C.C.G. Silva

Technological properties of bacteriocin-producing lactic acid bacteria isolated from Pico cheese an artisanal cow's milk cheese

Evaluate technologically relevant properties from bacteriocin‐producing strains to use as starter/adjunct cultures in cheese making.

Genetic diversity, safety and technological characterization of lactic acid bacteria isolated from artisanal Pico cheese

A total of 114 lactic acid bacteria were isolated at one and 21 days of ripening from a traditional raw cows milk cheese without the addition of starter culture, produced by three artisanal cheese-makers in Azores Island (Pico, Portugal). Identification to species and strain level was accomplished by16S rRNA gene and PFGE analysis. Carbohydrate utilization profiles were obtained with the relevant API kits. Isolates were evaluated according to safety and technological criteria. The most frequently observed genus identified by 16S rRNA sequencing analysis was Enterococcus, whereas API system mostly identified Lactobacillus. The highest percentages of antibiotic resistance were to nalidixic acid (95%), and aminoglycosides (64-87%). All isolates were sensitive to several beta-lactam antibiotics and negative for histamine and DNase production. Gelatinase activity was detected in 49.1% of isolates, 43% were able to degrade casein and 93% were α-hemolytic. Most enterococci presented virulence genes, such as gelE, asaI, ace. Diacetyl production was found to be species dependent and one strain (Leu. citreum) produced exopolysaccharides. Selected strains were further studied for technological application and were found to be slow acid producers in milk and experimental cheeses, a desirable trait for adjunct cultures. Two strains were selected on the basis of technological and safety application as adjunct cultures in cheese production and presented the best cheese aroma and flavor in consumer preference tests. This is the first effort to characterize Pico cheese LAB isolates for potential application as adjunct cultures; the results suggest the potential of two strains to improve the quality of this traditional raw milk product.

Effect of corn supplementation of grass finishing of Holstein bulls on fatty acid composition of meat lipids

Finishing Holstein young bulls exclusively on pasture generally results in very lean carcass and meat, but corn supplementation is expected to simultaneously improve carcass traits and intramuscular lipids (IML). The expected increase in IML would allow for a larger 18:2c9,t11 (CLA) deposition in meat without affecting the n-3 PUFA present in LM phospholipids (PL). Holstein bulls (n = 33) with initial BW of 423 ± 52.4 kg reared exclusively on pasture were assigned to 1 of 3 finishing period (85 d) diets: finished exclusively on pasture (P0) or finished on pasture and individually supplemented with 4 (P4) or 8 kg/d (P8) of ground corn. Final BW (546 ± 56.3 kg) was not affected (P > 0.05) by corn supplementation, but ADG increased (P < 0.01) with the increasing corn supplementation level from 1.23 kg/d for P0 to 1.44 kg/d for P4 and to 1.67 kg/d for P8. Subcutaneous fat depth in P0 bulls was 0.8 mm and increased (P < 0.001) in both P4 (2.9 mm) and P8 (2.7 mm) bulls, but no difference (P = 0.73) was observed between P4 and P8 bulls. Similarly, the IML increased with corn supplementation, from 1.84 g/100 g muscle in P0 to 2.96 in P4 and to 3.24 in P8, but no difference (P = 0.55) was found between P4 and P8 bulls. Corn supplementation decreased (P < 0.01) 18:1t11 in neutral lipids (NL) but not 18:2c9,t11 (P > 0.34). The 18:1t10 (mg/g total NL fatty acid [FA] ± SEM) were 2.5 ± 0.13 in P0, 5.5 ± 1.68 in P4, and 14.8 ± 3.18 in P8 bulls, being greater in P8 compared with P4 (P = 0.02). Total FA in muscle PL and SFA were unaffected, but increasing corn supplementation resulted (P < 0.001) in an increase of 18:2n-6 in PL by replacement of mostly the 18:1c9 and 18:3n-3. Notably, the total number of cis double bonds present in FA of PL remained constant (P = 0.74) with corn supplementation. Compared with P0, corn supplementation maintained (P > 0.05) the high n-3 PUFA content in meat (mg/100 g meat) and increased the 18:2c9,t11 (P = 0.028) and 18:1c9 (P < 0.001). However, increasing corn supplementation from 4 to 8 kg/d increased the 18:1t10 (P = 0.031) and had no effect on 18:2c9,t11. Therefore, supplementing grass-finished Holstein bulls with moderate amounts of ground corn (4 kg/d) increased carcass fat cover and IML, maintained n-3 PUFA, and increased 18:2c9,t11 content in meat, whereas greater corn supplementation (8 kg/d; P8) resulted in no further improvements.

