Stefania Carpino

Improvement of the Streptomyces griseus method for degradable protein in ruminant feeds

Abstract The methods of Krishnamoorthy et al. [Krishnamoorthy, U., Sniffen, C.J., Stern, M.D., Van Soest, P.J., 1983. Evaluation of a mathematical model of rumen digestion and an in vitro simulation of rumen proteolysis to estimate the rumen undegraded nitrogen content of feedstuffs. Br. J. Nutr. 50, 555–568] and Roe et al. [Roe, M.B., Sniffen, C.J., Chase, L.E., 1990. Proc. Cornell Nutrition Conference, Dept. of Animal Science, Cornell University, Ithaca, NY, pp. 81–88], and a new one presented in this paper have been compared, and a revised procedure is proposed using a fixed ratio of enzyme to true protein (TP) determined by tungstic acid precipitation. A comparison of the three methods shows that they are statistically different. The ratio of enzyme has also significant effect ( p >0.01) on the estimate of degradable nitrogen when compared to the fixed concentration of enzyme in the original method. The effect of buffer pH (6.7 vs. 8) on degradation of protein sources was also statistically significant ( p >0.05) with somewhat higher degradation at pH 8.

Forage quality of native pastures in a Mediterranean area

Three field sites in the Hyblean region of Sicily were studied for their forage quality and botanical composition. A total of 70 plant species were analyzed. Another 36 were rare and of insufficient quantity for analysis. Forty-three bulk samples combining all existing species were also prepared and analyzed. The chemical analyses included were dry matter, ash, neutral-detergent fiber (NDF), acid-detergent fiber (ADF), lignin, total nitrogen, nonprotein nitrogen (NPN), soluble nitrogen and degradable protein. Quality varied over the growing season declining with forage maturity. The nutritional quality was high overall. Nongrass nonlegume species dominated. The most abundant species of high quality was a Compositae, Calendula arvensis, which represented approximately 22.8% of the available forage.

Uncommonly thorough hydrolysis of peptides during ripening of Ragusano cheese revealed by tandem mass spectrometry.

Ragusano is a pasta filata cheese produced from raw milk in Sicily. The proteolysis was extensively analyzed after stretching (day 0), at 4 and 7 months of ripening through soluble nitrogen, urea-PAGE, and peptide identification by tandem mass spectrometry. After stretching, 123 peptides were identified: 72 arising from β-casein, 34 from α(s1)-casein, and 17 from α(s2)-casein. The main protein splitting corresponded to the action of plasmin, chymosin, cathepsin D, cell envelope proteinase, and peptidase activities of lactic acid bacteria. Unlike other types of cheeses, <10% residual β- and α(s)-caseins remained intact at 7 months, indicating original network organization based on large casein fragments. The number of identified soluble peptides also dramatically decreased after 4 and 7 months of ripening, to 47 and 25, respectively. Among them, bioactive peptides were found, that is, mineral carrier, antihypertensive, and immunomodulating peptides and phosphopeptides.

A large factory-scale application of selected autochthonous lactic acid bacteria for PDO Pecorino Siciliano cheese production.

The main hypothesis of this study was that the autochthonous lactic acid bacteria (LAB) selected for their dairy traits are able to stabilize the production of PDO (Protected Denomination of Origin) Pecorino Siciliano cheese, preserving its typicality. The experimental plan included the application of a multi-strain lactic acid bacteria (LAB) culture, composed of starter (Lactococcus lactis subsp. lactis CAG4 and CAG37) and non starter (Enterococcus faecalis PSL71, Lactococcus garviae PSL67 and Streptococcus macedonicus PSL72) strains, during the traditional production of cheese at large scale level in six factories located in different areas of Sicily. The cheese making processes were followed from milk to ripened cheeses and the effects of the added LAB were evaluated on the microbiological, chemico-physical and sensorial characteristics of the final products. Results highlighted a high variability for all investigated parameters and the dominance of LAB cocci in bulk milk samples. The experimental curds showed a faster pH drop than control curds and the levels of LAB estimated in 5-month ripened experimental cheeses (7.59 and 7.27 Log CFU/g for rods and cocci, respectively) were higher than those of control cheeses (7.02 and 6.61 Log CFU/g for rods and cocci, respectively). The comparison of the bacterial isolates by randomly amplified polymorphic DNA (RAPD)-PCR evidenced the dominance of the added starter lactococci over native milk and vat LAB, while the added non starter LAB were found at almost the same levels of the indigenous strains. The sensory evaluation showed that the mixed LAB culture did not influence the majority of the sensory attributes of the cheeses and that each factory produced cheeses with unique characteristics. Finally, the multivariate statistical analysis based on all parameters evaluated on the ripened cheeses showed the dissimilarities and the relationships among cheeses. Thus, the main hypothesis of the work was accepted since the quality parameters of the final cheeses were stabilized, but all cheeses maintained their local typicality.

Survival of potential probiotic lactobacilli used as adjunct cultures on Pecorino Siciliano cheese ripening and passage through the gastrointestinal tract of healthy volunteers

In the present study, two lactobacilli strains, Lactobacillus rhamnosus H25 and Lactobacillus paracasei N24, used as adjunct cultures, were evaluated for their heat resistance both with and without prior heat adaptation and for their survival, at industrial scale, during the production and ripening of the Pecorino Siciliano cheese. In addition, the viability and persistence of the lactobacilli strains after passage through the gastrointestinal tract of healthy volunteers were evaluated by using rep-PCR analysis of viable cells. Both strains exhibited good heat resistance and survival throughout cheese production and ripening, and positively influenced the physico-chemical, the microbiological and the sensorial characteristics of the final product. In addition, the molecular typing of the lactobacilli isolates, retrieved from fecal samples of healthy volunteers during and after 15 days of the experimental cheese administration, revealed a high survival of the strains, highlighting their persistence during passage into the GI tract. In conclusion, this study proposes the two adjunct cultures as potential probiotic candidate deliverable by cheese.

