Contribution of omics to biopreservation: Toward food microbiome engineering.

Biopreservation is a sustainable approach to improve food safety and maintain or extend food shelf life by using beneficial microorganisms or their metabolites. Over the past 20 years, omics techniques have revolutionised food microbiology including biopreservation. A range of methods including genomics, transcriptomics, proteomics, metabolomics and meta-omics derivatives have highlighted the potential of biopreservation to improve the microbial safety of various foods. This review shows how these approaches have contributed to the selection of biopreservation agents, to a better understanding of the mechanisms of action and of their efficiency and impact within the food ecosystem. It also presents the potential of combining omics with complementary approaches to take into account better the complexity of food microbiomes at multiple scales, from the cell to the community levels, and their spatial, physicochemical and microbiological heterogeneity. The latest advances in biopreservation through omics have emphasised the importance of considering food as a complex and dynamic microbiome that requires integrated engineering strategies to increase the rate of innovation production in order to meet the safety, environmental and economic challenges of the agri-food sector.

Effect of the Manufacturing Process on the Microbiota, Organoleptic Properties and Volatilome of Three Salmon-Based Products.

Lightly preserved seafood products, such as cold-smoked fish and fish gravlax, are traditionally consumed in Europe and are of considerable economic importance. This work aimed to compare three products that were obtained from the same batch of fish: cold-smoked salmon (CSS) stored under vacuum packaging (VP) or a modified atmosphere packaging (MAP) and VP salmon dill gravlax (SG). Classical microbiological analyses and 16S rRNA metabarcoding, biochemical analyses (trimethylamine, total volatile basic nitrogen (TVBN), biogenic amines, pH, volatile organic compounds (VOCs)) and sensory analyses (quantitative descriptive analysis) were performed on each product throughout their storage at a chilled temperature. The three products shared the same initial microbiota, which were mainly dominated by Photobacterium, Lactococcus and Lactobacillus genera. On day 28, the VP CSS ecosystem was mainly composed of Photobacterium and, to a lesser extent, Lactococcus and Lactobacillus genera, while Lactobacillus was dominant in the MAP CSS. The diversity was higher in the SG, which was mainly dominated by Enterobacteriaceae, Photobacterium, Lactobacillus and Lactococcus. Although the sensory spoilage was generally weak, gravlax was the most perishable product (slight increase in amine and acidic off-odors and flavors, fatty appearance, slight discoloration and drop in firmness), followed by the VP CSS, while the MAP CSS did not spoil. Spoilage was associated with an increase in the TVBN, biogenic amines and spoilage associated VOCs, such as decanal, nonanal, hexadecanal, benzaldehyde, benzeneacetaldehyde, ethanol, 3-methyl-1-butanol, 2,3-butanediol, 1-octen-3-ol, 2-butanone and 1-octen-3-one. This study showed that the processing and packaging conditions both had an effect on the microbial composition and the quality of the final product.

Genotypic and phenotypic characterization of the food spoilage bacterium Brochothrix thermosphacta.

Microbial food spoilage is responsible for significant economic losses. Brochothrix thermosphacta is one of the major bacteria involved in the spoilage of meat and seafood. Its growth and metabolic activities during food storage result in the production of metabolites associated with off-odors. In this study, we evaluated the genotypic and phenotypic diversity of this species. A collection of 161 B. thermosphacta strains isolated from different foods, spoiled or not, and from a slaughterhouse environment was constituted from various laboratory collections and completed with new isolates. A PCR test based on the rpoB gene was developed for a fast screening of B. thermosphacta isolates. Strains were typed by MALDI-TOF MS, rep-PCR, and PFGE. Each typing method separated strains into distinct groups, revealing significant intra-species diversity. These classifications did not correlate with the ecological origin of strains. The ability to produce acetoin and diacetyl, two molecules associated with B. thermosphacta spoilage, was evaluated in meat and shrimp juices. The production level was variable between strains and the spoilage ability on meat or shrimp juice did not correlate with the substrate origin of strains. Although the B. thermosphacta species encompasses ubiquitous strains, spoiling ability is both strain- and environment-dependent.

