CHARACTERIZATION OF LYOPHILIZED LIPOSOMES PRODUCED WITH NON-PURIFIED SOY LECITHIN: A CASE STUDY OF CASEIN HYDROLYSATE MICROENCAPSULATION

A commercial casein hydrolysate was microencapsulated in liposomes produced with non-purified soy lecithin, cryoprotected with two different disaccharides and lyophilized. The encapsulation efficiency of casein hydrolysate ranged from 30 to 40%. The powders were analyzed by differential scanning calorimetry (DSC), scanning electron micrography (SEM), infrared spectroscopy (FTIR) and wide angle X-ray diffraction (WAXD). DSC data revealed the presence of an exothermal transition in empty lyophilized liposomes, which was ascribed to the presence of a quasicrystalline lamellar phase (intermediary characteristics between the L-beta and L-c phases). The addition of peptides to the liposomal system caused the disappearance of this exothermic phenomenon, as they were located in the polar headgroup portion of the bilayer, causing disorder and preventing the formation of the quasicrystalline phase. Infrared data indicated the presence of the peptides in the lyophilized formulations and showed that the cryoprotectants interacted effectively with the polar heads of phospholipids in the bilayer.

Characterization of Staphylococcus aureus isolates in milk and the milking environment from small-scale dairy farms of Sao Paulo, Brazil, using pulsed-field gel electrophoresis

This research aimed to evaluate the occurrence of Staphylococcus aureus isolates in milk and in the milking environment of 10 small-scale farms (<400 L/d) located in the regions of Franca and Ribeirao Preto, state of Sao Paulo, Brazil. Two-hundred twenty samples of milk were collected from individual cows, along with 120 samples from bulk tank milk, 389 samples from milking equipment and utensils (teat cups, buckets, and sieves), and 120 samples from milkers' hands. Fifty-six Staph. aureus strains were isolated from 849 analyzed samples (6.6%): 12 (5.5%) from milk samples of individual cows, 26 (21.7%) from samples of bulk tank milk, 14 (3.6%) from samples collected from equipment and utensils, and 4 (3.3%) from samples from milkers' hands. Pulsed-field gel electrophoresis typing of the 56 Staph. aureus isolates by SmaI restriction enzyme resulted in 31 profiles (pulsotypes) arranged in 12 major clusters. Results of this study indicate a low incidence, but wide distribution of Staph. aureus strains isolated from raw milk collected from individual cows and surfaces of milkers' hands and milking equipment in the small-scale dairy farms evaluated. However, the high percentage of bulk milk samples found with Staph. aureus is of public health concern because raw, unprocessed milk is regularly consumed by the Brazilian population.

The effects of extrusion conditions and the addition of volatile compounds and flavour enhancers to corn grits on the retention of the volatile compounds and texture of the extrudates

Corn grits that were supplemented with isovaleraldehyde, ethyl butyrate, butyric acid and flavour enhancers were extruded under different processing conditions. Volatile compounds retained in the extrudates were isolated by dynamic headspace and analysed using gas chromatographymass spectrometry. The expansion ratio, density and cut force to break down the extrudates were evaluated and aroma intensity was assessed using a multisample difference test. Butyric acid showed the greatest retention (96.4%), regardless of the extrusion conditions. All compounds were better retained when samples were extruded at 20% feed moisture and 90 degrees C processing temperature (2.981.0%), conditions that also resulted in greater aromatic intensity (moderate to moderate-strong intensity). The addition of volatile compounds reduced the expansion ratio and cut force, whereas the addition of flavour enhancers increased the expansion ratio but reduced ethyl butyrate and butyric acid retention.

