Indirect competitive enzyme-linked immunosorbent assay for the detection of native and heat-denatured bovine β-lactoglobulin in ewes' and goats' milk cheese

An indirect competitive enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of native and heat-denatured bovineβ-lactoglobulin (hd-β-lg) in goats' and ewes' milk cheese. Polyclonal antibodies raised in chicken against hd-β-lg were purified by immunadsorption chromatography on native bovine, ovine and caprineβ-lactoglobulins conjugated to CNBr-activated Sepharose. Although lactoglobulins from ewe, goat and cow have very similar amino acid sequences, crossreactivity was eliminated completely by this procedure. A commercially available goat-anti-chicken alkaline phosphatase conjugate was used as a secondary antibody to detect the anti-hd-β-lg antibodies bound to immobilized hd-β-lg. The detection limit of the assay was 100 ng/ml bovine native lg or hd-β-lg or 0.1–0.2% of cows' milk equivalent in cheese. Within a method validation study of the European Union, the ELISA test was successfully applied for the detection of native and heat-denatured bovine whey proteins in ewes' and goats' milk cheeses.     

Effect of probiotic co-cultures on physico-chemical and biochemical properties of small ruminants’ fermented milk

Small ruminants' fermented probiotic milk is an alternative to fermented cows' milk, especially because of the monounsaturated/polyunsaturated fatty acid profiles. The technological and biochemical potential of Bifidobacterium and Lactobacillus co-cultures, with or without inulin, on goats' and ewes' milk was assessed. Microbial stability, lactose consumption, organic acid production, proteolytic parameters and conjugated linoleic acid (CLA) production in situ, were followed in ewes' and goats’ fermented milk (EFM and GFM, respectively) over 21 days at 4 °C; technological feasibility for probiotic fermented milk production was shown. In EFM, all co-cultures presented high viable cell numbers (>7.0 log cfu mL⁻¹) throughout storage, presenting faster acidification capacities and higher CLA isomer levels than in GFM. Inulin had no impact on probiotic growth, yet contributed to storage stability. CLA isomers and proteolysis indices were co-culture dependent traits: for example, co-culture of Bifidobacterium animalis B94 with Lactobacillus acidophilus L10 registered the best CLA-production in GFM.     

Effect of preservatives on milk composition determination

The main objective of our study was to determine the effect of different concentration of four different preservatives, potassium dichromate, Azidiol, Bronopol and Microtabs II, on the results of laboratory analyses of milk using mid-infrared spectrometry. The final concentrations of the preservatives in the raw cows' milk samples were 0.005%, 0.01%, 0.05%, 0.1%, 0.5% and 1% respectively. We analysed unpreserved, fresh bulk raw cows' milk 2 and 24 h after milking. The effect of preservatives on the composition of the milk was studied 24 h after preservation. The experiment was replicated 10 times. Each experiment was carried out after the calibration of the mid-infrared spectrophotometer using calibration samples preserved with 0.02% Bronopol. We found that the concentration of the preserving agent had a significant effect (P < 0.005) on the results of the laboratory analyses; therefore, the proper concentration of preservative has to be used for routine sample preservation.     

Evaluation of fatty acid composition in commercial infant formulas on the Chinese market: A comparative study based on fat source and stage

The fatty acid compositions of 180 commercial infant, follow-on and growing-up formulas with different fat sources (plant-oil, cows' milk, goats' milk) on the Chinese market were analysed and compared with mature human milk. Fatty acid composition in formulas was dependent on fat source. The plant-oil formulas were more similar to mature human milk, while the cows' milk and goats' milk formulas contained higher levels of saturated (C4:0–C10:0) and lower levels of oleic acid and long-chain polyunsaturated fatty acids than those of mature human milk. There was little difference among formulas for the three stages except for the goats' milk formulas. The major fatty acids of formulas were similar to those of mature human milk, while levels of some micro-fatty acids in formulas, such as nervonic acid and long-chain polyunsaturated fatty acids, were significantly lower than in mature human milk.     

