Fermentation – based food: influence of processing on product quality

Fermented foods and beverages represent an important part of human nutrition in every food culture around the world. Some of the most popular fermented products derive from grain, fruit and vegetables and are alcoholic based, for instance the notable variety of traditional beers and wines. Originally, fermentation can be regarded as a biological method of food preservation, but it is increasingly understood that some fermented foods also promote human health through the contribution of microorganism leading to further properties unlike to the starting food materials, beyond basic nutrition. Indeed, there is evidence that some fermented foods provide beneficial effects through direct microbial/ probiotic action and indirectly via the production of metabolites and breakdown of complex proteins. Kefir, for example, can help to relieve gastrointestinal disorders and reduce the symptoms of lactose intolerance besides having antioxidant, antimicrobial, anti-tumor and immunomodulation effects. Moderate wine consumption has undeniable health benefits as a decrease in the probability of cardiovascular disease, to delay the onset of noninsulin-dependent diabetes mellitus, combat hypertension, and reduce the frequency of certain cancers and several other diseases. In the last years, there is a growing interest in winemaking without added of sulfites, owing to the perception that sulfites may cause negative health effects. The absence of sulfites in wine can be regarded as a quality factor, because consumers are attracted to foods without added additives, which are considered as healthy and genuine. Several methods for sulfites removal or reduction during winemaking have been studied, but the complete substitution with a product performing the same roles without the disadvantages of sulfites has been to date unsuccessful. Conversely, the wine production without addion of sulfites is becoming increasingly feasible due to technological improvement in winemaking. Furthermore, in the last years, there is a increased interest in the world wine market of red wines with fruity characteristics and old techniques developed some decades ago such as thermovinification, are becoming more widespread. Thermovinification is a winemaking process consisting of heating grapes to improve the extraction of grape anthocyanins and polysaccharides, responsible for color and roundness, whereas it reduces the tannins extraction, thus exogenous tannins are often added. Within fermented foods, milk kefir is becoming more popular in European countries due to various health benefits linked for example to bioactive peptides. In particular, phosphopeptides are a subgroup of mulitphosphorylated bioactive peptides, which for instance can enhance the absorption of minerals in the gastrointestinal tract because binding and solubilizing bivalent metal ions such as Ca2+. Formation of phosphopeptides during the manufacture of kefir is affected of production technology. Finally, a great attention is paid to offer an alternative to conventional dairy-based drink, such as soy drink kefir, which could be a substitute food for vegans and people suffering from lactose intolerance or allergies. The purpose of the present Ph.D. work was the study of influence of several technological parameters on the composition of two fermented foods: wine and kefir. In the first part, the work was focused on the study of several factors affecting the quality of white and red wines, whereas, in the second part, Ph.D. research regarded the influence of some technological parameters on the production of phosphopeptides in kefir. Finally, the characterization of microbiological and peptide profiles of kefir prepared with kefir grains from soy-drink was achieved. As concerning wine, a study of the evolution in bottle of white wine produced by reductive winemaking, with and without sulfites, under several experimental conditions, was carried out over 15 months. Dark storage at 12 °C and 30 °C were compared to investigate the temperature effect, meanwhile uncontrolled temperature and light conditions were selected to simulate inadequate storage conditions. Results showed that volatile acidity and the absorbance at 420 nm increased at 15 months of storage particularly in both samples without sulfur dioxide addition and stored at 30°C and uncontrolled temperature and light conditions. Total SO2 and free SO2 decreased with time in wine samples with sulfites, with the exception of wine stored at 12°C, due to SO2 action as antioxidant. Seventeen phenolic compounds were determined by HPLC-DAD-MS/MS analysis including hydroxycinnamate derivatives and minor compounds resulting from oxidation processes. At 15 months, the sample without sulfites addition and stored at the constant temperature of 30°C was the most browned, followed by samples stored at inadequate temperature and light conditions and at constant temperature of 12°C both without sulfites added. Besides the oxidation compounds, the absence of sulfites seems to have favored the presence of cis forms of hydroxycinnamate derivatives and caffeic acid derivatives. Furthermore, the combination of inadequate conditions of light and