Gases and volatile compounds associated with micro-organisms in blown pack spoilage of Brazilian vacuum-packed beef

This study correlated the composition of the spoilage bacterial flora with the main gaseous and volatile organic compounds (VOCs) found in the package headspace of spoiled, chilled, vacuum-packed meat. Fifteen chilled, vacuum-packed beef samples, suffering from blown pack spoilage, were studied using 16S rRNA clone sequencing. More than 50% of the bacteria were identified as lactic acid bacteria (LAB), followed by clostridia and enterobacteria. Fifty-one volatile compounds were detected in the spoiled samples. Although the major spoilage compounds were identified as alcohols and aldehydes, CO2 was identified as the major gas in the spoiled samples by headspace technique. Different species of bacteria contribute to different volatile compounds during meat spoilage. LAB played an important role in blown pack deterioration of the Brazilian beef studied.

Protein turnover, amino acid requirements and recommendations for athletes and active populations

Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

Níveis de células somáticas sobre a proteólise do queijo Mussarela

Objetivou-se, com a realização deste trabalho, avaliar o efeito do nível de células somáticas sobre a microbiota e a proteólise do queijo Mussarela, durante o período de armazenamento. Foram selecionadas vacas com contagem de células somáticas ≤200mil células/mL; de > 200 a ≤400mil células/mL; de >400mil células/mL a ≤750mil células/mL e >750mil células/mL e que não tinham recebido tratamento com antimicrobianos nos dias que antecederam a coleta da matéria-prima. Os queijos produzidos foram avaliados após 1; 15 e 30 dias de armazenamento para a contagem de coliformes a 35ºC, coliformes a 45ºC, psicrotróficos e bactérias ácido lácticas. Paralelamente, foram determinados os índices de extensão e profundidade da proteólise. O experimento completo foi repetido quatro vezes e o delineamento experimental foi em blocos aleatórios. Na análise estatística, utilizou-se a análise de variância seguida do teste de Tukey, considerando p<0,05 como probabilidade mínima aceitável para diferença entre as médias. O leite com elevada contagem de células somáticas apresentou concentração menor de proteína e maior de nitrogênio não proteico. Observou-se diminuição das bactérias ácido lácticas no queijo elaborado com leite composto de células somáticas >750mil células/mL. Não obstante, ocorreu um aumento significativo na extensão e profundidade da proteólise durante o período de armazenamento, resultados observados nos queijos fabricados com o leite com células somáticas >400mil células/mL. Portanto, para se produzir um queijo Mussarela de boa qualidade torna-se necessário o controle da matéria prima, e esta deve apresentar células somáticas inferiores a 400mil células/mL.

Muscle glycogen resynthesis during recovery from cycle exercise: no effect of additional protein ingestion

[EN] In the present study, we have investigated the effect of carbohydrate and protein hydrolysate ingestion on muscle glycogen resynthesis during 4 h of recovery from intense cycle exercise. Five volunteers were studied during recovery while they ingested, immediately after exercise, a 600-ml bolus and then every 15 min a 150-ml bolus containing 1) 1.67 g. kg body wt(-1). l(-1) of sucrose and 0.5 g. kg body wt(-1). l(-1) of a whey protein hydrolysate (CHO/protein), 2) 1.67 g. kg body wt(-1). l(-1) of sucrose (CHO), and 3) water. CHO/protein and CHO ingestion caused an increased arterial glucose concentration compared with water ingestion during 4 h of recovery. With CHO ingestion, glucose concentration was 1-1.5 mmol/l higher during the first hour of recovery compared with CHO/protein ingestion. Leg glucose uptake was initially 0.7 mmol/min with water ingestion and decreased gradually with no measurable glucose uptake observed at 3 h of recovery. Leg glucose uptake was rather constant at 0.9 mmol/min with CHO/protein and CHO ingestion, and insulin levels were stable at 70, 45, and 5 mU/l for CHO/protein, CHO, and water ingestion, respectively. Glycogen resynthesis rates were 52 +/- 7, 48 +/- 5, and 18 +/- 6 for the first 1.5 h of recovery and decreased to 30 +/- 6, 36 +/- 3, and 8 +/- 6 mmol. kg dry muscle(-1). h(-1) between 1.5 and 4 h for CHO/protein, CHO, and water ingestion, respectively. No differences could be observed between CHO/protein and CHO ingestion ingestion. It is concluded that coingestion of carbohydrate and protein, compared with ingestion of carbohydrate alone, did not increase leg glucose uptake or glycogen resynthesis rate further when carbohydrate was ingested in sufficient amounts every 15 min to induce an optimal rate of glycogen resynthesis.