Effect of Sicilian pasture feeding management on content of α-tocopherol and β-carotene in cow milk.

This study was performed to evaluate α-tocopherol and β-carotene contents of pasture milk under ordinary Sicilian farming conditions. Fourteen dairy farms were allocated into 2 balanced groups on the basis of cultivated (CULT) or spontaneous (SPO) pasture type feeding. Bulk milk per farm was collected 4 times from February through April at 3-wk intervals. Pasture botanical and diet composition, diet nutritional quality, milk yield and composition were estimated each time. Pasture intake levels were calculated based on feed analyses, hay and concentrate amounts fed, and milk yield and chemical composition. According to pasture intake, the farms were split into low pasture intake (LPI; <29.5% of dry matter) and high pasture intake (HPI; >29.5% of dry matter) groups. Milk samples per farm were analyzed for α-tocopherol and β-carotene contents by HPLC. The SPO group had higher levels of α-tocopherol and β-carotene in milk (0.7 and 0.3 mg/L, respectively) and milk fat (19.0 and 7.5 mg/kg fat, respectively) compared with the CULT group in milk (0.5 and 0.2 mg/L, respectively) and milk fat (14.6 and 4.9 mg/kg, respectively). High pasture intake compared with LPI increased α-tocopherol in milk fat (18.0 and 16.0 mg/kg of fat, respectively). However, only in the SPO (not in CULT), HPI compared with LPI increased milk α-tocopherol (0.8 vs. 0.6 mg/L, respectively), milk β-carotene (0.3 vs. 0.2 mg/L, respectively), and milk fat β-carotene (8.4 vs. 6.6 mg/kg, respectively). Results may be related to the different botanical composition of the respective pasture types and pasture intake. Spontaneous pasture compared with CULT contained a higher mass proportion of Asteraceae, Fabaceae, Cruciferae, Euphorbiaceae, and Malvaceae plants. Milk and milk fat α-tocopherol levels were higher on test-days (TD)-1, TD-2, and TD-4 compared with TD-3. For HPI farms, milk fat β-carotene content was higher on the first 2 TD compared with the last 2 TD. These differences could be related to plant biological stage. On Sicilian dairy farms, the highest milk α-tocopherol and β-carotene contents may be obtained feeding high levels of SPO pasture in the spring.

The Microfloras and Sensory Profiles of Selected Protected Designation of Origin Italian Cheeses

Approximately 39 Italian cheeses carry protected designation of origin (PDO) status. These cheeses differ in their manufacturing technology and the microbial flora which comprise the finished products. The evolution of lactic microflora in cheeses with PDO status is of particular interest because the biochemical activities of these organisms participate in cheesemaking and may play an acknowledged role in the development of organoleptic characteristics during ripening. Nonstarter lactic acid bacteria (NSLAB) constitute complex microbial associations that are characterized by the occurrence of various species and many biotypes as a result of a number of selective conditions persisting during the manufacturing process and different ecological niches. The evolution of different species during ripening of Fiore Sardo showed that, when present, Lactobacillus paracasei persists and dominates the microflora of the cheese in the last period of ripening, suggesting that this species, more resistant to the constraints of the mature cheese, could be involved in proteolysis and in other enzymatic processes occurring during cheese ripening. In contrast, the stretching step typical of pasta filata cheese, such as Ragusano, induced a simplification of the raw milk profiles, allowing the persistence only of some predominant species, such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. lactis, Lactococcus lactis, and Streptococcus macedonicus, after the stretching step. Lactobacillus plantarum and L. paracasei were isolated from ripened Castelmagno PDO cheese samples with the highest frequencies. These species, generally absent in the milk, occur in dairy ecosystems and dominate the bacterial flora of many ripened semihard cheeses. In PDO long-ripened Italian cheese such as Parmigiano Reggiano, the NSLAB population is mainly formed by L. paracasei, Lactobacillus rhamnosus, and Pediococcus acidilactici. Lactobacillus helveticus, L. delbrueckii subsp. lactis, and L. delbrueckii subsp. bulgaricus were also detected. Continued insight into the microbial populations of traditional Italian cheeses will allow continued production of characteristic, high-quality cheeses which have been enjoyed for many centuries.

Microscopy of a Goatskin Bag Cheese “Bouhezza”

Scanning electron microscopy and confocal laser scanning microscopy (CLSM) were used to visualize changes in the microstructure of a goatskin bag cheese “Bouhezza” during manufacturing and ripening. The exceptional cheesemaking process is based on the successive addition of salted fermented milk “Lben” in the goatskin bag for few weeks and then adding raw milk at the end of ripening. The SEM observation shows a proteinic aggregates organised in an open space matrix in which lipids and whey are entrapped in the proteinic mass. We observe the limits of micelles where caseins are not completely fused between them. During the manufacturing the semi-liquid structure of the Lben change to a gel structure and the formed cheese grows rich out of proteins and its structure becomes denser, specially marked enter the first and the third week of manufacturing. Then and to 6 weeks, the structure of cheese does not changed and the proteinic network became just more dense. This structure allows the continuous draining of the whey through the perforations of the goatskin. The CLSM shows the same observation. Fat globules were less in the first weeks of manufacturing and at the end their distribution appears to be homogenous in the protein matrix and are in form of visible small and large globules.