Salmon Gravlax Biopreservation With Lactic Acid Bacteria: A Polyphasic Approach to Assessing the Impact on Organoleptic Properties, Microbial Ecosystem and Volatilome Composition.

Seafood and fishery products are very perishable commodities with short shelf-lives owing to rapid deterioration of their organoleptic and microbiological quality. Microbial growth and activity are responsible for up to 25% of food losses in the fishery industry. In this context and to meet consumer demand for minimally processed food, developing mild preservation technologies such as biopreservation represents a major challenge. In this work, we studied the use of six lactic acid bacteria (LAB), previously selected for their properties as bioprotective agents, for salmon dill gravlax biopreservation. Naturally contaminated salmon dill gravlax slices, with a commercial shelf-life of 21 days, were purchased from a French industrial company and inoculated by spraying with the protective cultures (PCs) to reach an initial concentration of 106 log CFU/g. PC impact on gravlax microbial ecosystem (cultural and acultural methods), sensory properties (sensory profiling test), biochemical parameters (pH, TMA, TVBN, biogenic amines) and volatilome was followed for 25 days of storage at 8°C in vacuum packaging. PC antimicrobial activity was also assessed in situ against Listeria monocytogenes. This polyphasic approach underlined two scenarios depending on the protective strain. Carnobacterium maltaromaticum SF1944, Lactococcus piscium EU2229 and Leuconostoc gelidum EU2249, were very competitive in the product, dominated the microbial ecosystem, and displayed antimicrobial activity against the spoilage microbiota and L. monocytogenes. The strains also expressed their own sensory and volatilome signatures. However, of these three strains, C. maltaromaticum SF1944 did not induce strong spoilage and was the most efficient for L. monocytogenes growth control. By contrast, Vagococcus fluvialis CD264, Carnobacterium inhibens MIP2551 and Aerococcus viridans SF1044 were not competitive, did not express strong antimicrobial activity and produced only few organic volatile compounds (VOCs). However, V. fluvialis CD264 was the only strain to extend the sensory quality, even beyond 25 days. This study shows that C. maltaromaticum SF1944 and V. fluvialis CD264 both have a promising potential as bioprotective cultures to ensure salmon gravlax microbial safety and sensorial quality, respectively.

Influence of lactate and acetate removal on the microbiota of French fresh pork sausages.

The microbiota of fresh French pork sausages were characterised in five batches of comminuted pork meat that were equally divided into two formulations either containing the acid-based preservatives lactate and acetate, or no preservatives. Conventional microbiological analysis and high-throughput 16S rDNA amplicon sequencing methods were performed on meat batches packed under modified atmosphere (70% oxygen and 30% carbon dioxide) during chilled storage. In addition, meat pH and colour, and gas composition of the packages were monitored until the end of the shelf-life. During storage, the population of mesophilic and lactic acid bacteria increased from 4 log CFU/g to 8 log CFU/g after 15 days of chilled storage, both with and without preservatives. Despite similar changes of the physical and chemical parameters, such as pH and package gas composition, spoilage was delayed in the meat containing the preservatives, suggesting that lactate and acetate are effective against spoilage. Metagenetic analysis showed that at the end of the shelf-life, the species distribution differed between both the formulations and the batches. Lactic acid bacteria were shown to dominate both with and without preservatives; however, samples containing no preservatives were characterised by the presence of an increased population of Brochothrix spp. and Pseudomonas spp. whereas, Leuconostoc mesenteroides/pseudomesenteroides and Lactobacillus curvatus/graminis were more abundant in the meat with preservatives.

Bioprotective Effect of Lactococcus piscium CNCM I-4031 Against Listeria monocytogenes Growth and Virulence.

