a commercial protective culture (cPC) composed of a Lactiplantibacillus plantarum bacteriocin producer designed to inhibit L. monocytogenes growth in cheese. The in vitro antilisterial activities of n.74 autochthonous LAB and a cPC were tested against 51 L. monocytogenes strains using an agar well diffusion assay. In addition, 16S rRNA sequencing of LAB isolates with antilisterial activity was conducted and strains of Lactobacillus helveticus, Lactobacillus delbrueckii subsp. indicus, Lactobacillus delbrueckii subsp. sunkii, Lactobacillus delbrueckii subsp. lactis and Enterococcus faecalis were identified. In this study, 33.6% (74/220) bacterial strains isolated from milk had characteristics compatible with thermophilic LAB, of which 17.6% (13/74) had in vitro antilisterial activity. These results demonstrate that raw sheep milk can be considered an important source of autochthonous thermophilic LAB that can be employed as protective cultures during the manufacturing of Sardinian PDO cheeses to improve their food safety. The use of bioprotective cultures should be seen as an additional procedure useful to improve cheese safety along with the correct application of good hygienic practices during manufacturing and the post-processing stages.

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Microbial stability of fresh pasta depends on heat treatment, storage temperature, proper preservatives, and atmosphere packaging. This study aimed at improving the microbial quality, safety, and shelf life of fresh pasta using modified atmosphere composition and packaging with or without the addition of bioprotective cultures (Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium spp., and Bacillus coagulans) into semolina. Three fresh pasta variants were made using (i) the traditional protocol (control), MAP (20:80 CO2:N2), and barrier packaging, (ii) the experimental MAP (40:60 CO2:N2) and barrier packaging, and (iii) the experimental MAP, barrier packaging, and bioprotective cultures. Their effects on physicochemical properties (i.e., content on macro elements, water activity, headspace O2, CO2 concentrations, and mycotoxins), microbiological patterns, protein, and volatile organic compounds (VOC) were investigated at the beginning and the end of the actual or extended shelf-life through traditional and multi-omics approaches. We showed that the gas composition and properties of the packaging material tested in the experimental MAP system, with or without bioprotective cultures, positively affect features of fresh pasta avoiding changes in their main chemical properties, allowing for a storage longer than 120 days under refrigerated conditions. These results support that, although bioprotective cultures were not all able to grow in tested conditions, they can control the spoilage and the associated food-borne microbiota in fresh pasta during storage by their antimicrobials and/or fermentation products synergically. The VOC profiling, based on gas-chromatography mass-spectrometry (GC-MS), highlighted significant differences affected by the different manufacturing and packaging of samples. Therefore, the use of the proposed MAP system and the addition of bioprotective cultures can be considered an industrial helpful strategy to reduce the quality loss during refrigerated storage and to increase the shelf life of fresh pasta for additional 30 days by allowing the economic and environmental benefits spurring innovation in existing production models.

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Sardinian fermented sausage "Salsiccia Sarda" is a Mediterranean-style, semi-dry, fermented, RTE product, representing the main pork meat product in Sardinia (Italy). The high variability that characterizes the technological processes applied in different production plants results in sausages with different chemico-physical features sometimes permissive for the growth of Listeria monocytogenes. In order to guarantee the hygienic-sanitary quality of the final product and to innovate the manufacturing process, the main objective of this study was to evaluate the use of different commercial protective cultures to control L. monocytogenes growth in the Sardinian fermented sausage. In the first step, in vitro tests were carried out to evaluate the effectiveness of five freeze-dried bioprotective cultures availabe on the market in limiting the growth of L. monocytogenes. The two protective cultures that showed the best in vitro results were selected for a challenge test on artificially contaminated Sardinian fermented sausages. Moreover, the protective culture that showed the best results in inhibiting the growth of L. monocytogenes according to in vitro and challenge test experiments, was included into real production settings and validated in three producing plants. As a result, it was observed that protective cultures represent an important technological innovation for the Sardinian fermented sausage processing plants as they allow to control L. monocytogenes growth without altering the composition, the microflora and the chemical-physical characteristics of the product, thus ensuring safety and quality. Protective cultures also showed to reduce Enterobacteriaceae mean levels at the end of ripening and not to affect the natural concentration of lactic acid bacteria and coagulase-negative staphylococci.

