Effetto della temperatura di conservazione del latte, dalla raccolta all'affioramento, sulla microflora del latte, del sieroinnesto e del formaggio Trentingrana

This PhD research is part of a project addressed to improve the quality of Grana Trentino production. The objectives were to evaluated if milk storage and collection procedures may affect cheese-making technology and quality. Actually the milk is collected and delivered to the cheese factory just after milking in 50 L cans without refrigeration or in tanks cooled at 18 °C. This procedure is expensive (two deliveries each day) and the milk quality is difficult to preserve as temperatures are not controlled. The milk refrigeration at the farm could allow a single delivery to the dairy. Therefore it could be a good strategy to preserve raw milk quality and reduce cheese spoilage. This operation may, however, have the drawbacks of favouring the growth of psychrotrophic bacteria and changing the aptitude of milk to coagulation. With the aim of studying the effect on milk and cheese of traditional and new refrigerated technologies of milk storage, two different collection and creaming technologies were compared. The trials were replicated in three cheese factories manufacturing Grana Trentino. Every cheese-making day, about 1000 milk liters were collected from always the same two farms in the different collection procedures (single or double). Milk was processed to produce 2 wheels of Grana trentino every day. During the refrigerated trials, milk was collected and stored at the farm in a mixed tank at 12 or 8 °C and then was carried to the dairy in truck once a day. 112 cheese making day were followed: 56 for traditional technology and 56 for the refrigerated one. Each one of these two thechnologies lead to different ways of creaming: long time in the traditional one and shorter in the new one. For every cheese making day we recorded time, temperatures and pH during the milk processing to cheese. Whole milk before ceraming, cream and skim milk after creaming, vat milk and whey were sampled during every cheese-making day for analysis. After 18 months ripening we opened 46 cheese wheels for further chemical and microbiological analyses. The trials were performed with the aim of: 1 estimate the effect of storage temperatures on microbial communities, physico-chemical or/and rheological differences of milk and skim milk after creaming. 2 detect by culture dependent (plate counts) and indipendent (DGGE) methodolgies the microbial species present in whole, skimmed milk, cream and cheese sampled under the rind and in the core; 3 estimate the physico-chemical characteristics, the proteolytic activity, the content of free aminoacids and volatile compounds in 18 months ripened Grana Trentino cheeses from different storing and creaming of milk technologies. The results presented are remarkable since this is the first in-deep study presenting microbiological and chemical analysis of Grana Trentino that even if belonging to Grana Padano Consortium, it is clearly different in the milk and in the manufacturing technology.

Ricerca di Escherichia coli produttori di verocitotossine e definizione di obiettivi di performance nella produzione primaria di alimenti di origine animale

La presenza di Escherichia coli produttori di verocitotossine (VTEC o STEC) rappresenta una tra le più importanti cause di malattia alimentare attualmente presenti in Europa. La sua presenza negli allevamenti di animali destinati alla produzione di alimenti rappresenta un importante rischio per la salute del consumatore. In conseguenza di comuni contaminazioni che si realizzano nel corso della macellazione, della mungitura i VTEC possono essere presenti nelle carni e nel latte e rappresentano un grave rischio se la preparazione per il consumo o i processi di lavorazione non comportano trattamenti in grado d’inattivarli (es. carni crude o poco cotte, latte non pastorizzato, formaggi freschi a latte crudo). La contaminazione dei campi coltivati conseguente alla dispersione di letame o attraverso acque contaminate può veicolare questi stipiti che sono normalmente albergati nell’intestino di ruminanti (domestici e selvatici) e anche prodotti vegetali consumati crudi, succhi e perfino sementi sono stati implicati in gravi episodi di malattia con gravi manifestazioni enteriche e complicazioni in grado di causare quadri patologici gravi e anche la morte. Stipiti di VTEC patogeni ingeriti con gli alimenti possono causare sintomi gastroenterici, con diarrea acquosa o emorragica (nel 50% dei casi), crampi addominali, febbre lieve e in una percentuale più bassa nausea e vomito. In alcuni casi (circa 5-10%) l’infezione gastroenterica si complica con manifestazioni tossiemiche caratterizzate da Sindrome Emolitico Uremica (SEU o HUS) con anemia emolitica, insufficienza renale grave e coinvolgimento neurologico o con una porpora trombotica trombocitopenica. Il tasso di mortalità dei pazienti che presentano l’infezione da E. coli è inferiore all’1%. I dati forniti dall’ECDC sulle infezioni alimentari nel periodo 2006-2010 hanno evidenziato un trend in leggero aumento del numero di infezioni a partire dal 2007. L’obiettivo degli studi condotti è quello di valutare la prevalenza ed il comportamento dei VTEC per una analisi del rischio più approfondita.