Listeria monocytogenes is a Gram-positive pathogen occurring in many refrigerated ready-to-eat foods. It is responsible for foodborne listeriosis, a rare but severe disease with a high mortality rate (20-30%). Lactococcus piscium CNCM I-4031 has the capacity to prevent the growth of L. monocytogenes in contaminated peeled and cooked shrimp and in a chemically defined medium using a cell-to-cell contact-dependent mechanism. To characterize this inhibition further, the effect of L. piscium was tested on a collection of 42 L. monocytogenes strains. All strains were inhibited but had different sensitivities. The effect of the initial concentration of the protective and the target bacteria revealed that the inhibition always occurred when L. piscium had reached its maximum population density, whatever the initial concentration of the protective bacteria. Viewed by scanning electron microscopy, L. monocytogenes cell shape and surface appeared modified in co-culture with L. piscium CNCM I-4031. Lastly, L. monocytogenes virulence, evaluated by a plaque-forming assay on the HT-29 cell line, was reduced after cell pre-treatment by the protective bacteria. In conclusion, the bioprotective effect of L. piscium toward L. monocytogenes growth and virulence was demonstrated, and a hypothesis for the inhibition mechanism is put forward.

Next generation microbiological risk assessment meta-omics: The next need for integration.

The development of a multi-omics approach has provided a new approach to the investigation of microbial communities allowing an integration of data, which can be used to better understand the behaviour of and interactions between community members. Metagenomics, metatranscriptomics, metaproteomics and metabolomics have the potential of producing a large amount of data in a very short time, however an important challenge is how to exploit and interpret these data to assist risk managers in food safety and quality decisions. This can be achieved by integrating multi-omics data in microbiological risk assessment. In this paper we identify limitations and challenges of the multi-omics approach, underlining promising potentials, but also identifying gaps, which should be addressed for its full exploitation. A view on how this new way of investigation will impact the traditional microbiology schemes in the food industry is also presented.

Complete Genome Sequence of Lactococcus piscium CNCM I-4031, a Bioprotective Strain for Seafood Products.

Lactococcus piscium CNCM I-4031 is a psychotrophic foodborne lactic acid bacterium showing potential interest for the biopreservation of seafood products due to its inhibition properties toward pathogenic and spoilage bacteria. The analysis of its genome will provide a better understanding of the mechanisms of interaction between these bacteria.

Improving simultaneously the quality and safety of cooked and peeled shrimp using a cocktail of bioprotective lactic acid bacteria.

Tropical shrimp is of considerable economic importance in the world but is highly perishable due to microbial and chemical degradation. Biopreservation is a food preservation technology based on the addition of "positive" bacteria able to kill or prevent the growth of undesirable microorganisms. Two strains of lactic acid bacteria (LAB) have previously been selected for a biopreservation strategy: Lactococcus piscium CNCM I-4031, for its ability to prevent the sensory deterioration of seafood and Carnobacterium divergens V41, which inhibits growth of Listeria monocytogenes. The objective was to test the association of the two strains to improve both the quality and safety of shrimp. In a first trial, the two LAB were inoculated alone or in a cocktail in cooked and peeled shrimp (CPS) Penaeus vannamei at 5×105CFU/g. Chemical, sensory and microbiological analyses by culture-dependent and -independent methods were performed during storage under modified atmosphere packaging (MAP) at 8°C. The results were compared to a non-inoculated batch. In a second trial, the same experiments were repeated in the presence of 102CFU/g of L. monocytogenes RF191. The microbiota of CPS was composed of LAB, Shewanella spp. and Enterobacteriaceae. Brochothrix thermosphacta was not detected. L. piscium and C. divergens reached 108 and 109CFU/g, respectively, in 7days and did not inhibit each other when co-inoculated. L. piscium reduced L. monocytogenes by 1Log (CFU/g) for 28days. C. divergens had an immediate listericidal effect lasting 7days. A regrowth of L. monocytogenes was then observed but the count was always 2 to 5Log (CFU/g) lower than in the control. No additional or synergic effect between protective strains was observed and the cocktail had the same inhibitory effect as C. divergens alone. C. divergens was very effective at preventing the sensory deterioration of CPS. This may be related to the inhibition of Shewanella and Enterobacteriaceae. However, the panelists could detect the presence of C. divergens during the first 10days of storage, with slight unpleasant odors and flavors. L. piscium improved the sensory quality of CPS for 14days only. In co-culture, L. piscium eliminated the off-odors and flavors released by C. divergens in the early stage of storage and the co-culture allowed maintaining a good quality of CPS throughout the storage. Therefore, the use of a cocktail of L. piscium CNCM I-4031 and C. divergens V41 is recommended in a strategy of biopreservation of shrimp.