Characterisation of the anti-microbial activity of bovine milk ribonuclease4 and ribonuclease5 (angiogenin)

Two members of the ribonucleaseA (RNaseA) family were identified in cows' milk. RNase5, already known to be present, was found at concentrations between 3 and 19 μg mL^?1. RNase4, which was not previously known to be in milk, was found at concentrations between 1 and 4 μg mL^?1. These proteins were purified from milk. Purified RNase5 suppressed the growth of Candida albicans, particularly its hyphal form, but not any other microbes tested. The concentrations required to suppress growth were higher than those found in milk. RNase4 had no growth suppression activity. Both RNases reduced the number of viable C. albicans cells, with RNase5 being the more potent. This activity was abrogated by physiological concentrations of NaCl. Although both RNases bound to C. albicans cell membrane, their mode of action was not through membrane lysis or permeabilization. Together, these results suggest that the bovine milk RNases do not have intrinsic anti-microbial activity in milk.     

MINERAL CONTENT IN GOATS' MILKS

Concentrations of eight essential minerals were determined in 78 samples of goats'milk from the Canary Islands. The following mean values (ranges) were found: Se (μ/L), 19.98 (9.2‐31.2); Fe (mg/L), 0.52 (0.19‐1.17); Cu (mg/L), 0.17 (0.08‐0.31); Zn (mg/L), 3.31 (1.6‐4.6); Na (mg/L), 514 (289‐906); K (mg/L), 1585 (1212‐2160); Ca (mg/L), 1533 (1118‐1962); and Mg (mg/L), 157 (101‐210). Comparing with data for human milk and cows' milk from the Canary Islands, the mean concentrations of Se, K and Mg arranged in according the following sequence: goats' milk > cows' milk > human milk. Cows' milk had the highest (P<0.05) mean concentration for Ca and Zn, while humans had the highest (P < 0.05) mean concentration for Cu. The milk of goats milked once per day had significantly higher concentrations of Se and lower Mg and Zn than the milk of goats milked twice per day. While the concentrations of Na and Mg increased significantly from March to August, the concentrations of Se, Fe and Cu in goats' milk decreased significantly from March to June.     

Effect of Bifidobacterium bifidum fermented milk on Helicobacter pylori and serum pepsinogen levels in humans

Helicobacter pylori infection is an important risk factor for gastric diseases. Some probiotics are useful for suppressing H. pylori infection. Bifidobacterium bifidum YIT 4007 can improve the experimental gastric injury in rats and the disease stages on the gastric mucosa in peptic ulcer patients. We evaluated the fermented milk using a clone (BF-1) having the stronger ability to survive in the product than this parent strain to clarify the in vitro suppressive effect of BF-1 on H. pylori and the in vivo efficacy of BF-1 fermented milk on H. pylori and gastric health. In the mixed culture assay of BF-1 and H. pylori, the number of pathogens was decreased such that it was not detected after 48 h in the Brucella broth with a decrease in pH values. In the cell culture experiment with human gastric cells, the H. pylori infection-induced IL-8 secretion was suppressed by the preincubation of BF-1. In a human study of 12-wk ingestion (BF-1 group, n = 40; placebo group, n = 39) with a randomized double-blind placebo-control design, the H. pylori urease activity and gastric situation were evaluated using a urea breath test (UBT) and the serum pepsinogen (PG) levels as biomarkers for inflammation or atrophy, respectively. In the H. pylori-positive subjects, the difference (ΔUBT) of the UBT value from the baseline value in the BF-1 group (n = 34) was lower than that in the placebo group (n = 35) at 8 wk. The baseline UBT values showed a negative correlation with ΔUBT values at 8 and 12 wk in the BF-1 group but not in the placebo. In the PG-positive subjects classified by the PG test method, the BF-1 group was lower in ΔUBT values than the placebo group at 8 and 12 wk. In the active gastritis class by PG levels, the BF-1 group was lower in their ΔUBT values than the placebo at 8 and 12 wk. The PG I levels in the BF-1 group were lower than the placebo at 12 wk. The PG II levels in the BF-1 group did not change during the ingestion period, but the placebo was increased. The PG I/II ratios slightly decreased from baseline at 12 and 20 wk in the BF-1 and placebo groups. These patterns were also observed in the H. pylori-positive subjects. The improving rates of upper gastrointestinal symptomatic subjects and total symptom numbers in the BF-1 group were higher than those in the placebo. These results indicate that BF-1 fermented milk may affect H. pylori infection or its activity, gastric mucosal situation, and the emergence of upper gastrointestinal symptoms.     