Advances in methods to detect, isolate and quantify foodborne pathogens

Foodborne diseases impact human health and economies worldwide in terms of health care and productivity loss. Prevention is necessary and methods to detect, isolate and quantify foodborne pathogens play a fundamental role, changing continuously to face microorganisms and food production evolution. Official methods are mainly based on microorganisms growth in different media and their isolation on selective agars followed by confirmation of presumptive colonies through biochemical and serological test. A complete identification requires form 7 to 10 days. Over the last decades, new molecular techniques based on antibodies and nucleic acids allow a more accurate typing and a faster detection and quantification. The present thesis aims to apply molecular techniques to improve official methods performances regarding two pathogens: Shiga-like Toxin-producing Escherichia coli (STEC) and Listeria monocytogenes. In 2011, a new strain of STEC belonging to the serogroup O104 provoked a large outbreak. Therefore, the development of a method to detect and isolate STEC O104 is demanded. The first objective of this work is the detection, isolation and identification of STEC O104 in sprouts artificially contaminated. Multiplex PCR assays and antibodies anti-O104 incorporated in reagents for immunomagnetic separation and latex agglutination were employed. Contamination levels of less than 1 CFU/g were detected. Multiplex PCR assays permitted a rapid screening of enriched food samples and identification of isolated colonies. Immunomagnetic separation and latex agglutination allowed a high sensitivity and rapid identification of O104 antigen, respectively. The development of a rapid method to detect and quantify Listeria monocytogenes, a high-risk pathogen, is the second objective. Detection of 1 CFU/ml and quantification of 10–1,000 CFU/ml in raw milk were achieved by a sample pretreatment step and quantitative PCR in about 3h. L. monocytogenes growth in raw milk was also evaluated.

Sviluppo di sensori elettrochimici per la determinazione di amine biogene e microrganismi patogeni in alimenti di origine animale

La possibilità di monitorare la presenza di residui di farmaci veterinari e contaminanti biologici negli alimenti può trarre beneficio dall’uso di metodi di screening affidabili e di facile utilizzo. A tal fine, sono in fase di sviluppo molteplici applicazioni di biosensori in grado di coniugare sistemi di rilevamento biologico-specifici con trasduttori elettronici o ottici capaci di rilevare, amplificare, elaborare e misurare il segnale derivante dall’interazione tra un substrato costituito da enzimi, anticorpi o apteni e contaminanti ambientali o alimentari. Lo sviluppo di biosensori permette di rilevare la presenza di quantità residuali di un determinato analita in varie matrici sia animali che alimentari. Per questo Progetto di Ricerca sono state messe a punto tecniche di analisi elettrochimiche per rilevare quantitativamente la presenza di istamina e di batteri istaminogeni in campioni di pesce e determinare la presenza di ceppi di Escherichia coli nel latte crudo. Sono stati condotti anche degli studi riguardanti la presenza di residui di farmaci veterinari negli alimenti. Lo scopo di queste ricerche era quello di: • Sviluppare diversi tipi di sensori elettrochimici ed immunoenzimatici e valutare le loro potenzialità come metodi di analisi rapida. • Validare i risultati mediante comparazione con metodi analitici di riferimento. • Avviare uno studio per lo sviluppo di biosensori basato sulla valutazione del rischio

Life Cycle Thinking for sustainability assessment and decision-making in selected food supply chains in Costa Rica

The Agenda 2030 contains 17 integrated Sustainable Development Goals (SDGs). SDG 12 for Sustainable Consumption and Production (SCP) promotes the efficient use of resources through a systemic change that decouples economic growth from environmental degradation. The Food Systems (FS) pillar in SDG 12 entails paramount relevance due to its interconnection to many other SDGs, and even when being a crucial world food supplier, the Latin American and Caribbean (LAC) Region struggles with environmental and social externalities, low investment in agriculture, inequity, food insecurity, poverty, and migration. Life Cycle Thinking (LCT) was regarded as a pertinent approach to identify hotspots and trade-offs, and support decision-making process to aid LAC Region countries as Costa Rica to diagnose sustainability and overcome certain challenges. This thesis aimed to ‘evaluate the sustainability of selected products from food supply chains in Costa Rica, to provide inputs for further sustainable decision-making, through the application of Life Cycle Thinking’. To do this, Life Cycle Assessment (LCA), Life Cycle Costing (LCC), and Social Life Cycle Assessment (S-LCA) evaluated the sustainability of food-waste-to-energy alternatives, and the production of green coffee, raw milk and leafy vegetables, and identified environmental, social and cost hotspots. This approach also proved to be a useful component of decision-making and policy-making processes together with other methods. LCT scientific literature led by LAC or Costa Rican researchers is still scarce; therefore, this research contributed to improve capacities in the use of LCT in this context, while offering potential replicability of the developed frameworks in similar cases. Main limitations related to the representativeness and availability of primary data; however, future research and extension activities are foreseen to increase local data availability, capacity building, and the discussion of potential integration through Life Cycle Sustainability Assessment (LCSA).