Applicability of Lactococcus hircilactis and Lactococcus laudensis as dairy cultures.

The aim of this study was to evaluate whether Lactococcus hircilactis and Lactococcus laudensis can be used as starter cultures. To this end, the two lactococci were characterized for traits of technological and functional interest. Tests in milk included growth at 20, 25, 30, and 37 °C, flavor production, antioxidant (AO) activity, folate and exopolysaccharide (EPS) production. At 30 °C, which resulted the best growth temperature for both strains, Lc. hircilactis and Lc. laudensis lowered the pH of the milk to 4.8 and 5.5, respectively, after 24 h of incubation. Sugar and organic acid composition indicated a higher lactose utilization, coupled with a higher lactate accumulation, by Lc. hircilactis, while galactose was completely consumed by both species. Both strains showed a Cit- phenotype after growth in a selective medium containing citrate as the sole carbon source. Nevertheless, a small amount of citrate was used by both lactococci when grown in milk. The two strains were characterized by a different flavor production, showed high AO activity, and produced small amounts of EPS (~30 mg/L). Lactococcus laudensis showed a weak proteolytic activity while Lc. hircilactis was able to accumulate folate at levels four times higher than uninoculated milk. When the two lactococci were tested as starter cultures in small-scale cheesemaking trials, cheeses resulted of satisfying quality and contained amounts of ethanol, acetic acid, diacetyl and acetoin higher than controls, obtained using a commercial culture. The application of Lc. hircilactis and Lc. laudensis as aromatic cultures in cheesemaking is proposed.

Prototheca blaschkeae subsp. brasiliensis subsp. nov., isolated from cow milk.

A strain of an achlorophyllic alga, named PR24T, was isolated from cow milk samples from the state of Minas Gerais, Brazil. Based on 18S rDNA, 28S rRNA, D1/D2 region of the LSU rDNA and SSU rRNA gene sequence similarities, this strain was found to be a member of the genus Prototheca and closely related to Protothecablaschkeae SAG2064T. However, the novel strain could easily be distinguished from recognized Prototheca species by internal transcribed spacer, species-specific PCR, single-strand conformation polymorphism-PCR analysis and phenotypic characteristics. The inability to grow in Sabouraud broth at pH 4.0 and the different cellular fatty acid composition clearly distinguished PR24T from the reference strain of P. blaschkeae. The combination of genotypic and phenotypic data indicates that strain PR24T represents a subspecies of P. blaschkeae, for which the name Prototheca blaschkeae subsp. brasiliensis subsp. nov. is proposed. The respective type strain is PR24T (=DSM 103592T=IHEM 26958T).

Can the development and autolysis of lactic acid bacteria influence the cheese volatile fraction? The case of Grana Padano.

In this study, the relationship between the dynamics of the growth and lysis of lactic acid bacteria in Grana Padano cheese and the formation of the volatile flavor compounds during cheese ripening was investigated. The microbial dynamics of Grana Padano cheeses that were produced in two different dairies were followed during ripening. The total and cultivable lactic microflora, community composition as determined by length heterogeneity-PCR (LH-PCR), and extent of bacterial lysis using an intracellular enzymatic activity assay were compared among cheeses after 2, 6 and 13months of ripening in two dairies. The evolution of whole and lysed microbiota was different between the two dairies. In dairy 2, the number of total cells was higher than that in dairy 1 in all samples, and the number of cells that lysed during ripening was lower. In addition, at the beginning of ripening (2months), the community structure of the cheese from dairy 2 was more complex and was composed of starter lactic acid bacteria (Lactobacillus helveticus and Lactobacillus delbrueckii) and NSLAB, possibly arising from raw milk, including Lactobacillus rhamnosus/Lactobacillus casei and Pediococcus acidilactici. On the other hand, the cheese from dairy 1 that ripened for 2months was mainly composed of the SLAB L. helveticus and L. delbrueckii. An evaluation of the free-DNA fraction through LH-PCR identified those species that had a high degree of lysis. Data on the dynamics of bacterial growth and lysis were evaluated with respect to the volatile profile and the organic acid content of the two cheeses after 13months of ripening, producing very different results. Cheese from dairy 1 showed a higher content of free fatty acids, particularly those deriving from milk fat lipolysis, benzaldehyde and organic acids, such as pGlu and citric. In contrast, cheese from dairy 2 had a greater amount of ketones, alcohols, hydrocarbons, acetic acid and propionic acid. Based on these results, we can conclude that in the first cheese, the intracellular enzymes that were released from lysis were mainly involved in aroma formation, whereas in the second cheese, the greater complexity of volatile compounds may be associated with its more complex microbial composition caused from SLAB lysis and NSLAB (mainly L. rhamnosus/L. casei) growth during ripening.

