Elena Franciosi

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.

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.

The bacterial biota of laboratory-reared edible mealworms (Tenebrio molitor L.): From feed to frass

Tenebrio molitor represents one of the most popular species used for the large-scale conversion of plant biomass into protein and is characterized by high nutritional value. In the present laboratory study, the bacterial biota characterizing a pilot production chain of fresh T. molitor larvae was investigated. To this end, different batches of fresh mealworm larvae, their feeding substrate (wheatmeal) and frass were analyzed by viable microbial counts, PCR-DGGE and Illumina sequencing. Moreover, the occurrence of Coxiella burnetii, Pseudomonas aeruginosa and Shiga toxin-producing E. coli (STEC) was assessed through qualitative real-time PCR assays. Microbial viable counts highlighted low microbial contamination of the wheatmeal, whereas larvae and frass were characterized by high loads of Enterobacteriaceae, lactic acid bacteria, and several species of mesophilic aerobes. Spore-forming bacteria were detected to a lesser extent in all the samples. The combined molecular approach used to profile the microbiota confirmed the low microbial contamination of wheatmeal and allowed the detection of Enterobacter spp., Erwinia spp., Enterococcus spp. and Lactococcus spp. as dominant genera in both larvae and frass. Moreover, Klebsiella spp., Pantoea spp., and Xenorhabdus spp. were found to be in the minority. Entomoplasmatales (including Spiroplasma spp.) constituted a major fraction of the microbiota of one batch of larvae. From the real-time PCR assays, no sample was positive for either C. burnetii or STEC, whereas P. aeruginosa was detected in one sample of frass. Based on the overall results, two sources of microbial contamination were hypothesized, namely feeding with wheatmeal and vertical transmission of microorganisms from mother to offspring. Since mealworms are expected to be eaten as a whole, the overall outcomes collected in this laboratory study discourage the consumption of fresh mealworm larvae. Moreover, microbial loads and the absence of potential pathogens known to be associated with this insect species should be carefully assessed in order to reduce the minimum risk for consumers, by identifying the most opportune processing methods (e.g., boiling, frying, drying, etc.).

A new resource from traditional wines: characterisation of the microbiota of “Vino Santo” grapes as a biocontrol agent against Botrytis cinerea

Abstract The microflora of grapes involved in the production of a traditional Italian straw wine, “Vino Santo Trentino”, was evaluated as a biocontrol agent against Botrytis cinerea, one of the main diseases affecting fruit and grapes. The microbiota was described using plate counts and genotypic characterisation (sequencing of 16S rRNA for bacteria and 26s rRNA for yeast), allowing identification of yeasts belonging to the Hanseniaspora, Metschnikowia, Cryptococcus and Issatchenkia genera and bacteria (Bacillus, Microbacterium, Acetobacter and Gluconobacter spp.). The distribution of these species is related to the extent of B. cinerea infection. 7 isolates were able to halt the growth of B. cinerea in antagonistic cultures grown in Petri plates, using both synthetic growth and grape juice media. Technological characterisation of potential biocontrol agents, performed with the help of flow cytometry and HPLC-ECD, demonstrated that these microorganisms did not represent a risk for wine production due to their low resistance to ethanol, low pH and the absence of off-flavours. This ensures that the biocontrol agents disappear during winemaking and excludes a negative impact on the quality of wines. In conclusion, the microflora associated with dried grapes is a precious source of biocontrol agents against B. cinerea, both in terms of preventing disease in the vineyard and in control of the grape drying process for the production of straw wines.

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.

Evaluation of autochthonous lactic acid bacteria as starter and non-starter cultures for the production of Traditional Mountain cheese

Lactococcus lactis subsp. lactis 68, Streptococcus thermophilus 93 and Lactobacillus rhamnosus BT68, previously isolated from Traditional Mountain (TM) cheese, were tested for the production of four experimental mountain cheeses, with the aim to assess their effectiveness in leading the TM-cheese-making process. Lactococcus lactis subsp. lactis 68 and Streptococcus thermophilus 93 were used as starter cultures, whereas Lactobacillus rhamnosus BT68 was used as non-starter culture. Three control (CTRL) cheeses were manufactured without adding any starter, according to the traditional cheese-making process; nine, cheeses were produced inoculating the vat milk with the starters (ST), starter and low concentration of non-starter culture (STLC), starter and high concentration of non-starter culture (STHC). Samples of vat milk, cheese after 24 h and 7 months ripening were processed for microbiological counts. Mesophilic cocci were dominant in all 24 h-cheese samples, while a dominance of both cocci and lactobacilli was observed after 7 months ripening. The total genomic DNA was extracted, and a fragment of V1-V3 region was amplified and pyrosequenced. Lactococci and streptococci were the most abundant species, and Lc. lactis ssp. lactis 68 affected the proliferation of milk-resident Lc. lactis ssp. cremoris, during the early fermentation. Lb. rhamnosus BT68 showed to be responsible in reducing the abundance of other Lactobacillus species. Moreover, it likely competed against Sc. thermophilus 93 for the same energetic sources, when added in concentration higher than 5 × 103 CFU/mL milk. The sensorial and fatty acid (FA) composition analysis were performed on cheese samples at the end of ripening, demonstrating that the inoculated cheeses had better sensorial characteristics (aspect, smell, taste, texture) than CTRL cheeses, and that Lb. rhamnosus BT68 at high concentration is related to the increase of short chain fatty acids and conjugated linoleic acid in cheese after 7 months ripening.