Influence of lamb rennet paste containing probiotic on proteolysis and rheological properties of pecorino cheese.

Abstract Pecorino cheeses made from heat-treated ewes' milk using traditional lamb rennet paste (RP), lamb rennet paste containing Lactobacillus acidophilus (LA-5; RPL), and lamb rennet paste containing a mix of Bifidobacterium lactis (BB-12) and Bifidobacterium longum (BB-46; RPB) were characterized for proteolytic and rheological features during ripening. Consumer acceptance of cheeses at 60 d of ripening was evaluated. Lactobacillus acidophilus and Bifidobacterium mix displayed counts of 8 log 10 cfu/g and 9 log 10 cfu/g, respectively, in cheese during ripening. The RPB cheese displayed a greater degradation of casein (CN) matrix carried out by the enzymes associated to both Bifidobacterium mix and endogenous lactic acid microflora, resulting in the highest values of non-CN N and water-soluble N and the highest amount of α s -CN degradation products in cheese at 60 d of ripening. The RPL cheese displayed intermediate levels of lactic acid bacteria and of N fractions. The percentage of γ -CN in RP and RPL cheeses at 60 d was 2-fold higher than in the cheese curd of the same groups, whereas the mentioned parameter was 3-fold higher in RPB cheese than in the corresponding fresh curd according to its highest plasmin content. The lower hardness in RPB at the end of ripening could be ascribed to the greater proteolysis observed in cheese harboring the Bifidobacterium mix. Although differences in proteolytic patterns were found among treatments, there were no differences in smell and taste scores

Technical Note: A Simple Salting-Out Method for DNA Extraction from Milk Somatic Cells: Investigation into the Goat CSN1S1 Gene

Abstract In this study, a sensitive, rapid, and toxic solvent-free method to extract DNA from milk somatic cells was implemented for characterization of the goat α S1 -casein gene ( CSN1S1 ). Methods reported for purification of DNA from milk often involve organic extraction, overnight incubation, or use of expensive commercial kits. The present method was implemented for goat milk and is based on a salting-out protocol. The method yielded an amount of DNA suitable for PCR-RFLP without the need for sample enrichment. The PCR-RFLP of DNA extracted from milk produced amplified and digested products of correct size, comparable with those obtained using PCR-RFLP of DNA extracted from blood. Therefore, milk can be used as an alternative source of DNA, making sample collection easier and reducing stressful practices such as capture, handling, and venipuncture in animal management

Consumer acceptance and sensory evaluation of Monti Dauni Meridionali Caciocavallo cheese

Twelve Caciocavallo cheeses were collected from 6 factories (A, B, C, D, E, F) located in the Monti Dauni Meridionali area (Southern Italy) that adopted different protocols for cheese production. A total of 160 consumers were involved in the sensory evaluation of Caciocavallo cheese after 180 d of ripening. Cheese attributes were used to describe the flavor, texture, and appearance of cheeses. The highest scores for the shiny attribute were assigned to cheeses B, C, and E, whereas color intensity was the highest in cheeses B, D, and F. Strength, salty, and piquant attributes were higher in cheeses F and A because of the use of raw milk (F), rennet paste (A), and percentage of salt in the brine (A, F). Consumers perceived a more granular structure during the second half of chewing of Caciocavallo cheese F, as evidenced by the highest value for the grainy attribute. A positive correlation was found between overall flavor and odor intensity and water-soluble nitrogen, low molecular weight peptides, and free fatty acids and between piquant and butyric and caproic acids. A principal components analysis applied to the sensory attributes accounted for 65% of the total variance. The score plot showed that cheeses F and A were located in a well-defined zone of the plot, with cheeses in this zone displaying higher levels of strength, piquant, and salty attributes. The preference test assigned 40% of the preference to Caciocavallo cheese A, 38% to cheese F, 9% to cheese E, 8% to cheese D, and 7% to cheeses B and C. Sensory evaluation of Monti Dauni Meridionali Caciocavallo cheeses is a useful analysis to highlight the principal attributes able to influence consumers' liking that are related to biochemical features of the cheese

Probiotic in lamb rennet paste enhances rennet lipolytic activity, and conjugated linoleic acid and linoleic acid content in Pecorino cheese.