Prediction of indicators of cow diet composition and authentication of feeding specifications of Protected Designation of Origin cheese using mid-infrared spectroscopy on milk

The ability of mid-infrared spectroscopy (MIR) to predict indicators (1) of diet composition in dairy herds and (2) for the authentication of the cow feeding restrictions included in the specification of 2 Protected Designation of Origin (PDO) cheeses (Cantal and Laguiole) was tested on 7,607 bulk milk spectra from 1,355 farms located in the Massif Central area of France. For each milk sample, the corresponding cow diet composition data were obtained through on-farm surveys. The cow diet compositions varied largely (i.e., from full grazing for extensive farming systems to corn silage-based diets, which are typical of more intensive farming systems). Partial least square regression and discriminant analysis were used to predict the proportion of different feedstuffs in the cows' diets and to authenticate the cow feeding restrictions for the PDO cheese specifications, respectively. The groups for the discriminant analysis were created by dividing the data set according to the threshold of a specific feedstuff. They were issued based on the specifications of the restriction of the PDO cheese. The pasture proportion in the cows' diets was predicted by MIR with an coefficient of determination in external validation (R²V) = 0.81 and a standard error of prediction of 11.7% dry matter. Pasture + hay, corn silage, conserved herbage, fermented forage, and total herbage proportion in the cows' diets were predicted with a R²V >0.61 and a standard error of prediction <14.8. The discrimination models for pasture presence, pasture ≥50%, and pasture ≥57% in the cows' diets achieved an accuracy and specificity ≥90%. A sensitivity and precision ≥85% were also observed for the pasture proportion discrimination models, but both of these indexes decreased at increasing thresholds from 0 to 50, and 57% pasture in the cows' diets. An accuracy ≥80% was also observed for pasture + hay ≥72%, herbage ≥50%, pasture + hay ≥25%, absence of fermented herbage, absence of corn silage, and corn silage ≤30% in the cows' diets, but for several models, either the sensitivity or precision was lower than the accuracy. Models built on the simultaneous respect of all the criteria of the feeding restrictions of PDO cheese specifications achieved an accuracy, specificity, sensitivity, and precision >90%. Both the regression and discriminant MIR models for bulk milk can provide useful indicators of cow diet composition and PDO cheese specifications to producers and consumers (farmers, dairy plants).     

Antimicrobial activity of pediocin-producing Lactococcus lactis on Listeria monocytogenes,Staphylococcus aureus and Escherichia coli O157:H7 in cheese

The antimicrobial activity of two pediocin-producing transformants obtained from wild strains of Lactococcus lactis on the survival of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 during cheese ripening was investigated. Cheeses were manufactured from milk inoculated with the three pathogens, each at approximately 6 log cfu mL⁻¹. Pediococcus acidilactici 347 (Ped⁺), Lc. lactis ESI 153, Lc. lactis ESI 515 (Nis⁺) and their respective pediocin-producing transformants Lc. lactis CL1 (Ped⁺) and Lc. lactis CL2 (Nis⁺, Ped⁺) were added at 1% as adjuncts to the starter culture. After 30 d, L. monocytogenes, S. aureus and E. coli O157:H7 counts were 5.30, 5.16 and 4.14 log cfu g⁻¹ in control cheese made without adjunct culture. On day 30, pediocin-producing derivatives Lc. lactis CL1 and Lc. lactis CL2 lowered L. monocytogenes counts by 2.97 and 1.64 log units, S. aureus by 0.98 and 0.40 log units, and E. coli O157:H7 by 0.84 and 1.69 log units with respect to control cheese. All cheeses made with nisin-producing LAB exhibited bacteriocin activity throughout ripening. Pediocin activity was only detected throughout the whole ripening period in cheese with Lc. lactis CL1. Because of the antimicrobial activity of pediocin PA-1, its production in situ by strains of LAB growing efficiently in milk would extend the application of this bacteriocin in cheese manufacture.     

