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The use of protective microbial strains in combination with modified atmosphere packaging (MAP) and refrigerated storage on the shelf life of tuna burgers was investigated. Preliminary, the protective ability of three lactic acid bacteria (LAB) cultures (Lactobacillus casei, Lactobacillus paracasei and Lactobacillus plantarum) have been assessed on ready-to-cook tuna fish burgers. Among them, L. paracasei showed the best preserving performance and significantly controlled both aerobic mesophilic bacteria and Pseudomonas spp. growth. Subsequently, the efficacy of the selected LAB culture under MAP conditions (5% O2 and 95% CO2) was assessed evaluating microbial and sensory quality, as well as volatile aldehydes content. Results indicated that the shelf life of burgers containing L. paracasei and packaged under MAP was 4 days longer than the control (shelf life about 6 days) and that the applied procedure represents an effective approach for the mild preservation of fish products.

Introduction Lactobacillus plantarum and Lactobacillus paracasei are species generally used as starters in food fermentation and/or as probiotics. The growth cardinal values of these strains, characteristic parameters independent from the food matrix, can be exploited in predictive microbiology to set the appropriate food processing/manufacturing conditions. The aim of this study was to transfer available and validated mathematical models used to ensure food safety to technological microflora in order to assess the impact of environmental conditions on bacterial growth for further industrial applications. Material and methods A total of 4 strains of L. plantarum isolated from sourdough and table olives as well as 4 strains of L. paracasei strains isolated from table olives and probiotic human isolates were investigated to determine growth cardinal values. For that purpose strains were grown in liquid medium and incubated at nine temperature levels from 5.5 to 40°C. The growth was automatically monitored by a Bioscreen C using the turbidimetry method or determined manually after static incubation. Maximum growth rates (?max) for each temperature were obtained fitting data by the Rosso model. To estimate the cardinal growth values, the ?max values relevant to each temperature were fitted to the growth cardinal model using Sym'Previus decision making tool (www.symprevius.eu). Results Cardinal values were determined for Lactobacillus plantarum strains with the following average values for Tmin 2.05±0.54°C, Topt 33.74±0.63 °C and Tmax 39.79±0.50°C. In the case of L. paracasei strains, a wider diversity was obtained with average values for Topt 34.90±1.70 °C and Tmax 38.99±1.72°C. The Tmin values were lower than 0°C for three out of four strains. The ?opt values ranged from 0.778 and 0.90 h-1 for L. plantarum strains and from 0.553 and 0.654 h-1 for L. paracasei strains. Discussion The determination of growth cardinal values is useful to assess variability in bacterial growth abilities. This preliminary study also demonstrates the transfer of already available mathematical models to technological and probiotic strains. Indeed several user friendly tools are available to facilitate the practical use recognized mathematical models for growth prediction. The generic approach used in Sym'Previus is applicable to pathogenic, spoilage and technological microflora to further predict the impact of environmental conditions on bacterial growth. Besides the importance of using real life strains, this study further underlines the importance of characterized collection for the selection of the bacterial strain to be used in challenge test to optimize process/shelf-life and ensure food safety and quality.

Baby lettuce leaves are generally produced for the unwashed ready-to-eat market. The aim of this research was to predict sensory and microbiological aspects of this product based on physicochemical properties during storage at 4 and 10°C. Products were analysed at sampling times chosen on the basis of five sensory visual quality (VQ) levels. Samples scoring VQ5 and VQ4 were considered acceptable (Ac), whereas the remaining scores were defined as unacceptable (UAc). Each VQ level was then characterized for physico-chemical (colour, ammonium, antioxidant activity, electrolytic leakage, phenols, chlorophyll, respiratory activity) and microbiological (total viable count, Pseudomonas spp., Enterobacteriaceae, lactic acid bacteria, yeast and moulds) parameters. UAc samples also proved unsatisfactory from a microbiological point of view (total viable count >=107 CFU g-1). Partial least squares (PLS) regression analysis allowed us to identify colour change (?E*) and total chlorophyll (TC) as suitable variables to predict the microbial load (TVC) associated with each sensory VQ level. The model obtained showed R2CV=0.94, RMSECV=0.41 and a relative error of 5.61%. In conclusion, the use of these parameters as quality indicators could be a new strategy for discriminating green leafy vegetables into acceptable or unacceptable products.