Cheeses manufactured using traditional lamb rennet paste, lamb rennet paste containing Lactobacillus acidophilus, and lamb rennet paste containing a mix of Bifidobacterium lactis and Bifidobacterium longum were characterized for the lipolytic pattern during ripening. Lipase activity of lamb rennet paste, lamb rennet containing Lb. acidophilus, and lamb rennet containing a mix of bifidobacteria was measured in sheep milk cream substrate. Rennet paste containing probiotics showed a lipase activity 2-fold greater than that displayed by traditional rennet. Total free fatty acid (FFA) in sheep milk cream was lower in lamb rennet paste (981 microg/g of milk cream) than in lamb rennet containing Lb. acidophilus (1,382.4 microg/g of milk cream) and in lamb rennet containing a mix of bifidobacteria (1,227.5 microg/g of milk cream) according to lipase activity of lamb rennet paste. The major increase of FFA in all cheeses occurred during the first 30 d of ripening with the greatest values being observed for C16:0, C18:0 C18:1. At 60 d of ripening all cheeses showed a reduction in the amount of free fatty acids; in particular, total free fatty acids underwent a decrease of more than 30% from 30 to 60 d in cheeses manufactured using traditional lamb rennet paste, whereas the same parameter decreased 10% in cheeses manufactured using lamb rennet paste containing Lb. acidophilus and cheeses manufactured using lamb rennet paste containing a mix of B. lactis and B. longum. Cheese containing Lb. acidophilus was characterized by the greatest levels of total conjugated linoleic acids (CLA) 9-cis, 11-trans CLA and 9-trans, 11-trans CLA, whereas cheese containing bifidobacteria displayed the greatest levels of free linoleic acid. Rennet pastes containing viable cells of Lb. acidophilus and a mix of B. lactis and B. longum were able to influence the amount of FFA and CLA in Pecorino cheese during ripening

Biochemical patterns in ovine cheese: influence of probiotic strains.

This study was undertaken to evaluate the effect of lamb rennet paste containing probiotic strains on proteolysis, lipolysis, and glycolysis of ovine cheese manufactured with starter cultures. Cheeses included control cheese made with rennet paste, cheese made with rennet paste containing Lactobacillus acidophilus culture (LA-5), and cheese made with rennet paste containing a mix of Bifidobacterium lactis (BB-12) and Bifidobacterium longum (BB-46). Cheeses were sampled at 1, 7, 15, and 30 d of ripening. Starter cultures coupled with probiotics strains contained in rennet paste affected the acidification and coagulation phases leading to the lowest pH in curd and cheese containing probiotics during ripening. As consequence, maturing cheese profiles were different among cheese treatments. Cheeses produced using rennet paste containing probiotics displayed higher percentages of alpha(S1)-I-casein fraction than traditional cheese up to 15 d of ripening. This result could be an outcome of the greater hydrolysis of alpha-casein fraction, attributed to higher activity of the residual chymosin. Further evidence for this trend is available in chromatograms of water-soluble nitrogen fractions, which indicated a more complex profile in cheeses made using lamb paste containing probiotics versus traditional cheese. Differences can be observed for the peaks eluted in the highly hydrophobic zone being higher in cheeses containing probiotics. The proteolytic activity of probiotic bacteria led to increased accumulation of free amino acids. Their concentrations in cheese made with rennet paste containing Lb. acidophilus culture and cheese made with rennet paste containing a mix of B. lactis and B. longum were approximately 2.5 and 3.0 times higher, respectively, than in traditional cheese. Principal component analysis showed a more intense lipolysis in terms of both free fatty acids and conjugated linoleic acid content in probiotic cheeses; in particular, the lipolytic pattern of cheeses containing Lb. acidophilus is distinguished from the other cheeses on the basis of highest content of health-promoting molecules. The metabolic activity of the cheese microflora was also monitored by measuring acetic, lactic, and citric acids during cheese ripening. Cheese acceptability was expressed for color, smell, taste, and texture perceived during cheese consumption. Use of probiotics in trial cheeses did not adversely affect preference or acceptability; in fact, panelists scored probiotic cheeses higher in preference over traditional cheese, albeit not significantly

Rennet paste from lambs fed a milk substitute supplemented with Lactobacillus acidophilus: effects on lipolysis in ovine cheese.