Role of caloric content on gastric emptying in humans

[EN] 1. This study examined the effects of caloric content (caloric density and the nature of calories) on the rate of gastric emptying using the double-sampling gastric aspiration technique. Four test meals of 600 ml (glucose, 0.1 kcal ml-1; pea and whey peptide hydrolysates, both 0.2 kcal ml-1; milk protein, 0.7 kcal ml-1) were tested in six healthy subjects in random order on four separate occasions. 2. The glucose solution was emptied the fastest with a half-time of 9.4 +/- 1.2 min (P < 0.05) and the milk protein the slowest with a half-time of 26.4 +/- 10.0 min (P < 0.05); the pea peptide hydrolysate and whey peptide hydrolysate solutions had half-times of emptying of 16.3 +/- 5.4 and 17.2 +/- 6.1 min, respectively. The rates of gastric emptying for the peptide hydrolysate solutions derived from different protein sources were not different. 3. Despite the lower rate of gastric emptying for the milk protein solution, the rate of caloric delivery to the duodenum during the early phase of the gastric emptying process was higher than that for the other three solutions (46.3 +/- 6, 63.5 +/- 22, 62.5 +/- 19 and 113.8 +/- 25 cal min-1 kg-1 for the glucose, pea peptide hydrolysate, whey peptide hydrolysate and milk protein meals, respectively; P < 0.05). The caloric density of the test solutions was linearly related to the half-time of gastric emptying (r = 0.96, P < 0.05) as well as to the rate at which calories were delivered to the duodenum (r = 0.99, P < 0.001). 4. This study demonstrates that the rate of gastric emptying is a function of the caloric density of the ingested meal and that a linear relationship exists between these variables. Furthermore, the nature of the calories seems to play a minor role in determining the rate of gastric emptying in humans.

Biometanazione della frazione organica di rifiuti solidi urbani da raccolta indifferenziata mediante codigestione con refluo zootecnico o addizione di solfuro di sodio

Il presente elaborato è stato finalizzato allo sviluppo di un processo di digestione anaerobica della frazione organica dei rifiuti solidi urbani (FORSU oppure, in lingua inglese OFMSW, Organic Fraction of Municipal Solid Waste) provenienti da raccolta indifferenziata e conseguente produzione di biogas da impiegarsi per il recupero energetico. Questo lavoro rientra nell’ambito di un progetto, cofinanziato dalla Regione Emilia Romagna attraverso il Programma Regionale per la Ricerca Industriale, l’Innovazione e il Trasferimento Tecnologico (PRRIITT), sviluppato dal Dipartimento di Chimica Applicata e Scienza dei Materiali (DICASM) dell’Università di Bologna in collaborazione con la Facoltà di Ingegneria dell’Università di Ferrara e con la società Recupera s.r.l. che applicherà il processo nell’impianto pilota realizzato presso il proprio sito di biostabilizzazione e compostaggio ad Ostellato (FE). L’obiettivo è stato la verifica della possibilità di impiegare la frazione organica dei rifiuti indifferenziati per la produzione di biogas, e in particolare di metano, attraverso un processo di digestione anaerobica previo trattamento chimico oppure in codigestione con altri substrati organici facilmente fermentabili. E’ stata inoltre studiata la possibilità di impiego di reattori con biomassa adesa per migliorare la produzione specifica di metano e diminuire la lag phase. Dalla sperimentazione si può concludere che è possibile giungere allo sviluppo di metano dalla purea codigerendola assieme a refluo zootecnico. Per ottenere però produzioni significative la quantità di solidi volatili apportati dal rifiuto non deve superare il 50% dei solidi volatili complessivi. Viceversa, l’addizione di solfuri alla sola purea si è dimostrata ininfluente nel tentativo di sottrarre gli agenti inibitori della metanogenesi. Inoltre, l’impiego di supporti di riempimento lavorando attraverso processi batch sequenziali permette di eliminare, nei cicli successivi al primo, la lag phase dei batteri metanogeni ed incrementare la produzione specifica di metano.