Fate of Listeria monocytogenes in Gouda microcheese: No growth,and substantial inactivation after extended ripening times

This challenge study demonstrates that Listeria monocytogenes does not grow in Gouda cheese: during the first 8 weeks of ripening no growth was observed and between 8 and 52 weeks viable numbers declined significantly in a well-established Gouda microcheese system. Cheese milk was artificially contaminated just prior to addition of the starter culture. Three individual L. monocytogenes strains were used, including strains originating from cheese, a cheese plant environment and a reference strain. During curd formation, viable numbers of L. monocytogenes increased by 0.5 log cfu g⁻¹, resulting from entrapment in the curd. No growth was observed during the first 8 weeks of ripening. A significant decline in the viable numbers of L. monocytogenes was observed in Gouda cheese that was ripened for longer than 8 weeks. Two factors that could possibly control the fate of L. monocytogenes in Gouda cheese were lactic acid and water activity.     

Prediction of the percentages of cows', goats' and ewes' milk in “Iberico” cheese by electrophoretic analysis of whey proteins

Stepwise multiple linear regression (SMLR) and principal components regression (PCR) have been used to predict the percentages of cows', goats' and ewes' milk in “Iberico” cheese, using the results obtained by electrophoretic analysis (PAGE and IEF) of whey proteins, using standard cheeses. Similar predictions of the percentages of milks from the three species were obtained when either SMLR or PCR were applied to the electrophoretic data, i.e. the optical intensity of the electrophoretic bands (PAGE or IEF) of the whey proteins. The root mean square error of prediction in cross-validation (RMSEPCV) was lower than 4% in all cases.     

The combined effects of fat content,calcium chloride,and coagulant concentration on the development of cheese curd structure

The effects of differing content levels of calcium chloride (approximately 200 and 400 μg Ca per 100 g milk protein) and of a microbial coagulant (3200 and 6400 μL per 100 L of milk; 950 IMCU (international milk coagulating units) mL⁻¹) on the coagulation of cows' milk with various fat levels (0.02–3.77%, w/w) was studied. Non-linear regression analysis was used to evaluate dynamic factors (lag time, t_lag; maximum coagulation rate, C_max; time for the maximum coagulation rate, t_max). Increasing fat content in the milk at constant calcium chloride and coagulant contents had no significant and clear effects on the t_lag, C_max, and t_max values. Increased levels of calcium chloride or microbial coagulant led to a significant decrease in t_lag and t_max, and conversely increased C_max. Therefore, milk fat content had no significant effect on gel development; however, levels of calcium chloride and coagulant significantly influenced gel structure.     

Peptidase and proteinase activity ofLactococcus lactis,Lactobacillus casei andLactobacillus plantarum

The proteolytic system of several non-commercial strains of lactococci and lactobacilli that were isolated directly from traditional-Spanish, semi-hard, goats' milk cheese was studied. The aminopeptidase, X-prolyldipeptidyl aminopeptidase, dipeptidase and proteinase activity of these new strains was measured for the cytoplasmic, cell-wall/membrane and spontaneously released fractions. The aminopeptidase activity was exclusively intracellular and higher forLactobacillus casei subsp.casei than forLactococcus lactis subsp.lactis. Lactobacillus plantarum showed higher dipeptidase activity thanL. casei. The highest level of proteinase activity was recorded for the cell-wallmembrane fraction ofLactococcus lactis subsp.lactis IFPL 359, and was higher on β-casein than on α_s-casein for all the strains studied. These results suggest some different contribution of these strains to the proteolysis of cheese during ripening and they seem to complement each other when used together in the starter culture.     

