Agostino Cavazza

Immobilization of yeast and bacteria cells in alginate microbeads coated with silica membranes: procedures, physico-chemical features and bioactivity

Salt alginate beads are used to entrap yeast or bacteria cells and exploited as protective environment for deposition of silica gel membranes. This inorganic layer is obtained through different methods: by dipping the beads in a silica sol (method A), by further consolidating them with tetraethoxysilane in an apolar solvent (method B), and by coating the beads with methyltriethoxysilane in the gas phase (method C). Physical–chemical and biological features are investigated. The pyrolysis study elucidates the features of the polymeric organic layer. Elemental analysis and 29Si solid state NMR prove the presence and the condensation degrees of the silica membranes. The bioactivity is studied by evaluating both glucose (Saccharomyces cerevisiae) and L-malic acid (Oenococcus oeni) fermentations. The fermentation performance is discussed considering possible limitations of mass transport across the silica gel layer. Method A produces an inorganic layer made by of 57.3% Si(OSi)4 and 42.7% HO–Si(OSi)3 units). In this case a linear relation is found among different contact times between silica sol and beads and both silica amount and average thickness. Method B appears to be successful in building up the layer, but it is also detrimental to cell viability. Method C is very efficient in terms of mass deposit, cell viability maintenance and leakage reduction. In this case the membrane is constituted by H3C–Si(OSi)2OH and H3C–Si(OSi)3 units in an almost 1 : 1 ratio.

A Factory-Scale Application of Secondary Adjunct Cultures Selected from Lactic Acid Bacteria During Puzzone di Moena Cheese Ripening

The lactic acid populations of 2 seasonal Puzzone di Moena cheeses made from winter and summer raw cows milk were characterized at different ripening times. Lactic acid bacteria (LAB) were isolated on selective media and subjected to genetic typing and identification. The species most frequently found during ripening were Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, and Pediococcus pentosaceus. The different strains recognized by random amplification of polymorphic DNA-PCR were characterized for their acidifying and proteolytic activities to select nonstarter LAB to be used as secondary adjunct cultures (SAC). For each of the 3 above species, a strain showing weak acidification and high proteolytic capacity was selected. The 3 strains (Lb. paracasei ssp. paracasei P397, Lb. plantarum P399, and P. pentosaceus P41) constituted a mixed SAC used at 2 levels of concentration (10(3) and 10(4) cfu/mL) in experimental cheese making at dairy factory-scale. The analysis of volatile organic compounds as well as sensory analyses showed that the preferred level of SAC inoculation was 10(3) cfu/mL.

Changes in psychrotrophic microbial populations during milk creaming to produce Grana Trentino cheese

The aim of this study was to study the psychrotrophic microbiota developing during milk creaming of Grana Trentino cheese-making. 138 isolates from raw whole milk, cream and skim milk samples were screened by Randomly amplified polymorphic DNA PCR biotyping and representative strains of each biotype were characterised by partial 16S rRNA gene sequencing and enzymatic activity. Pseudomonadaceae were commonly isolated in cream samples while Streptococcaceae and Enterobacteriaceae in milk samples. Moraxellaceae and Flavobacteriaceae were found in both cream and milk samples. More than 80% of psychrotrophic isolates could grow at 37°C. All Flavobacteriaceae and half of Pseudomonadaceae biotypes displayed proteolytic activity on milk agar even at low temperatures such as 10°C. All Streptococcaceae and some of Enterobacteriaceae displayed acidifying activity and almost all Acinetobacter spp. (Moraxellaceae) displayed lipolytic activity towards tributyrin. Even if psychrotrophic bacteria is not the dominant microbial group in raw milk, their total number increases during creaming and becomes one of the most present group together with Lactic Acid Bacteria. Their enzymatic activities may be key players in determining milk quality for cheese making.

Selection of a new highly resistant strain for malolactic fermentation under difficult conditions.

Malolactic fermentation (MLF) is a biological process that contributes to wine quality, but it is frequently affected by various vinification conditions. Resistance to four wine-limiting factors was studied with respect to 10 Oenococcus oeni strains in order to select a suitable strain for performing reliable MLF in difficult wines. Resistance to low fermentation temperature, high SO2 and/or ethanol concentration, and low pH were assayed in laboratory tests. A pool of the most resistant strains was used in a set of laboratory MLFs. At the end of fermentation, the dominant strains were identified by RAPD-PCR. The PN4 strain was found to be dominant in the majority of cases and under the most detrimental wine conditions, and it was therefore chosen as the single-strain inoculum for the subsequent MLF trials. The effectiveness of the PN4 strain was confirmed in a series of MLFs carried out in three different countries under experimental and industrial conditions. It accomplished MLF in wines with up to 15.8% ethanol, pH as low as 3.0, 60 mg/L of free SO2, and in fermentation temperatures below 17oC. Our findings indicate that the O. oeni PN4 strain could be an effective starter, guaranteeing regular and reliable MLF fermentation.

Biodiversity and γ-aminobutyric acid production by lactic acid bacteria isolated from traditional alpine raw cow's milk cheeses.