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Currently, consumption of spontaneously fermented milks is common in Algeria, making it a feasible source of diverse lactic acid bacteria (LAB) with the potential to be used as adjunct cultures to improve quality and safety of fermented dairy products. In this context, to select eligible indigenous strains which could be applied as bioprotective and/or starter cultures, the present study aimed to characterize the genomic variability, biotechnological potential, and safety of thirty-eight LAB isolated from Algerian dairy and farm sources of western Algeria. The isolates were unequivocally identified by 16S rRNA gene and fingerprint-based methods. The following species were identified: Enterococcus faecium (n = 15), Enterococcus durans (n = 2), Enterococcus hirae (n = 2), Enterococcus lactis (n = 1), Lactiplantibacillus plantarum (n = 6), Lactococcus lactis (n = 4), Levilactobacillus brevis (n = 3), Lacticaseibacillus paracasei (n = 3), Lacticaseibacillus rhamnosus (n = 1), and Pediococcus acidilactici (n = 1). Among the strains, three of them, L. lactis LGMY8, Lb. plantarum LGMY30 and Lb. paracasei LGMY31 were safe and showed some valuable biotechnological properties, such as high acidification, proteolytic activity, EPS production, and inhibition of undesirable bacteria that made them powerful candidates to be used as starter.

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In this study, the inhibitory capacity of Lactobacillus sakei strain L115 against Listeria monocytogenes has been assayed at 4, 8, 11, 15 and 20 °C in broth culture. Besides, the use of predictive microbiology models for describing growth of both microorganisms in monoculture and coculture has been proposed. A preliminary inhibitory test confirmed the ability of Lb. sakei strain L115 to prevent the growth of a five-strain cocktail of L. monocytogenes. Next, the growth of microorganisms in isolation, i.e. in monoculture, was monitored and kinetic parameters maximum specific growth rate (µsp;max) and maximum population density (Nmax) were estimated by fitting the Baranyi model to recorded data. Inhibition coefficients (α) were calculated for the two kinetic parameters tested (µsp:max and Nmax) to quantify the percentage of reduction of growth when the microorganisms were in coculture in comparison with monoculture. The kinetic parameters were input into three interaction models, developed based on modifications of the Baranyi growth model, namely Jameson effect, new modified version of the Jameson effect and Lotka-Volterra models. Two approaches were utilized for simulation, one using the monoculture µsp;max, under the hypothesis that the growth potential is similar under monoculture and coculture conditions provided the environmental conditions are not modified, and the other one, based on adjusting the monoculture kinetic parameter by applying the corresponding α to reproduce the observed µsp;max under coculture conditions, assuming, in this approach, that the existence of a heterogeneous population can change the growth potential of each microbial population. It was observed that in coculture, µsp;max of L. monocytogenes decreased (e.g., α = 31% at 4 °C) and the Nmax was much lower than that of monoculture (e.g., α = 36% at 4 °C). The best simulation performance was achieved applying α to adjust the estimated monoculture growth rate, with the modified Jameson and Lotka-Volterra models showing better fit to the observed microbial interaction data as demonstrated by the fact that 100% data points fell within the acceptable simulation zone (±0.5 log CFU/mL from the simulated data). More research is needed to clarify the mechanisms of interaction between the microorganisms as well as the role of temperature.

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The enterohemorrhagic Escherichia (E.) coli (EHEC) is a pathogen of great concern for public health and the meat industry all over the world. The high economic losses in meat industry and the high costs of the illness highlight the necessity of additional efforts to control this pathogen. Previous studies have demonstrated the inhibitory activity of Enterococcus mundtii CRL35 towards EHEC, showing a specific proteomic response during the co-culture. In the present work, additional studies of the EHEC-Ent. mundtii interaction were carried out: i) differential protein expression of E. coli O157:H7 NCTC12900 growing in co-culture with Ent. mundtii in a meat environment, ii) the reciprocal influence between these two microorganisms in the adhesion to extracellular matrix (ECM) proteins and iii) the possible induction of the phage W933, coding for Shiga toxin (Stx1), by Ent. mundtii CRL35. Proteomic analysis showed a significant repression of a number of E. coli NCTC12900 proteins in co-culture respect to its single culture, these mostly related to the metabolism and transport of amino acids and nucleotides. On the other hand, statistically significant overexpression of EHEC proteins involved in stress, energy production, amino acid metabolism and transcription was observed at 30 h respect to 6 h when EHEC grew in co-culture. Data are available via ProteomeXchange with identifier PXD014588. Besides, EHEC showed a decreased adhesion capacity to ECM proteins in the presence of the bioprotective strain. Finally, Ent. mundtii CRL35 did not induce the lytic cycle of W933 bacteriophage, thus indicating its potential safe use for eliminating this pathogen. Overall, this study expands the knowledge of EHEC- Ent. mundtii CRL35 interaction in a meat environment, which will certainly contribute to find out effective biological strategies to eliminate this pathogen.

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In the context of growing consumer demand for clean label foods, antifungal cultures offer alternatives to chemical preservatives for the reduction of food fungal spoilage. Selected binary combinations of lactobacilli strains were recently successfully used to inhibit Penicillium commune and Mucor racemosus in four dairy products. Our aim was to identify the compounds most likely involved in their antifungal activity. Four chromatographic methods, targeting 56 antifungal compounds as well as volatiles, were combined. Overall, 53 antifungal compounds were detected, of which 33 were in significantly higher amounts in at least one product inoculated with an antifungal culture compared to the controls. They were present at concentrations below their MIC and thus could act in synergy. Among them, the most commonly identified were acetic, hydroxyphenyllactic, phenyllactic, 3-phenylpropanoic, 3-(4-hydroxyphenyl)propanoic and 5-oxopyrrolidine-2-carboxylic acids, diacetyl, acetoin, and an unidentified volatile. This extensive study contributes to improve the knowledge about the action mode of antifungal lactobacilli.