Meat is subjected to chemical and microbiological deterioration, caused by oxidative degradation of meat fat and by the presence of microbial populations, particularly Pseudomonas spp.. Therefore spoilage can be monitored through their growth and changes in TBARS, thiols and carbonyls. The aim of the current study is to evaluate the correlation between chemical and microbiological data by PCA analysis and predict meat spoilage by pseudomonads contamination under isothermal and dynamic conditions or by chemical parameters in high and low fat ground meat by multivariate statistical analysis. Beef ground meat containing a total fat amount of about 5% or 15% of fat was divided into 150 g portions, stored under isothermal conditions (0, 5, 10 and 15°C) and checked for the presence of Pseudomonas spp., Brochothrix spp., total viable count (TVC). Water loss, pH, thiols, carbonyls, TBARS, metmyoglobin, deoxymioglobin, oxymioglobin and colour changes were determined on all samples. All data were subjected to PCA analysis to evaluate the correlation between microbiological and chemical variables. Subsequently, a square root model was fitted to the growth rates of pseudomonads and used to predict spoilage of ground meat under isothermal and dynamic conditions. Additionally, multivariate statistical analysis was applied to fit a PLS (Partial Least Square Regression) model, aiming at predicting microbial counts of minced meat, based on chemical data, regardless of time-temperature storage profile. Growth models allow to predict the evolution of food spoilage during storage as a function of extrinsic conditions food characteristics. Results by the PCA analysis showed that, among microbiological and chemical indexes monitored during refrigerated storage, a positive correlation was found between increase in TBARS (0.70) and levels of pseudomonads, which were inversely correlated with thiols (-0.71) and oxymioglobin (-0.81). Moreover, results demonstrated that the fat level of ground meat did not influence the growth rate of pseudomonads allowing the application of a single model for predicting the growth of pseudomonads and time to spoilage of ground meat given dynamic temperature conditions, regardless of fat content. PLS analysis was performed considering all the microbiological parameters as Y-variables, while the chemical data were used as X-variables. The model (R2CV 0.81, RMSECV of 0.68 for pseudomonads prediction) can allow to use chemical data to predict the microbial load of ground meat during refrigerated shelf life, regardless of fat content and time-temperature conditions. Moreover, the PLS model can allow to discriminate acceptable (TVC 7 log cfu/g; TBARS 0.13-1.33 mg/kg; thiols 52.94-73.06 nmol/mg protein) samples. Knowledge of perishability of food matrices associated to the storage history provide important information on the quality and edibility of food.