Lactococcus hircilactis sp. nov. and Lactococcus laudensis sp. nov., isolated from milk.

Two strains of lactic acid bacteria, designated 117(T) and 4195(T), were isolated from goat milk in Valtellina, Italy and from cow milk in Valle Trompia, Italy, respectively, and characterized taxonomically by a polyphasic approach. The strains were Gram-stain-positive, coccoid, non-spore-forming and catalase-negative bacteria. Morphological, physiological and phylogenetic data indicated that these isolates belonged to the genus Lactococcus. Strain 117(T) was closely related to Lactococcus fujiensis, Lactococcus lactis subsp. lactis, L. lactis subsp. cremoris, L. lactis subsp. hordniae, L. lactis subsp. tructae and Lactococcus taiwanensis, showing 93-94% and 82-89% 16S rRNA and rpoB gene sequence similarities, respectively. Strain 4195(T) was closely related to Lactococcus chungangensis, Lactococcus raffinolactis, Lactococcus plantarum and Lactococcus piscium, showing 92-98% and 86-99% 16S rRNA and rpoB gene sequence similarities, respectively. Based on this evidence and the data obtained in the present study, the milk isolates represent two novel species of the genus Lactococcus, for which the names Lactococcushircilactis sp. nov., and Lactococcuslaudensis sp. nov. are proposed. The respective type strains are 117(T) ( = LMG 28352(T) = DSM 28960(T)) and 4195(T )( = LMG 28353(T) = DSM 28961(T)).

Phospholipids in milk fat: composition, biological and technological significance, and analytical strategies.

Glycerophospholipids and sphingolipids are quantitatively the most important phospholipids (PLs) in milk. They are located on the milk fat globule membrane (MFGM) and in other membranous material of the skim milk phase. They include principally phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, while sphingomyelin is the dominant species of sphingolipids There is considerable evidence that PLs have beneficial health effects, such as regulation of the inflammatory reactions, chemopreventive and chemotherapeutic activity on some types of cancer, and inhibition of the cholesterol absorption. PLs show good emulsifying properties and can be used as a delivery system for liposoluble constituents. Due to the amphiphilic characteristics of these molecules, their extraction, separation and detection are critical points in the analytical approach. The extraction by using chloroform and methanol, followed by the determination by high pressure liquid chromatography (HPLC), coupled with evaporative light scattering (ELSD) or mass detector (MS), are the most applied procedures for the PL evaluation. More recently, nuclear magnetic resonance spectrometry (NMR) was also used, but despite it demonstrating high sensitivity, it requires more studies to obtain accurate results. This review is focused on milk fat phospholipids; their composition, biological activity, technological properties, and significance in the structure of milk fat. Different analytical methodologies are also discussed.

Cholesterol removal capability of lactic acid bacteria and related cell membrane fatty acid modifications.

Milk and dairy products play an important role in a healthy diet because of their high nutritional value, even if they represent a source of lipids and cholesterol. Nowadays, some commercially hypocholesterolemic products are available, which contain lactic acid bacteria (LAB). Therefore, the aims of this study were to test and compare the cholesterol removal abilities of different LAB species and to investigate the capacity of the cholesterol to change the cellular fatty acid composition of microorganisms. Fifty-eight strains of dairy LAB were studied for their ability to remove cholesterol during 24 h of growth. Two of them, L. plantarum 885 and L. acidophilus LA-5®, showed the higher reduction capability. For these strains, the cellular fatty acid composition was studied. They showed a different behaviour, which appeared related to the needs of the cells to maintain the characteristics of membrane fluidity, but was dependent upon their original fatty acid composition. Further studies are required to better characterise the LAB strains to be used to develop fermented dairy products with reduced cholesterol content or be able to induce hypocholesterolemic effects. It will also be interesting to investigate the possible modifications of the cell membrane caused by cholesterol and its possible involvement in cell metabolism.

Characterization of two Agrostis-Festuca alpine pastures and their influence on cheese composition.

Recently, there has been a renewed interest in mountain farming, and several studies have been carried out on milk and cheese obtained in the unique environmental conditions of the Alps, a 1300 km mountain chain, located in the north of Italy. In this paper, the influence, on some cheese constituents, of two very similar mountain grasslands, both dominated by Festuca - Agrostis , was investigated. The two pastures were located in the same area in the southeastern Italian alpine region and differed in sunshine orientation and exposure. Milk obtained from cows grazing on these pastures was used to produce a semi-hard traditional cheese. The differences observed between the cheeses of the two areas for both some hydrocarbons (1-phytene and 2-phytene) and trans-fatty acids can be explained by a different rumen environment created by the botanical composition of the two pastures. The multidisciplinary approach can be considered a successful strategy, suitable for studying markers of authenticity.