The present work was undertaken to evaluate the effects of Lactobacillus acidophilus supplementation of a milk substitute on the features of lamb rennet paste used for cheese making. Lipolysis in cheese manufactured with rennet paste from lambs receiving supplemented milk was also evaluated. Lambs were subjected to 3 different feeding regimens (mother suckling, MS; artificial rearing, AR; and artificial rearing with 7 log10 cfu/mL of Lb. acidophilus supplementation of the milk substitute, ARLb) and slaughtered at 20 and 40 d of age for each feeding treatment. Abomasa of the lambs were processed to rennet paste. Microbial loads, enzymatic activities (chymosin, pepsin, and lipases), and renneting characteristics of the lamb rennet paste were determined. Free fatty acids and conjugated linoleic acids were detected in cheese at 60 d of ripening. Addition of 7 log10 cfu/mL of Lb. acidophilus to the milk substitute was carried out successfully. Total recovery of viable cells was recorded in milk supplied daily to the lambs in the ARLb group. The ARLb rennet had greater amounts of lactobacilli than did the MS or AR rennet, irrespective of the slaughter age of the lambs, and the ARLb rennet had higher concentrations of lactococci when lambs were slaughtered at 40 d of age. Chymosin and lipase activities were also higher in ARLb rennet than in MS or AR rennet from lambs slaughtered at an older age. Milk supplementation of ARLb lambs resulted in improved coagulating ability of the rennet and enhanced cheese lipolysis after 60 d of ripening. A reduction of all free fatty acids was observed in all cheeses when passing from 20 to 40 d of slaughter of the lambs. Conjugated linoleic acids were more abundant in ARLb cheeses at both 20 and 40 d. Therefore, supplementation of the milk substitute with Lb. acidophilus improved the enzymatic features of rennet and the healthful and nutritional characteristics of it the ovine cheese. Moreover, the addition of lactobacilli to the milk substitute made it possible to increase the slaughter age of lambs without detrimental effects on rennet characteristics

Stress-induced changes in immune response of dairy ewes

two groups of 16 Comisana ewes were selected from a group of 30, and divided, according to their cortisol secretion after isolation in a novel environment, into HC ewes, having a cortisol secretion >90 ng/mL, and LC ewes having a cortisol secretion <80 ng/mL. Blood samples were collected immediately before and immediately after isolation, and 60, 120, 300min and then 24h and 48h after isolation, to evaluate the percentage of lymphocyte T-helper (CD4+) and lymphocyte T-cytotoxic (CD8+) by flow cytometry analysis, and IL-1β and IL-6 concentrations by ELISA. The day before the isolation test, the ewes were milked in the afternoon at 1500h (-19h from isolation), and then milked for the three days starting from the day of the isolation test (at 0, 5, 24, 29, 48 and 53h from isolation) to evaluate milk cortisol secretion, and IL-1β and IL-6 concentrations. Results suggest that the levels of cortisol secretions can influence the immune competence of dairy ewes and cytokines concentrations. Milk cytokine concentrations, and not milk cortisol concentrations, can be considered indicators of the magnitude of the hypothalamic-pituitary-adrenal axis activation

Contribution of macrophages to proteolysis and plasmin activity in ewe bulk milk.