Studi ed approfondimenti sulla composizione in acidi grassi del latte bovino di razza Reggiana con diverso fenotipo per k-caseina e Beta-lactoglobulina

The aim of the present study was to examine the association between milk protein polymorphism and fatty acids profiles of bovine milk. Milk samples were collected from each of 55 Reggiana cows during early, mid and late lactation, respectively, in two farms within the production area of Parmigiano Reggiano cheese. Identification and quantification of fatty acids were performed by gas chromatography. Milk fatty acid composition using cows of differing κ-casein (κ-Cn) and β-lactoglobulin (β-Lg) phenotypes was investigated. Statistically significant results regarding the associations between milk fatty acid composition and κ-Cn phenotype were found, in particular, κ-Cn BB seems to influence de novo fatty acid synthesis in the mammary gland. Also κ-Cn AB seems to have the same effect. Proportions of C10:0 (2,29a AA; 2,53b AB; 2,59b BB), C12:0 (2,77a AA; 3,17b AB; 3,20b BB) and C14:0 (9,22a AA; 10,25b AB; 10,27b BB) were higher in the milk from cows with κ-Cn phenotype AB and BB vs κ-Cn phenotype AA (p<0,05). Conversely C18:0 (7,84b AA; 7,20a,b AB; 6,94a BB) and C18:1 (19,19b AA; 16,81a AB; 16,79a BB) were lower in the milk from cows with κ-Cn phenotype AB and BB vs κ-Cn phenotype AA. The association between milk fatty acid composition and β-Lg phenotype was not statistically significant, except for some fatty acids. In particular, C12:0 (3,05a AA; 3,04a AB; 3,33b BB) was higher in the milk from cows with β-Lg phenotype BB vs β-Lg phenotype AA and AB (p<0,05). Concentrations of C18:0 (6,93a AA; 7,86b AB; 6,59a BB) and C18:1 (16,74a,b AA; 18,24b AB; 16,07a BB) were lower in the milk from cows with β-Lg phenotype AA and BB vs β-Lg phenotype AB (p<0,05). Moreover this research, carried out in farms within the Parmigiano Reggiano cheese district, analysed also the size distribution of fat globules in bulk milk of Reggiana and Frisona breed cows. In particular, the size distribution of individual milk fat globules of Reggiana cows with differing κ-Cn phenotypes was considered. From first observations, no statistically significant differences were observed.

Application of different instrumental techniques to the selective evaluation of bio-sensible compounds in foodstuff of animal and vegetable origin

This PhD thesis describes the application of some instrumental analytical techniques suitable to the study of fundamental food products for the human diet, such as: extra virgin olive oil and dairy products. These products, widely spread in the market and with high nutritional values, are increasingly recognized healthy properties although their lipid fraction might contain some unfavorable components to the human health. The research activity has been structured in the following investigations: “Comparison of different techniques for trans fatty acids analysis” “Fatty acids analysis of outcrop milk cream samples, with particular emphasis on the content of Conjugated Linoleic Acid (CLA) and trans Fatty Acids (TFA), by using 100m high-polarity capillary column” “Evaluation of the oxidited fatty acids (OFA) content during the Parmigiano-Reggiano cheese seasoning” “Direct analysis of 4-desmethyl sterols and two dihydroxy triterpenes in saponified vegetal oils (olive oil and others) using liquid chromatography-mass spectrometry” “Quantitation of long chain poly-unsatured fatty acids (LC-PUFA) in base infant formulas by Gas Chromatography, and evaluation of the blending phases accuracy during their preparation” “Fatty acids composition of Parmigiano Reggiano cheese samples, with emphasis on trans isomers (TFA)”