Safety and technological properties of bacteriocinogenic enterococci isolates from Tunisia

To investigate the safety and technological traits of previously isolated bacteriocinogenic enterococci strains for potential use as starter/adjunct cultures in foods.

Short communication: Latin-style fresh cheese enhances lactic acid bacteria survival but not Listeria monocytogenes resistance under in vitro simulated gastrointestinal conditions.

Different studies in humans have provided evidence about the health benefits of probiotics. However, most probiotic strains do not maintain good viability in the harsh conditions of the gastrointestinal tract (GIT). In the present study, Latin-style fresh cheese produced with potential probiotic bacteria was tested to evaluate this cheese type as a food carrier for the delivery of viable microorganisms after exposure to simulated GIT conditions. The resistance of 28 lactic acid bacteria (LAB) strains and Listeria monocytogenes upon exposure to acidic conditions (pH 2.5) and bile and pancreatic enzymes (0.3% of bile salts and 0.1% of pancreatin) was evaluated in vitro. When compared with fresh cultures, fresh cheese greatly improved LAB survival to simulated GIT conditions, as no loss of viability was observed in either acidic conditions (pH 2.5) or bile salts and pancreatin environment over a 3-h period. In opposition, L. monocytogenes did not survive after 1h under acidic conditions. These data demonstrated that Latin-style fresh cheese could play an important role in probiotic protection against gastrointestinal juices, enhancing delivery within the gut and thereby maximizing potential health benefits of LAB.

Application of Bacteriocins and Protective Cultures in Dairy Food Preservation

In the last years, consumers are becoming increasingly aware of the human health risk posed by the use of chemical preservatives in foods. In contrast, the increasing demand by the dairy industry to extend shelf-life and prevent spoilage of dairy products has appeal for new preservatives and new methods of conservation. Bacteriocins are antimicrobial peptides, which can be considered as safe since they can be easily degraded by proteolytic enzymes of the mammalian gastrointestinal tract. Also, most bacteriocin producers belong to lactic acid bacteria (LAB), a group that occurs naturally in foods and have a long history of safe use in dairy industry. Since they pose no health risk concerns, bacteriocins, either purified or excreted by bacteriocin producing strains, are a great alternative to the use of chemical preservatives in dairy products. Bacteriocins can be applied to dairy foods on a purified/crude form or as a bacteriocin-producing LAB as a part of fermentation process or as adjuvant culture. A number of applications of bacteriocins and bacteriocin-producing LAB have been reported to successful control pathogens in milk, yogurt, and cheeses. One of the more recent trends consists in the incorporation of bacteriocins, directly as purified or semi-purified form or in incorporation of bacteriocin-producing LAB into bioactive films and coatings, applied directly onto the food surfaces and packaging. This review is focused on recent developments and applications of bacteriocins and bacteriocin-producing LAB for reducing the microbiological spoilage and improve safety of dairy products.

Consumption of high energy maize diets is associated with increased soluble collagen in muscle of Holstein bulls

The aim was to investigate whether energy supplementation of gazing bulls can influence collagen concentration and solubility in three different muscles: longissimus dorsi (LD), semitendinosus (ST) and supraspinatus (SS). Thirty three Holstein bulls aged 15 months and reared on grass, were randomly assigned to three treatment groups for 85 days: fed grass ad libitum (G1-control); fed grass ad libitum and supplemented with 4 kg day(-1) of ground maize (G2); fed grass ad libitum and supplemented with 8 kg day(-1) of ground maize (G3). Total collagen content did not differ (P>0.05) among treatments, however, soluble (heat-labile) collagen was significantly (P<0.001) higher in meat from G3 (34%, 23% and 25% for LD, ST and SS, respectively), compared with G1-control (24%, 18% and 17% in LD, ST and SS, respectively). Overall, these results suggest that meat tenderness of grazing bulls may be improved by supplementing with ground maize (8 kg day(-1)).