A total of 225 bulk sheep milk samples were collected from 5 intensively managed flocks during early, mid, and late lactation to assess the contribution of macrophages to the regulation of the plasmin-plasminogen system. Samples were analyzed for composition, somatic cell counts, milk renneting characteristics, and for plasmin (PL), plasminogen (PG), and plasminogen activators (PA) activities. Isolation of macrophages from milk was performed using a magnetic positive separation and mouse antiovine macrophage antibody; separated cells were lysed by several freeze-thaw cycles, and activity of urokinase PA (u-PA) was determined. Plasmin activity decreased during lactation (42.06 +/- 0.66, early; 31.29 +/- 0.66, mid; 28.19 +/- 0.66 U/mL, late). The reduction in PL activity recorded in the mid and late lactation milk matched the increase in PG:PL ratio. The activity of PA increased throughout lactation; the highest value being recorded in the late lactation milk (260.20 +/- 8.66 U/mL). Counts of isolated and concentrated macrophages were higher in early and mid lactation milk (3.89 +/- 0.08 and 3.98 +/- 0.08 log10 cells/mL, respectively) than in late lactation milk (3.42 +/- 0.08 log10 cells/mL). Stage of lactation did not influence the activity of u-PA detected in isolated macrophages. The activity of u-PA associated with isolated milk macrophages only minimally contributed to total PA activity detected in milk. Proteolytic enzymes, associated with isolated macrophages, act on alpha-casein hydrolysis, as shown by urea-PAGE electrophoresis analysis. Somatic cell counts did not exceed 600,000 cells/mL, and this threshold can be considered a good index of health status of the flock and of the ability of milk to being processed. Our results lend support to the hypothesis that macrophages in ewe bulk milk from healthy flocks only slightly contribute to the activation of the PL-PG system

Effects of Somatic Cell Count and Stage of Lactation on the Plasmin Activity and Cheese-Making Properties of Ewe Milk

The experiment was conducted from March to July 2002 using 5 intensively managed flocks of Southern Italy. In each flock, 2 groups of 50 ewes were created. The groups were designated LSCC (low somatic cell count [SCC]) when their milk SCC was lower than 500,000/mL and HSCC (high SCC) when their milk SCC was higher than 1,000,000/mL. Bulk milk and whey samples were analyzed for fat, total protein, lactose, casein, and whey protein contents. Renneting properties of milk were also determined. Moisture, NaCl, and nitrogen fractions were determined in fresh cheese curds. In addition, plasmin (PL) and plasminogen (PG) activities in milk and cheese were monitored. The proteolytic activity of plasmin by urea-polyacrylamide gel electrophoresis and the white blood cell (WBC) differentials were determined. The HSCC resulted in higher pH values in milk and in higher moisture and lower fat contents in fresh cheese curds. Moreover, a lower recovery of fat and whey proteins was obtained from the HSCC than from the LSCC raw milk. The crude protein and casein contents were higher in the HSCC than in the LSCC curds during early and midlactation; an opposite trend was observed in late lactation. Plasmin and PG activities underwent more marked fluctuations in the LSCC than in the HSCC curds through lactation. The results of this experiment demonstrate that the PL activity in ewe milk is markedly influenced by the SCC, although SCC is not the only parameter for predicting PL and PG evolution in ewe milk. The LSCC milk resulted in a higher proteolytic potential of Canestrato pugliese cheese curds

Technical note: Rapid method for determination of amino acids in milk.

A rapid method for measurement of amino acids in milk was developed and validated. The method included a first step of milk protein hydrolysis, followed by the derivatization and separation of amino acids by HPLC. Six combinations of hydrolysis agent and temperature-time conditions were compared with a reference method; derivatization procedures as well as HPLC separation were improved. Hydrolysis of milk samples with 6 N HCl at 160 degrees C for 60 min resulted in no significantly differences compared with the reference method but allowed the analysis of a greater number of milk samples in a short time. In addition, this method was characterized by high precision, low repeatability uncertainty, and high accuracy for all amino acids evaluated; the recovery mean value of the single amino acids was 98.38%. The proposed method is, therefore, accurate, simple, rapid, and suitable for large numbers of milk samples