Studio della maturazione di formaggi pecorino stagionati in stabilimento e in grotta

Aims: Ripening evaluation of two different Pecorino cheese varieties ripened according either to a traditional method in plant and in cave. Different ripening features have been analyzed in order to evaluate the cave as possible ripening environment with the aim of obtaining a peculiar product which could also establish an added value to the cultural heritage of the local place in which it has been originally manufactured. Methods and Results: Chemical-physical features of Pecorino cheese have been initially analyzed into two different ripening environments and experimentations, among which: pH, weight reduction and subsequent water activity. Furthermore, the microbial composition has been characterized in relationship with the two different ripening environments, undertaking a variety of microbial groups, such as: lactic bacteria, staphylococci, yeasts, lactococci, enterobacteria, enterococci. Besides, an additional analysis for the in-cave adaptability evaluation has been the identification of biogenic amines inside the Pecorino cheese (2-phenilethylamine, putrescine, cadaverine, hystidine, tyramine, spermine and spermidine). Further analysis were undertaken in order to track the lipid profile evolution, reporting the concentration of the cheese free fatty acids in object, in relation with ripening time, environment and production. In order to analyse the flavour compounds present in Pecorino cheese, the SPME-GC-MS technique has been widely employed. As a result, it is confirmed the trend showed by the short-chain free fatty acids, that is to say the fatty acids which are mostly involved in conveying a stronger flavor to the cheese. With the purpose of assessing the protheolytic patterns of the above-mentioned Pecorino cheese in the two different ripening environments and testing methods, the technique SDS-PAGE has been employed into the cheese insoluble fraction, whereas the SDS-PAGE technique has been carried out into the cheese soluble portion. Furthermore, different isolated belonging to various microbial groups have been genotypically characterized though the ITS-PCR technique with the aim to identify the membership species. With reference to lactic bacillus the characterized species are: Lactobacillus brevis, Lactobacillus curvatus and Lactobacillus paraplantarum. With reference to lactococci the predominant species is Lactococcus lactis, coming from the employed starter used in the cheese manufacturing. With reference to enterococcus, the predominant species are Enterococcus faecium and Enterococcus faecalis. Moreover, Streptococcus termophilus and Streptococcus macedonicus have been identified too. For staphylococci the identified species are Staphyilococcus equorum, Staphylococcus saprophyfiticus and Staphylococcus xylosus. Finally, a sensorial analysis has been undertaken through on one side a consumer test made by inexperienced consumers, and on the other side through a panel test achieved by expert consumers. From such test Pecorino cheese ripened in cave were found to be more pleasant in comparison with Pecorino cheese ripened in plant. Conclusions: The proposed approach and the undertaken analysis showed the cave as preferential ripening environment for Pecorino cheese and for the development of a more palatable product and safer for consumers’ health.

Nuovi ingredienti funzionali per l'applicazione in campo alimentare

The Mediterranean diet is rich in healthy substances such as fibres, vitamins and phenols. Often these molecules are lost during food processing. Olive oil milling waste waters, brans, grape skins are some of the most relevant agri-food by-products in the Mediterranean countries. These wastes are still rich in extremely valuable molecules, such as phenolic antioxidants, that have several interesting health promoting properties. Using innovative environmental friendly technologies based in the rational use of enzymatic treatment is possible to obtain from agri-food by-products new ingredients containing antioxidants that can be used as functional ingredients in order to produce fortified foods. These foods, having health protecting/promoting properties, on top of the traditional nutritional properties, are attracting consumer’s attentions due to the increasing awareness on health protection through prevention. The use of these new ingredients in different food preparation was studied in order to evaluate the effects that the food processing might have on the antioxidant fraction, the effect of these ingredient on foods appearances as well as the impact in terms of taste and scent, crucial feature for the acceptability of the final product. Using these new ingredients was possible to produce antioxidant bred, pasta, cheese, cookies and ice-cream. These food products retains very well the antioxidant properties conferred by the added ingredients despite the very different treatments that were performed. The food obtained had a good palatability and in some cases the final product had also a good success on the market.