Alternaria species are ubiquitous and includes both pathogens and saprophytes that may damage crops and cause postharvest decay. Alternaria spp. are able to grow at low temperature and may responsible for spoilage during refrigerated transport and storage. Certain species are also capable of producing mycotoxins which can contaminate plant products. Due to their thin skin, tomatoes are particularly at risk of being infected by Alternaria, and rapid growth may occur in soft tissues, causing the well-known black mold. In that context, predictive models of mycotoxin synthesis may be helpful in determining the levels of these mycotoxins for conditions supporting growth of Alternaria spp. The objective of this study was to evaluate the effects of temperature and pH on the toxin production of an A. alternata strain in a tomato-based medium A full factorial design of six pH levels (ranging from 2 to 7) and 11 incubation temperatures (ranging from 3.2 to 37°C) was undertaken. Tomato-based agar plates at aw 0.99 were centrally inoculated with a standardized inoculum of 40 spores/5µL. Each experimental condition was tested in triplicate. For each of the 35 combinations supporting fungal development, mycotoxin accumulation was measured. Measurements of TeA, AOH and AME concentrations were performed on plates fully covered by the fungus, using HPLC. Quantification was performed by measuring peak areas at toxins retention time and comparing them with the relevant calibration curves. The experimental observations suggest that increasing pH induces a linear increase in the square root of mycotoxin synthesis, followed by a logistic decrease. The equation used to describe this relationship is based on the model of Bréand et al. (1997) and is defined by the following parameters: pHminTOX (minimum pH for toxin production), pHoptTOX (optimum pH for toxin production), TQoptTOX (toxin quantity at pHoptTOX) and TQminTOX (the toxin quantity at high pH values). Secondary models were developed to describe the relationship between temperature and the parameters (pHminTOX, pHoptTOX and TQoptTOX) while TQminTOX was assumed to be independent of temperature TeA has been described as the major mycotoxin produced by Alternaria spp. on tomatoes. Perhaps not surprisingly, only TeA could be measured in this work while AOH and AME have not been detected. TeA measurements show an important contribution of acidic pH to mycotoxin synthesis, in particular near pH 4. At similar pH levels, incubation at low temperatures (from 6.5°C to 12°C) resulted in higher TeA production than at temperatures above 20°C. The developed model for TeA synthesis describes accurately the experimental measurements (R2=0.98). The predicted maximum toxin quantity TQoptTOX increases from of 180 µg/g at 6.5°C to 290 µg/g at 12°C. It then decreases until reaching 70 µg/g at 30°C. The parameters pHminTOX and pHoptTOX were also found to be temperature dependent The model for toxin production can be used together with a model of fungal growth to highlight at-risk storage conditions for which mycotoxin production is at its highest although fungal growth is rather small

Contamination of bread ingredients with Bacillus spp. spores and their persistence in bread is source of concern mainly for its industrial production. The microbial eco-system of raw materials used for bread-making and in particular spore-forming bacteria play a role in the development of "bread rope spoilage". Heat resistant spores can survive the baking process and under warm and humid favorable conditions spore germination occurs and vegetative cells cause the rope spoilage, a deterioration process of bread texture. The chapter analyzes the factors contributing to the rope occurrence such as the complexity of the microbiota of sporeformers contaminating the raw materials, the physiological and genetic traits of the Bacillus strains belonging to several species responsible for the alteration including potential toxigenic species and the spore's heat resistance. The chapter also examines some strategies to prevent and/or control the rope spoilage, with particular regard to the application of predictive microbiology to evaluate the contamination risk and to the use of microbial biotechnologies as tools for controlling microbial growth to counteract the alterative phenomenon.