Enterococcus lactis sp. nov., from Italian raw milk cheeses.

Ten atypical Enterococcus strains were isolated from Italian raw milk cheeses. The 16S rRNA gene, phenylalanyl-tRNA synthase alpha subunit (pheS), RNA polymerase alpha subunit (rpoA) and the 16S-23S rRNA intergenic transcribed spacer (ITS) sequences, randomly amplified polymorphic DNA (RAPD) PCR and the phenotypic properties revealed that the isolates represent a novel enterococcal species. On the basis of 16S rRNA gene sequence analysis, the isolates were closely related to Enterococcus hirae ATCC 8043(T), Enterococcus durans CECT 411(T) and Enterococcus faecium ATCC 19434(T), with 98.8, 98.9 and 99.4% sequence similarity, respectively. On the basis of sequence analysis of the housekeeping gene pheS, the reference strain, BT159(T), occupied a position separate from E. faecium LMG 16198. The group of isolates could be easily differentiated from recognized species of the genus Enterococcus by 16S-23S rRNA ITS analysis, RAPD-PCR and phenotypic characteristics. The name Enterococcus lactis sp. nov. is proposed, with BT159(T) ( = DSM 23655(T) = LMG 25958(T)) as the type strain.

Hydrocarbon and fatty acid composition of cheese as affected by the pasture vegetation type.

The determination of the geographical origin of dairy products is an ongoing issue. In this paper the effects of botanical diversity of two pastures on the hydrocarbon and fatty acid composition of cheese fat were studied, over 2 years of experimentation. Two areas in the Italian southwestern Alpine region, dominated by Trifolium alpinum (T) and Festuca nigrescens (F) vegetation, respectively, were chosen, and milk obtained from cows grazing on these pastures was used to produce a semihard traditional cheese. Cheese samples showed a significantly different composition of most linear hydrocarbons, odd-chain (C15, C17, and C17:1) and unsaturated (trans-11,cis-15-C18:2, C18:3, C20:4n-6, C20:4n-3, and 20:5n-3) fatty acids, according to pasture type. The ratio between C(29) and C(27) linear hydrocarbons, unlike the absolute content of the single molecules, showed a good discriminating ability between the two pastures and was little affected by the natural variability due to the climatic and environmental factors.

Significance of the nonvolatile minor compounds of the neutral lipid fraction as markers of the origin of dairy products.

In the past few years several researchers have approached the problem of traceability of dairy products mainly by examining the volatile compounds that could be transferred from forage into milk and cheese. Our research focused on the study of the composition of the nonvolatile minor components of the neutral lipid fraction in mountain dairy products, obtained from animals feeding on pasture, and in milk and cheese samples produced from cows under intensive breeding, fed with concentrates and silages. Hydrocarbons were separated by silica gel column chromatography from the whole lipid matrix and analyzed by GC/MS. Among all the compounds detected, 1-phytene, 2-phytene, neophytadiene, and to a lesser extent the esters of phytol with C16 and C18 fatty acids seem to be promising tools for the recognition of the feeding system. The value of the sum of isoprenoid hydrocarbons (summation operator-hyd) of mountain dairy products (12.3-34.0 mg/kg) was always higher than that obtained from plain samples (1.3-6.4 mg/kg).

Comparison of solid-phase microextraction and purge-and-trap methods for the analysis of the volatile fraction of butter.

The volatile fraction of butter stored at three different temperatures was investigated to monitor quality during commercial shelf-life (90 days). Two different extraction techniques were compared: dynamic headspace (purge-and-trap), and static headspace (solid-phase microextraction, SPME). As expected, the dynamic extraction provided a generally higher amount of volatile compounds than that obtained by SPME, but, with reference to individual compounds, SPME seemed to provide better extraction for volatiles having a higher molecular mass. Despite the different performances, both methods were able to detect volatiles useful for evaluating changes during storage.

Volatile fraction of milk: comparison between purge and trap and solid phase microextraction techniques.

The aim of this research was to validate the results obtained previously by purge and trap (PT) and to investigate the ability of solid phase microextraction (SPME), a more rapid and less expensive technique, to discriminate drinking milk subjected to different heat treatments (i.e., pasteurization, ultrahigh temperature, "in-bottle" sterilization) and produced at different factories. The data obtained by both methods were processed by multivariate statistical analysis. PT and SPME showed comparable repeatability, although with different performances for the yield of extraction, and allowed the three milk categories to be distinguished. Within the chemical class of methyl ketones, 2-heptanone was found to be the most discriminating compound, and the possibility of using the concentration of this volatile as a marker for heat treatment was investigated.