Contribution of proteolytic activity associated with somatic cells in milk to cheese ripening

The effect of proteolytic enzymes from somatic cells on cheese quality was studied. In preliminary experiments, milk and two sodium caseinate systems (pH 6.5 and pH 5.2, the latter in the presence of 5% NaCl) were used as substrates to investigate the proteolytic activity of somatic cells recovered from mastitic milk. Urea-polyacrylamide gel electrophoretograms of hydrolysates suggested that somatic cell extracts contributed directly to proteolysis both in buffer and in milk, but that such activity was reduced by batch pasteurisation (63 °C for 30 min). Sodium caseinate was readily hydrolysed by somatic cell extracts; hydrolysis of α_s1-casein was greater at pH 5.2 and increased with level of somatic cells, suggesting that somatic cells contain proteolytic enzymes which are more active at acidic pH values. Subsequently, miniature Cheddar-type cheeses were made from batches of milk to which somatic cells were added (at levels of levels of 3×10⁵ or 6×10⁵ cells mL⁻¹), either before or after pasteurisation. Proteolysis during ripening of cheese (as measured by levels of pH 4.6-soluble nitrogen) increased with somatic cell addition, although this effect was reduced by pasteurisation after cell addition. Somatic cells may also have directly influenced cheese moisture content, which has been established as a principal indicator of quality of Cheddar-type cheese. Proteolytic enzymes of somatic cells from milk were shown to contribute directly to proteolysis in milk and cheese.     

Antiviral activity of donkey milk protein fractions on echovirus type 5

Antiviral activity of Ragusano donkeys' milk proteins was investigated for the effect on echovirus type 5, known to infect the gastrointestinal tract of humans. Three protein fractions were tested; casein (CN), whey protein (WP) and a low molecular whey protein fraction (LWP; <30,000 Da). The antiviral activity of WP and LWP was tested on echovirus type 5 at three concentrations (1, 5 and 10 mg mL^?1); CN was assessed only at the lower concentration. All donkey milk protein fractions showed significant inhibition on virus replication at the concentration of 1 mg mL^?1, and both WP and LWP fractions showed significant inhibition on the virus replication at all concentrations tested. The strongest antiviral effect was observed for the WP fraction. These findings show that the different whey proteins in donkey milk, probably acting in synergy, exert antiviral activity on echovirus 5 and might contribute to prevent gastrointestinal virus infections in humans.     

Potential impact of dairy yeasts on the typical flavour of traditional ewes' and goats' cheeses

The contribution of Debaryomyces hansenii, Kluyveromyces lactis and Kluyveromyces marxianus strains to the typical flavour of traditional ewes' and goats' cheeses was assessed. Fourteen yeast strains were grown in liquid medium mimicking cheese composition and volatile compounds were identified by gas chromatography-mass spectrometry. Yeasts were able to produce key volatile compounds characteristic of the cheeses from which they were isolated. Inter-species and inter-strain variations were observed. Under the conditions tested, D. hansenii produced the lowest levels of volatile compounds, with large intra-strain variations. Kluyveromyces strains primarily produced esters and alcohols. K. marxianus strains were associated with the production of acids, ethyl decanoate, 1-propanol and benzaldehyde, whereas K. lactis was correlated with the presence of ketones, ethyl acetate and secondary alcohols. In conclusion, this study shows the heterogeneous potential of dairy yeasts to contribute to final cheese flavour.     

Extruded linseeds,vitamin E and plant extracts in corn silage-based diets of dairy cows: Effects on sensory properties of raw milk and uncooked pressed cheese

Linseed supplementation of dairy cows' diet increases unsaturated fatty acid (FA) concentrations, which could lead to oxidised off-flavours in dairy products if antioxidant content is low. The FA profiles and sensory properties of milk and uncooked pressed cheese (Saint-Nectaire type) from four groups of six cows receiving a corn silage-based diet with no additional lipid (control), or supplemented with extruded linseeds (5% of additional fat-in-dry matter intake; EL), EL and vitamin E (7500 IU per cow per day of dl-α-tocopherol; ELE), or ELE and plant extracts rich in carotenoids and polyphenols (1% of dry matter intake) were compared. Feeding EL decreased milk and cheese 4- to 16-carbon saturated FA, and increased 18:1 cis-9, 18:3n-3 and total trans FA concentrations. Extruded linseeds did not induce oxidised off-flavours but led to a less firm and more meltable texture in cheese. Vitamin E supplementation increased α-tocopherol concentration, but did not affect sensory properties.     