The beneficial relationship between the host health and its gut microbiota has led in the last decades to the enormous increase of research and commercial interest in the development of probiotic preparations to manipulate microbiome in helping host physiology or preventing diseases [1]. Producers of functional foods are exploring technological solutions for developing foods supplemented with additional functional benefits but with high level of consumer satisfactions (health-orientated as well as taste-orientated). Many strains belonging to lactic acid bacterial (LAB) species are widely used as probiotics in commercial products since, additionally to functional properties conferred to foods, they may also act as starters establishing a mild fermentation that protects products from deterioration. Particularly, a L. paracasei strain (IMPC 2.1) has been studied and used for the development of innovative patented functional foods based on the association of that probiotic strain with vegetables (olives, artichokes, cabbage etc.) whose commercialization has been authorized by the Italian Ministry of Health [2]. Clinical trials performed on subjects suffering from constipation and healthy subjects demonstrated the efficacy of L. paracasei IMPC 2.1 carried by ready-to-eat artichokes in transiently colonize the human gut thus modulating potentially harmful bacteria, faecal enzyme activity and short chain fatty acid production as well as symptom profile [3]. Currently among probiotic foods, fish products have been rarely investigated as vehicles for probiotic strains in humans, even if they are suitable to sustain viable LAB populations [4]. Therefore our investigation aimed to evaluate the ability of the probiotic L. paracasei strain IMPC 2.1 to survive in marinated ready-to-eat swordfish fillets and to reach, viable, the human gut in an alternate day- based study. Probiotic ready-to-eat swordfish fillets (PR-RTE) were prepared by brining (3% NaCl) fillets for 2 days with live cells of the probiotic strain at 7 log CFU/g; then fillets were drained, seasoned with sunflower seed oil, spiced with parsley and stored under vacuum packing at 4°C in polyethylene trays (shelf life 4 months). The final product, containing more than 7 log CFU/g of the probiotic strain, showed physicochemical characteristics (protein profile and total free amino acid content, pH, aw) similar to those of control-RTE fillets (traditionally processed with an acidic marinade) along shelf life. The probiotic fillets were used in a human feeding study involving 8 volunteers to assess the suitability of the fish matrix in delivering viable cells of the probiotic strain [5]. Participants integrated their dietary intakes throughout the study period with a portion (100 g) of PR-RTE fillets containing about log 9 CFU of probiotic cells. The protocol was approved by a local Scientific and Ethics Committee. The dietary intervention study lasted 27 days and subjects consumed PR-RTE fillets on alternate days for 20 days, with a washout period of 7 days. A good compliance of PR-RTE fillets was registered from all participants. To evaluate the transient gut colonization of the strain, its presence was molecularly ascertained in faecal samples of subjects. Results demonstrated that after consuming 10 portions during 20 days, the probiotic strain was recovered in the faeces of all subjects at concentrations ranging from 6.15 to 7.47 log CFU/g. Interestingly, five subjects resulted to be colonized by the probiotic strain, at levels ranging from 6.30 to 7.74 log CFU/g of faeces, after eating 5 portions of the product (day 10). The probiotic colonization led to an increase, with respect to the start of the study, in the genetic diversity of faecal LAB based on the Shannon diversity index, even though no differences in LAB counts were observed. We can conclude that swordfish fillet is a suitable food carrier for the probiotic strain L. paracasei IMPC 2.1which was able to survive on the fish matrix during a 4-month storage period and to transiently colonize the gut of volunteers after thus providing consumers with a non conventional probiotic food to achieve a Functional Diet.

Le materie prime impiegate nella produzione di prodotti panari rappresentano la principale fonte di batteri sporigeni associati all'alterazione "pane filante" che si manifesta prevalentemente in condizioni di clima caldo umido e che determina rilevanti perdite economiche nel settore panario (Valerio et al., 2012, Int. J. Food Microbiol, 156, 278-285). Le spore, sopravvivendo al processo di cottura germinano e le cellule vegetative avviano un processo degradativo dell'amido e delle proteine del pane che porta alla formazione di esopolisaccaridi (slime). Nell'ambito del presente studio è stato condotto un test di screening su 176 isolati da materie prime per valutare quali fossero le specie in grado di causare l'alterazione nel pane. Bacillus amyloliquefaciens, insieme con B. subtilis e B. pumilus, sono risultate le principali specie in grado di causare il fenomeno alterativo. Inoltre, tra le specie identificate, i ceppi appartenenti al gruppo B. cereus rappresentano il 17%, e tra questi il 38% è risultato in grado di provocare l'alterazione. I ceppi del gruppo B. cereus appartengono ai gruppi filogenetici III e IV, associati ad elevato rischio di intossicazione, in particolare quelli del gruppo III, per i quali è stata evidenziata un'elevata resistenza termica delle spore durante un test che simula il profilo termico del processo di cottura del pane (De Bellis et al. 2015, Int. J. Food Microbiol., 197, 30-39). Per stimare il rischio di insorgenza dell'alterazione del pane durante la shelf-life, è stata impiegata la microbiologia predittiva (Sym'Previus tool): sono stati determinati i parametri cardinali e i limiti di crescita (growth/no growth boundaries) di tre ceppi di B. amyloliquefaciens isolati rispettivamente da pane con chiari sintomi di alterazione, da grano e da semola. Inoltre sono stati condotti challenge test inoculando le spore di uno dei ceppi nella fase di impasto: sono state preparate varie formulazioni del pane per valutarne l'effetto sulla crescita batterica e i risultati sperimentali sono stati confrontati con le simulazioni in silico. Infine, per stimare la probabilità di contaminazione del prodotto durante la shelf-life, sono state considerate due temperature di conservazione e la soglia di 5 log cfu/g che può causare l'alterazione e/o rappresentare un rischio per la salute del consumatore. La caratterizzazione del comportamento di crescita dei tre ceppi ha evidenziato una differenza nella probabilità di contaminazione e successiva alterazione del prodotto, dimostrando la necessità di considerare la variabilità biologica negli studi di microbiologia predittiva al fine di ottenere una stima realistica del rischio di contaminazione. In conclusione, questo studio ha dimostrato l'applicabilità degli strumenti di microbiologia predittiva, noti per la valutazione del rischio di contaminazioni alimentari da microrganismi patogeni, allo studio del comportamento di microrganismi alterativi per il controllo della qualità dei prodotti alimentari (Valerio et al., 2015 Food Microbiol., 45, 2-9).