Draft Genome Sequence of Carnobacterium divergens V41, a Bacteriocin-Producing Strain.

In this study, we present the draft genome sequence of Carnobacterium divergens V41. This strain was previously reported as producing divercin V41, a bacteriocin of interest for food biopreservation. Its genome revealed also the presence of a gene cluster putatively involved in polyketide production, which is unique in lactic acid bacteria.

Protective Effect of Carnobacterium spp. against Listeria monocytogenes during Host Cell Invasion Using In vitro HT29 Model.

The pathogenesis of listeriosis results mainly from the ability of Listeria monocytogenes to attach, invade, replicate and survive within various cell types in mammalian tissues. In this work, the effect of two bacteriocin-producing Carnobacterium (C. divergens V41 and C. maltaromaticum V1) and three non-bacteriocinogenic strains: (C. divergens V41C9, C. divergens 2763, and C. maltaromaticum 2762) was investigated on the reduction of L. monocytogenes Scott A plaque-forming during human infection using the HT-29 in vitro model. All Carnobacteria tested resulted in a reduction in the epithelial cell invasion caused by L. monocytogenes Scott A. To understand better the mechanism underlying the level of L. monocytogenes infection inhibition by Carnobacteria, infection assays from various pretreatments of Carnobacteria were assessed. The results revealed the influence of bacteriocin production combined with a passive mechanism of mammalian cell monolayers protection by Carnobacteria. These initial results showing a reduction in L. monocytogenes virulence on epithelial cells by Carnobacteria would be worthwhile analyzing further as a promising probiotic tool for human health.

Inhibition mechanism of Listeria monocytogenes by a bioprotective bacteria Lactococcus piscium CNCM I-4031.

Listeria monocytogenes is a pathogenic Gram positive bacterium and the etiologic agent of listeriosis, a severe food-borne disease. Lactococcus piscium CNCM I-4031 has the capacity to prevent the growth of L. monocytogenes in contaminated peeled and cooked shrimp. To investigate the inhibititory mechanism, a chemically defined medium (MSMA) based on shrimp composition and reproducing the inhibition observed in shrimp was developed. In co-culture at 26 °C, L. monocytogenes was reduced by 3-4 log CFU g(-1) after 24 h. We have demonstrated that the inhibition was not due to secretion of extracellular antimicrobial compounds as bacteriocins, organic acids and hydrogen peroxide. Global metabolomic fingerprints of these strains in pure culture were assessed by liquid chromatography coupled with high resolution mass spectrometry. Consumption of glucose, amino-acids, vitamins, nitrogen bases, iron and magnesium was measured and competition for some molecules could be hypothesized. However, after 24 h of co-culture, when inhibition of L. monocytogenes occurred, supplementation of the medium with these compounds did not restore its growth. The inhibition was observed in co-culture but not in diffusion chamber when species were separated by a filter membrane. Taken together, these data indicate that the inhibition mechanism of L. monocytogenes by L. piscium is cell-to-cell contact-dependent.

Development of a real-time PCR method coupled with a selective pre-enrichment step for quantification of Morganella morganii and Morganella psychrotolerans in fish products.

Histamine fish poisoning is common and due to toxic concentrations of histamine often produced by Gram-negative bacteria in fin-fish products with a high content of the free amino acid histidine. The genus Morganella includes two species previously reported to cause incidents of histamine fish poisoning. Morganella morganii and Morganella psychrotolerans are both strong producer of histamine. However, little is known about the occurrence and critical stages for fish contamination with these bacteria. To elucidate contamination routes of Morganella, specific real-time quantitative PCR (RTi qPCR) methods for quantification of M. morganii and M. psychrotolerans have been developed. Selective primers amplified a 110 bp region of the vasD gene for M. psychrotolerans and a 171 bp region of the galactokinase gene for M. morganii. These primer-sets showed high specificity as demonstrated by using purified DNA from 23 other histamine producing bacteria and 26 isolates with no or limited histamine production. The efficiency of the qPCR reactions on artificially contaminated fish samples were 100.8% and 96.3% respectively. The limit of quantification (LOQ) without enrichment was 4 log CFU/g. A quantitative enrichment step with a selective medium was included and improved the sensitivity of the methods to a LOQ of below 50 CFU/g in seafood. RTi qPCR methods with or without enrichment were evaluated for enumeration of Morganella species in naturally contaminated fresh fish and lightly preserved seafood from Denmark. These new methods will contribute to a better understanding of the occurrence and histamine production by Morganella species in fish products, information that is essential to reduce the unacceptably high frequency of histamine fish poisoning.