temperature favored hydrolysis reactions more than the effect due to the only high temperature. Finally, results concerning volatile profile showed that the presence of sulfites helps the typical aroma of young wines; contrarily the storage without sulfites accelerated the hydrolysis of acetate esters and contributed to increase furfural, 5-methyl-2-furancarboxaldehyde, and benzaldehyde contents. The appropriate storage in the dark, at low temperature seems to be the most important condition as improper storage conditions favor aroma degradation regardless sulfite addition. In the second work, the influence of thermovinification on ochratoxin A (OTA) content in red wines was studied. Two heating treatments were investigated (60-65°C for 2 h and 80-85°C for 30 min), both treatments applied with and without added tannins. To achieve this goal, a rapid and automatable method for the determination of OTA in wine using a microextraction by packed C18 sorbent followed by high performance liquid chromatography with fluorescence detection was developed and validated for a successful application in the context of wine production. Important experimental parameters, such as sample and eluent volumes, extraction mode, draw and dispense speeds, number of eluent passes up and down through the stationary phase, were optimized. The validation included the comparison of the sensitivities related to solvent-matched, matrix-matched and standard addition calibrations and the participation to a proficiency test in an inter-laboratory circuit. Matrix effects were also investigated. Accuracies relevant to real samples were estimated to range between 76 and 100%, at 0.2 μg/L, and between 84 and 108%, at 1.0 μg/L, in compliance with the EU Regulation 401/2006; the limits of detection and quantification were of 0.08 and 0.24 μg/L, respectively, i.e. much lower than the maximum level currently permitted for OTA in the European Union (2.0 μg/kg, corresponding to ca 2.0 μg/L). As concern thermovinification process, no significate differences on OTA concentration among the wines studied were found. As regards the second part of the thesis, first, the profiling of multi-phosphopeptides in kefir was carried on, and phosphopeptides were selectively enriched on hydroxyapatite and further analyzed with mass spectrometry analysis. Thus, 22 phosphopeptide sequences were identified in kefir, mainly derived by β-casein, and 9 of them showed the polar acidic motif pSpSpSEE (pS= phosphoserine), which is the most active in binding minerals. Then, the influence of temperature (18 or 25°C), pH (4.0 or 4.7) and the repeated use of grains with 2-step fermentation (back-slopping approach) on phosphopeptide profile in milk kefir prepared using kefir grains was studied. Microbiological characterization was carried out in each sample, considering both grains and kefir, before and after fermentation. The total lactic acid bacteria, acetic acid bacteria and yeasts are increased after fermentation steps and, in particular, microbial growth, at the investigated conditions, was affected only quantitatively by the fermentation temperature whereas both quantitatively and qualitatively by the final pH. The LC-ESI-QTOF-MS/MS study revealed a phosphopeptide profile of kefir that include 93 phosphopeptides with lengths ranging from 10 to 43 amino acids, and of which, 82 showed the typical acid motif “SSSEE”, crucial for exhibiting the bioactive properties of mineral binding. Furthermore, kefir processing, in particular pH, largely influenced the proteolysis of kefir grains microorganisms and the profile of microbial population. A reduced proteolysis rate was observed in kefir prepared with back-slopping approach, indicating a low reproducibility of proteolytic activity of kefir grains. Moreover, a sensory evaluation was carried out on the kefir samples and no statistically significant differences (p< 0.05) were observed in odor, taste and acidity between kefir products. Finally, the microbiological evaluation and a comprehensive analysis of peptide profile in kefir, prepared with kefir grains from soy-drink were performed. As concerning microbial population, only lactic acid bacteria and yeasts were found in soy drink and grains kefir; acetic acid bacteria were not detected. One hundred seventy-nine peptides were identified mainly derived from glycinin and lipoxygenase and ranged from 9 to 37 amino acids. The released peptides from the parent proteins are not distributed equally among the protein sequences, indeed, there were distinct parts in the proteins where more peptides are released than in other parts. However, the specificity of lactic acid bacteria proteinases in soy-based fermented foods has not been equally investigated as the proteolytic behavior on milk proteins. Results showed 62 bioactive sequences, encrypted within the investigated peptides, which are known for ace-inhibitory, antihypertensive, antioxidative, hypocholesterolemic and antimicrobial activities. All results described in the present PhD thesis contributed to increase the knowledge on wine and kefir, on relationships between quality and processing and potential health benefits, providing important tools to improve quality of wine and kefir productions.