Sistemi innovativi per il miglioramento della qualità e della sicurezza degli alimenti

Nell’attuale contesto, caratterizzato da un’elevata attenzione alla qualità e alla sicurezza degli alimenti e alle soluzioni tese a garantirli, l’implementazione di sistemi microelettronici per il controllo del prodotto attraverso supporti miniaturizzati e a basso costo può risultare un’opportunità strategica. Oggetto della ricerca di dottorato sono stati lo studio dell’utilizzo di sensori e strumentazione innovativi per la misurazione ed il controllo di parametri ambientali di conservazione di prodotti alimentari e per la loro identificazione mediante la tecnologia della radiofrequenza. Allo scopo è stato studiato il contesto in cui operano gli attori principali della filiera agroalimentare ed è stata sviluppata un’idea di etichetta progettata per essere in grado di emettere attivamente segnale di allarme in caso di necessità (etichetta RFID intelligente semi-passiva). Il prototipo di chip, realizzato in via sperimentale, è stato validato positivamente, sia come strumento di misura, sia in termini di prestazione nel caso studio del monitoraggio della conservazione di un prodotto alimentare in condizioni controllate di temperatura e radiazione luminosa. Le significative evidenze analitiche di reazioni di degradazione dello stato qualitativo del prodotto, quali analisi di pH e colore, raccolte durante il periodo di osservazione di 64 giorni, hanno trovato riscontro con le misure rilevate dal chip prototipo. I risultati invitano ad individuare un partner industriale, con il quale sperimentare l’applicazione della tecnologia proposta.

Produzione di bioidrogeno in dark fermentation da scarti dell'industria agroalimentale mediante l'impiego di batteri ipertermofili

La presente tesi di dottorato ha come argomento la produzione d’idrogeno per via fermentativa sfruttando il metabolismo anaerobico di particolari batteri estremofili del genere Thermotoga. In questo lavoro, svolto in seno al progetto Bio-Hydro, sfruttando reattori batch da 116 mL, è stato selezionato il ceppo migliore di Thermotoga fra i quatto ceppi testati: T. neapolitana. Una volta individuato il candidato batterico migliore è stato individuato il valore ottimale di pH (8.5 a t.amb) per la produzione d’idrogeno. Un intenso lavoro è stato svolto sul medium di coltura permettendone la minimizzazione e rendendolo così economicamente sostenibile per il suo utilizzo nel reattore da 19L; in questo caso il glucosio è stato completamente sostituito con due sottoprodotti agroindustriali individuati in precedenza, il melasso di barbabietola e il siero di latte. Sono stati poi eliminati i gravosi micronutrienti e le vitamine. È stata sfruttata la capacità di T. neapolitana di produrre biofilm e sono stati testati 4 diversi supporti in vetro sinterizzato e ceramici, tali test hanno permesso di individuare Biomax come supporto migliore. Sono stati svolti studi sul metabolismo di T. neapolitana volti ad individuare le concentrazioni inibenti di ogni substrato testato, l’inibizione da prodotto (idrogeno) e l’inibizione da ossigeno. Tutte queste prove hanno dato le conoscenze di base per la conduzione di esperienze su reattore da 19L. L’innovativo reattore di tipo SPCSTR è stato interamente studiato, progettato e costruito presso il DICMA. dell’Università di Bologna. La conduzione di esperienze batch su SPCSTR ha dato la possibilità di verificare il funzionamento del nuovo tipo d’impianto. Presso il Wageningen UR (NL), è stata svolta la selezione del miglior ceppo di Caldicellulosisruptor fra 3 testati e del miglior supporto per la produzione d’idrogeno; è stato poi costruito testato e condotto in continuo l’innovativo reattore CMTB.