Bacterial spoilers of food: behavior, fitness and functional properties.

Most food products are highly perishable as they constitute a rich nutrient source for microbial development. Among the microorganisms contaminating food, some present metabolic activities leading to spoilage. In addition to hygienic rules to reduce contamination, various treatments are applied during production and storage to avoid the growth of unwanted microbes. The nature and appearance of spoilage therefore depend on the physiological state of spoilers and on their ability to resist the processing/storage conditions and flourish on the food matrix. Spoilage also relies on the interactions between the microorganisms composing the ecosystems encountered in food. The recent rapid increase in publicly available bacterial genome sequences, as well as the access to high-throughput methods, should lead to a better understanding of spoiler behavior and to the possibility of decreasing food spoilage. This review lists the main bacterial species identified as food spoilers, their ability to develop during storage and/or processing, and the functions potentially involved in spoilage. We have also compiled an inventory of the available genome sequences of species encompassing spoilage strains. Combining in silico analysis of genome sequences with experimental data is proposed in order to understand and thus control the bacterial spoilage of food better.

Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage.

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.

Evaluation of the spoilage potential of bacteria isolated from spoiled cooked whole tropical shrimp (Penaeus vannamei) stored under modified atmosphere packaging.

The spoilage potential of isolates belonging to five bacterial groups/species (Shewanella baltica, Carnobacterium maltaromaticum, Aeromonas salmonicida, Vibrio sp., "other Gamma-Proteobacteria" [containing one strain of Pseudoalteromonas sp. and one strain of Psychrobacter sp.]) isolated from spoiled cooked and whole tropical shrimp stored under modified atmosphere packaging (MAP) was evaluated by inoculation into ionized cooked and peeled tropical shrimp followed by storage for 32 days at 8 °C. Microbial growth and sensory changes were monitored during the storage period. The major spoilage bacterial isolate groups were C. maltaromaticum and S. baltica. In order to characterize their spoilage potential further and to study the effect of their interactions, each of these two specific spoilage organisms (SSO) and one mixed-culture, C. maltaromaticum/S. baltica, were tested using a combination of complementary methods: molecular (PCR-TTGE), sensory, chemical, and conventional microbiological analyses. It was concluded that, in the mixed-culture-inoculated samples, both species groups imposed their spoilage characteristics.

Development of a rapid real-time PCR method as a tool to quantify viable Photobacterium phosphoreum bacteria in salmon (Salmo salar) steaks.

A specific real-time PCR quantification method combined with a propidium monoazide sample treatment step was developed to determine quantitatively the viable population of the Photobacterium phosphoreum species group in raw modified-atmosphere-packed salmon. Primers were designed to amplify a 350-bp fragment of the gyrase subunit B gene (gyrB) of P. phosphoreum. The specificity of the two primers was demonstrated by using purified DNA from 81 strains of 52 different bacterial species. When these primers were used for real-time PCR in pure culture, a good correlation (R(2) of 0.99) was obtained between this method and conventional enumeration on marine agar (MA). Quantification was linear over 5 log units as confirmed by using inoculated salmon samples. On naturally contaminated fresh salmon, the new real-time PCR method performed successfully with a quantification limit of 3 log CFU/g. A correlation coefficient (R(2)) of 0.963 was obtained between the PCR method and classic enumeration on MA, followed by identification of colonies (290 isolates identified by real-time PCR or by 16S rRNA gene sequencing). A good correlation with an R(2) of 0.940 was found between the new PCR method and an available specific conductance method for P. phosphoreum. This study presents a rapid tool for producing reliable quantitative data on viable P. phosphoreum bacteria in fresh salmon in 6 h. This new culture-independent method will be valuable for future fish inspection, the assessment of raw material quality in fish processing plants, and studies on the ecology of this important specific spoilage microorganism.