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Human infection by Enterohemorrhagic Escherichia (E.) coli (EHEC) occurs through the ingestion of contaminated foods such as milk, vegetable products, water-based drinks, and particularly minced meats. Indeed EHEC is a pathogen that threatens public health and meat industry. The potential of different Lactic Acid Bacteria (LAB) strains to control EHEC in a meat-based medium was evaluated by using a simple and rapid method and by analyzing the growth kinetics of co-cultures (LAB-EHEC) in a meat-based medium. The activity of LAB toward EHEC in co-cultures showed variable inhibitory effect. Although, LAB were able to control EHEC, neither the produced acid nor bacteriocins were responsible of the inhibition. The bacteriocinogenic Enteroccus (Ent.) mundtii CRL35 presented one of the highest inhibition activities. A proteomic approach was used to evaluate bacterial interaction and antagonistic mechanisms between Ent. mundtii and EHEC. Physiological observations, such as growth kinetics, acidification ability and EHEC inhibitory potential were supported by the proteomic results, demonstrating significant differences in protein expression in LAB: (i) due to the presence of the pathogen and (ii) according to the growth phase analyzed. Most of the identified proteins belonged to carbohydrate/amino acid metabolism, energy production, transcription/translation, and cell division. These results contribute to the knowledge of competition strategies used by Ent. mundtii during its co-culture with EHEC setting new perspectives for the use of LAB to control this pathogen in meat.

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The use of sourdough fermented with specific strains of antifungal lactic acid bacteria can reduce chemical preservatives in bakery products. The main objective of this study was to investigate the production of antifungal carboxylic acids after sourdough fermentation of quinoa and rice flour using the antifungal strains Lactobacillus reuteri R29 and Lactobacillus brevis R2Δ as bioprotective cultures and the non-antifungal L. brevis L1105 as a negative control strain. The impact of the fermentation substrate was evaluated in terms of metabolic activity, acidification pattern and quantity of antifungal carboxylic acids. These in situ produced compounds (n=20) were extracted from the sourdough using a QuEChERS method and detected by a new UHPLC-MS/MS chromatography. Furthermore, the sourdough was applied in situ using durability tests against environmental moulds to investigate the biopreservative potential to prolong the shelf life of bread. Organic acid production and TTA values were lowest in rice sourdough. The sourdough fermentation of the different flour substrates generated a complex and significantly different profile of carboxylic acids. Extracted quinoa sourdough detected the greatest number of carboxylic acids (n=11) at a much higher concentration than what was detected from rice sourdough (n=9). Comparing the lactic acid bacteria strains, L. reuteri R29 fermented sourdoughs contained generally higher concentrations of acetic and lactic acid but also the carboxylic acids. Among them, 3-phenyllactic acid and 2-hydroxyisocaproic acid were present at a significant concentration. This was correlated with the superior protein content of quinoa flour and its high protease activity. With the addition of L. reuteri R29 inoculated sourdough, the shelf life was extended by 2 days for quinoa (+100%) and rice bread (+67%) when compared to the non-acidified controls. The L. brevis R2Δ fermented sourdough bread reached a shelf life of 4 days for quinoa (+100%) and rice (+33%). However, the shelf life was similar to the chemically acidified control indicating that the preservation effect of the carboxylic acids seems to have a minor contribution effect on the antifungal activity in gluten-free breads.

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Cooked hams have gained an important position within the delicatessen market. Nowadays, consumers not only demand superior sensory properties but also request low levels of sodium and fat and the absence of conventional chemicals and preservatives used for the increase of the technological yield and shelf-life of the products. As a result, products that apply strict quality certificates or ''clean'' labels become increasingly important. However, such cooked hams suffer from a limited shelf-life. Besides some physicochemical effects, this is mainly due to microbial impact, despite the application of modified-atmosphere-packaging and chilling. Microbial spoilage is mostly due to the metabolic manifestation of lactic acid bacteria and Brochothrix thermosphacta, although Enterobacteriaceae and yeasts may occur too. Several preservation strategies have been developed to prolong the shelf-life of such vulnerable cooked meat products by targeting the microbial communities, with different rates of success. Whereas high-pressure treatments do not always pose a straightforward solution, a promising strategy relates to the use of bioprotective cultures containing lactic acid bacteria. The latter consist of strains that are deliberately added to the ham to outcompete undesirable microorganisms. Spoilage problems seem, however, to be specific for each product and processing line, underlining the importance of tailor-made solutions.

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