Probiotic strains used as starter cultures may combine the positive image of fermented foods with a functional appeal. Moreover, these bacteria can be used as bioprotective cultures to extend the shelf-life of ready-to-eat (RTE) foods and to control harmful microorganisms Indeed, new probiotic vegetable products - table olives, artichokes and cabbage - have been obtained after a mild fermentation process supported by the probiotic strain Lactobacillus paracasei LMGP22043 (Valerio et al. 2013; Lavermicocca et al. 2016). In this study the probiotic strain L. paracasei LMGP22043, was applied as bioprotective culture (7 log CFU/g) to preserve RTE artichokes and as a starter to ferment blanched white cabbage thus preserving its nutritional quality. The probiotic strain was used to pilot the fermentation of cabbage leading, after 71 h fermentation, to a final product containing about log 8 CFU/g live cells and the 35% of the total glucosinolates (GSs) detected before fermentation otherwise completely lost during conventional fermentation of sauerkraut (Sarvan et al. 2013). After 30 days of refrigerated vacuum packed storage, GSs and the probiotic concentration still persisted. Moreover, the bioprotective features of the probiotic strain were ascertained in RTE artichokes challenged (3 log CFU/g) with pathogens Listeria monocytogenes ATCC19115, Salmonella enterica subsp. enterica ATCC13311, or Escherichia coli ATCC8739 and stored at 4°C for 45 days. All pathogens decreased in the probiotic RTE product, whereas a longer survival was observed in the standard product (control). At the same time, the probiotic RTE product still contained an adequate amount (more than 7 log CFU/g) of live probiotic cells (Valerio et al. 2013). Therefore, studies demonstrated that vegetables can be processed to obtain functional products, using a probiotic strain which plays the double role of starter and bioprotective culture.

Baby lettuce leaves have recently been developed for the unwashed fresh-cut and ready-to-eat markets. The aim of this research was to characterize this product by sensory, physicochemical and microbiological analyses performed during storage at 4 and 10°C. Products were analysed at sampling times chosen on the basis of 5 sensory visual quality (VQ) levels. Samples scoring VQ5 and VQ4 were considered acceptable (Ac), whereas the remaining scores were defined as unacceptable (UAc). Each VQ level was then characterized for physicochemical (colour, ammonia, antioxidant activity, electrolytic leakage, phenols, chlorophyll, respiratory activity) and microbiological (total viable count, Pseudomonas spp, Enterobacteriaceae, lactic acid bacteria, yeast and moulds) parameters. UAc samples also proved unsatisfactory from a microbiological point of view (total viable count >=107 CFU/g). Partial Least Square (PLS) regression analysis allowed to identify colour change (?E*) and total chlorophyll (TC) as X-variables to predict the microbial load (TVC) (Y-variable) associated with each sensory VQ level. The model obtained showed R2CV=0.94, RMSECV=0.41 and a relative error of 5.61%. In conclusion, the use of these parameters as quality indicators could be a new strategy for discriminating green leafy vegetables into acceptable or unacceptable products.