“Nostrano-cheeses” are traditional alpine cheeses made from raw cows milk in Trentino-Alto Adige, Italy. This study identified lactic acid bacteria (LAB) developing during maturation of “Nostrano-cheeses” and evaluated their potential to produce γ-aminobutyric acid (GABA), an immunologically active compound and neurotransmitter. Cheese samples were collected on six cheese-making days, in three dairy factories located in different areas of Trentino and at different stages of cheese ripening (24 h, 15 days, and 1, 2, 3, 6, and 8 months). A total of 1,059 LAB isolates were screened using Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and differentiated into 583 clusters. LAB strains from dominant clusters (n = 97) were genetically identified to species level by partial 16S rRNA gene sequencing. LAB species most frequently isolated were Lactobacillus paracasei, Streptococcus thermophilus, and Leuconostoc mesenteroides. The 97 dominant clusters were also characterized for their ability in producing GABA by high-performance liquid chromatography (HPLC). About 71% of the dominant bacteria clusters evolving during cheeses ripening were able to produce GABA. Most GABA producers were Lactobacillus paracasei but other GABA producing species included Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosaceus, and Streptococcus thermophilus. No Enterococcus faecalis or Sc. macedonicus isolates produced GABA. The isolate producing the highest amount of GABA (80.0±2.7 mg/kg) was a Sc. thermophilus.

Dynamic changes in microbiota and mycobiota during spontaneous 'Vino Santo Trentino' fermentation.

Vino Santo is a sweet wine produced from late harvesting and pressing of Nosiola grapes in a small, well‐defined geographical area in the Italian Alps. We used metagenomics to characterize the dynamics of microbial communities in the products of three wineries, resulting from spontaneous fermentation with almost the same timing and procedure. Comparing fermentation dynamics and grape microbial composition, we show a rapid increase in a small number of wine yeast species, with a parallel decrease in complexity. Despite the application of similar protocols, slight changes in the procedures led to significant differences in the microbiota in the three cases of fermentation: (i) fungal content of the must varied significantly in the different wineries, (ii) Pichia membranifaciens persisted in only one of the wineries, (iii) one fermentation was characterized by the balanced presence of Saccharomyces cerevisiae and Hanseniaspora osmophila during the later phases. We suggest the existence of a highly winery‐specific ‘microbial‐terroir’ contributing significantly to the final product rather than a regional ‘terroir’. Analysis of changes in abundance during fermentation showed evident correlations between different species, suggesting that fermentation is the result of a continuum of interaction between different species and physical–chemical parameters.

Use of organo-silica immobilized bacteria produced in a pilot scale plant to induce malolactic fermentation in wines that contain lysozyme

The exploitation of organo-silica immobilized lactic acid bacteria (LAB) to perform malolactic fermentation (MLF) in wine is described. The immobilization of a large amount of Oenococcus oeni cell culture was achieved by a two-step process in an original pilot plant. Cells are entrapped in Ca-alginate microbeads, coated with an organo-silica membrane obtained by two treatments: the first a sol suspension of tetraetoxysilane, the second using methyltriethoxysilane in gas phase. The resulting material improves the physico-chemical features of alginate, avoids cell leakage during fermentation, and protects the cells from antimicrobial compounds. In MLFs carried out at the microvinification scale, the activity of immobilized cells did not differ from that of free cells, and no differences were found in the chemical composition of the wines obtained. The use of immobilized bacteria allowed: (1) simultaneous alcoholic and malolactic fermentations in must inoculated with free yeast and immobilized bacteria; (2) the sequential MLF of three wine lots with the same biomass of immobilized bacteria; (3) the achievement of MLF in a wine with lysozyme added to suppress wild LAB and their potential spoilage.

Does milk treatment before cheesemaking affect microbial and chemical traits of ripened cheese? Grana Trentino as a case study

The aim of this study was to evaluate the influence of different storage temperatures and delivery system of the milk on the microbiological and physicochemical characteristics of Grana Trentino, a long-ripened hard-cooked Italian cheese. In particular, 3 kinds of milk storage and delivery were studied: milk delivered to the dairy in the traditional manner without temperature control, milk delivered at 18°C, and milk stored at the farm and delivered at 12°C. Milk, natural whey starter, and cheeses after 18 mo of ripening were sampled for microbiological profiles, physicochemical analysis, and proteolysis evaluation, and a study of cheese volatile compounds through a solid-phase microextraction gas chromatography-mass spectrometry technique was performed. Milk microbiota was not affected by any of the treatments. At the end of ripening, free fatty acid and ester contents were significantly higher in cheeses from milk without temperature control. This was probably due to the milk delivery to the dairy in churns causing the fat globule membrane break during transport and, consequently, a greater release of fat and deeper lipolysis. Milk refrigeration at 12°C for 12h before delivery affected the distribution of nitrogen fractions in cheeses. Lower temperatures of milk storage favored a larger soluble nitrogen fraction and greater cheese proteolysis, probably caused by an enhanced plasmin activity. From this work, it is concluded that both milk temperature storage and transport system could affect cheese ripening, leading to significant differences in chemical compounds: if milk was delivered by churns, higher free fatty acid and ester content in cheeses was observed; if milk was stored at 12°C for 12h before delivery, greater cheese proteolysis was induced with consequent faster ripening.

Microbiological monitoring of raw milk from selected farm in the Trentingrana region

Abstract A study was conducted to monitor raw milk hygiene, and to understand the relationship between different bacterial groups found in raw milk collected from stables in the Trentingrana area. Twenty stables in Trento province were sampled. Each farmer provided one milk sample every 2 months for 8 months. The milk samples were examined for total bacterial count, coliforms, enterococci, psycrotrophic, acidificant and proteolytic microflora. The bacterial counts of four milk samples for each stable, examined over an 8-months period were averaged and expressed as mean bacterial count per milliliter. In most samples, coliforms and enterococci were below 2Log cfu/ml; this suggests that herds and milking hygiene were well controlled by all farmers. The milk refrigeration practice seems to have no influence on the growth of its microflora but to affect that of acidifying and proteolytic bacteria.