Characterization and Application of Antilisterial Enterocins on Model Fresh Cheese

Enterococcus faecalis strains isolated from an artisanal cheese were selected based on enterocin production against Listeria monocytogenes. The strains formed biofilms and presented high hydrophobic character and good autoaggregation and coaggregation capacity with L. monocytogenes. Strains L3A21M3 and L3B1K3 presented high survival under gastrointestinal conditions, were able to adhere to human intestinal cells (Caco-2 and HT-29), and blocked the adhesion and invasion of L. monocytogenes. The antilisterial activity of enterocins was not affected by pH (2 to 12), heating (100°C), and chemical and surfactant agents. However, strains L3A21M3 and L3A21M8 produced thermolabile enterocins, which were also sensible to extreme pH values. Enterocins exhibited a bacteriostatic mode of action against L. monocytogenes, and maximum production was observed during the stationary phase. Common enterocin structural genes were not detected by PCR amplification with specific primers, although an exhaustive screening was not performed. The enterocin produced by the L3B1K3 strain was purified and applied to model cheeses contaminated with L. monocytogenes. This enterocin reduced survival of L. monocytogenes on fresh cheeses in a dose-dependent manner. The highest dose tested (2,048 arbitrary units per g of cheese) was effective in reducing the pathogen counts to undetectable values throughout storage (6 to 72 h). These results suggest that these strains have great potential to be used as biopreservatives in the food industry and also as probiotics, with the potential to prevent L. monocytogenes gastrointestinal infection.

The effect of paddock size on the response of seasonal anoestrous ewes to the ram effect

Many aspects of the conditions required to maximize the ewe’s response to ram introduction in the late anoestrous season remain unclear. The aim of this research was to determine whether grazing space allowances could influence the efficacy of the ram effect. In August 1995, at Reading (latitude 51 ◦ 27 � N), following a 3-month isolation period from rams, two groups of nulliparous Mule ewes, aged 15 months, were introduced to four rams in a low (12 ewes/ha; treatment L, n = 124) or in a high stocking rate (84 ewes/ha; treatment H, n = 126). From the beginning of August until the end of August oestrous behaviour was recorded by daily checks of mating marks on ewes. Rams were removed and in October all ewes were scanned (day 50) for pregnancy. No significant differences were found in the parameters investigated. Eighty-two percent of the L and 75.4% of the H ewes exhibited oestrus, with a pronounced peak on day 23 following ram introduction and a compact concentration in the 21–25-day period. The oestrous synchronisation rate in this 5-day period was 69.4 and 68.3%, respectively for L and H. The mean interval from ram introduction to oestrus was 23.17 ± 2.4 days in L and 23.0 ± 2.2 days in the H group. Conception rates were 84.3 and 87.4% for L and H groups, respectively. These results suggest that the response of anoestrous ewes to the introduction of rams was not affected by grazing space allowances and that yearling Mule ewes respond well to the ram effect in the late anoestrus season.

Isolation and characterization of an exopolysaccharide-producing Leuconostoc citreum strain from artisanal cheese

High molar mass exopolysaccharides (EPS) produced from sucrose by lactic acid bacteria (LAB) are of great interest as natural additives to use in foods, medical and pharmaceutical industry. This study aimed to identify the EPS produced by Leuconostoc citreum L3C1E7 isolated from Pico cheese and characterize the strain for technological and probiotic potential. Purified EPS was isolated from the culture of L. citreum L3C1E7 by ethanol precipitation, with a yield of 520 mg ml−1. The EPS‐producing strain had a mucoid phenotype and average molecular weight of 5·88 × 106 Da. The structural characterization of the purified EPS was determined by 1H, 13C and two‐dimensional NMR spectroscopy. EPS was composed of alternating α‐(1→6)‐linked and α‐(1→3)‐linked D‐glucopyranyl units, suggesting the existence of an alternan. The strain was slow acidifying, produced diacetyl and displayed high esterase/lipase and aminopeptidase activities, which promote the desirable flavours in dairy products. Moreover, L. citreum showed moderate resistance to the adverse conditions of the gastrointestinal (GI) tract and high adhesion to GI cells. This work provides a better understanding of EPS produced by L. citreum and the potential application of EPS‐producing strain in food and/or as a probiotic culture.