Evaluation of the spoilage potential of bacteria isolated from spoiled raw salmon (Salmo salar) fillets stored under modified atmosphere packaging.

The spoilage potential of eight bacterial groups/species (Serratia spp., Hafnia alvei, Brochothrix thermosphacta, Carnobacterium maltaromaticum, Shewanella baltica, Lactococcus piscium, Photobacterium phosphoreum, "other Enterobacteriaceae" [containing one strain of Moellerella sp., Morganella sp. and Pectobacterium sp.]) isolated from spoiled raw salmon fillets stored under modified atmosphere packaging (MAP) was evaluated by inoculation into sterile raw salmon cubes followed by storage for 12days at 8°C. Microbial growth and sensory changes were monitored during the storage period. The dominant spoilage bacteria were C. maltaromaticum, H. alvei and P. phosphoreum. In order to further characterize their spoilage potential and to study the effect of their interactions, each of these 3 specific spoilage organisms (SSO) and two mixed-cultures, C. maltaromaticum/H. alvei and C. maltaromaticum/P. phosphoreum were tested in the sterile salmon model system using a combination of complementary methods: molecular (PCR-TTGE), sensory, chemical and conventional microbiological analyses. It was concluded that, in the mixed-culture inoculated samples, the dominant species determined the spoilage characteristics. The volatile fraction of P. phosphoreum inoculated samples was analyzed by solid-phase microextraction (SPME) followed by gas chromatography coupled to mass spectrometry (GC-MS). Among the specific volatile compounds present on P. phosphoreum spoiled inoculated samples, acetic acid was correlated with sensory analysis and can be proposed as a raw salmon spoilage marker.

Purification and characterization of a new bacteriocin active against Campylobacter produced by Lactobacillus salivarius SMXD51.

Strain SMXD51, isolated from chicken ceca and identified as Lactobacillus salivarius, produced a component that inhibits the growth of Gram-positive and Gram-negative bacteria and especially Campylobacter jejuni. The active peptide from the cell-free supernatant of Lb. salivarius SMXD51 was purified in three steps: (i) precipitation with 80% saturated ammonium sulfate, (ii) elution on a reversed phase SPE UPTI-CLEAN cartridge using different concentrations of acetonitrile, (iii) final purification by reversed phase HPLC on a C(18) column. The mode of action of this peptide of 5383.2 Da was identified as bactericidal, and its amino acid composition was established. This new bacteriocin SMXD51 appears potentially very useful to reduce Campylobacter in poultry prior to processing.

Influence of temperature, pH and NaCl concentration on the maximal growth rate of Brochothrix thermosphacta and a bioprotective bacteria Lactococcus piscium CNCM I-4031.

The maximum specific growth rate (µ(max)) of Brochothrix thermosphacta, a spoilage bacteria of cooked peeled shrimp, and Lactococcus piscium CNCM I-4031, a bioprotective strain, was investigated under different conditions of temperature, NaCl concentrations and pH. The basic modelling approach used was the Gamma concept (γ-concept) and the model developed was then adapted to shrimp. Cardinal growth parameters were quite similar for the two strains, except for NaCl. No NaCl was required for growth and the NaCl(max) was three-times higher for B. thermosphacta than for L. piscium (62 and 23 g l(-1) respectively). However, tolerance to NaCl was higher in seafood than in liquid broth, possibly due to presence of osmoltically active molecules. L. piscium and B. thermosphacta were psychrotolerant, with T(min) = -4.8 and -3.4 °C, T(opt) = 23.4 and 27.0 °C and T(max) = 27.2 and 30.8 °C respectively. The optimal pH was neutral and growth possible till pH = 4.8 for the two strains, assuming possible applications of the bioprotective strain in lightly marinated seafood. The µ(max) of B. thermosphacta in shrimp was a little higher than in L. piscium whatever the environmental conditions. Validation of the model showed that the γ-concept was suitable for predicting µ(max) of B. thermosphacta in shrimp. Data generated in this study can be used to adapt the model to other foods with few additional experiments and the effect of different parameters may be added in the future. The model was less accurate for the bioprotective strain and the effect of NaCl must be studied in more detail directly in the matrix.