Biochemical,microbial and sensory evaluation of white soft cheese made from cow and lupin milk

The aim of the present study was to evaluate some physicochemical, microbiological and sensory properties of fresh and matured (75 days) soft cheeses made with mixtures of cow milk and 0, 25, 50 and 75 mL/100 mL of lupin milk. A remarkable increase in cheese yield was observed with increasing the level of lupin milk to the mixture. Compared to cow milk cheese, the protein content was significantly (P ≤ 0.05) increased while ash was decreased with the increase in the level of lupin milk for both fresh and matured cheese. However, fat content, total solids and acidity were increased only for fresh cheese and decreased for mature one compared to that of cow milk. The pH showed significant (P ≤ 0.05) reduction when the levels of lupin milk increased for fresh cheese while for matured cheese it slightly decreased. The total bacterial count is within the range that naturally exists in milk containing foods. The others microorganisms such as fungi, mold, Escherichia coli, and Salmonella were not existed in both types of cheese. Regardless of cheese color, incorporation of lupin milk at low concentration (25 mL/100 mL) significantly (P ≤ 0.05) enhanced the taste, texture, flavor, and overall acceptability of both fresh and mature cheese.     

Production of conjugated linoleic acid enriched milk and dairy products from cows receiving grass silage supplemented with a cereal-based concentrate containing rapeseed oil

Opportunities for the production of milk and dairy products enriched with cis-9, trans-11 conjugated linoleic acid (CLA) were investigated. Eighteen mid-lactation cows were used in a continuous-design for 7 weeks. During the first week, cows received grass silage ad libitum supplemented with 10 kg per day of a cereal-based concentrate (control) that was replaced with a concentrate containing 50 g kg⁻¹ of rapeseed oil (RO). Changes in milk fatty acid composition were monitored on a weekly basis and milk produced was used to manufacture Edam cheese and butter. Inclusion of RO in the concentrate supplement increased the mean levels of trans-octadecanoic, monounsaturated, CLA and polyunsaturated fatty acid in the milk fat from 1.6, 25.7, 0.46 and 2.8 to 4.3, 35.3, 1.02 and 3.9 g 100 g⁻¹ total fatty acids, respectively. In contrast, the mean level of saturated fatty acids decreased from 71.4 to 60.7 g  100 g⁻¹ total fatty acids. Changes in milk fatty acid composition due to RO occurred within 7 days, with responses reaching a plateau after 21 days. Furthermore, the CLA concentrations in the milk fat from individual cows ranged between 0.37 and 0.65 and 0.43 and 2.06 g 100 g⁻¹ total fatty acids for the control and RO diet, respectively. CLA enriched milk was used successfully to manufacture of Edam cheese and butter with softer textures but with acceptable organoleptic and storage properties. Processing milk into butter or cheese had no effect on the CLA concentrations indicating that enrichment of dairy products is dependent on the content in raw milk fat.     

Ripening of Cheddar cheese made from blends of raw and pasteurised milk

Cheddar cheeses were made from pasteurised milk (P), raw milk (R) or pasteurised milk to which 10 (PR10), 5 (PR5) or 1 (PR1) % of raw milk had been added. Non-starter lactic acid bacteria (NSLAB) were not detectable in P cheese in the first month of ripening, at which stage PR1, PR5, PR10 and R cheeses had 10⁴, 10⁵, 10⁶ and 10⁷ cfu NSLAB g⁻¹, respectively. After ripening for 4 months, the number of NSLAB was 1–2 log cycles lower in P cheese than in all other cheeses. Urea–polyacrylamide gel electrophoretograms of water-soluble and insoluble fractions of cheeses and reverse-phase HPLC chromatograms of 70% (v/v) ethanol-soluble as well as -insoluble fractions of WSF were essentially similar in all cheeses. The concentration of amino acids were pro rata the number of NSLAB and were the highest in R cheese and the lowest in P cheese throughout ripening. Free fatty acids and most of the fatty acid esters in 4-month old cheeses were higher in PR1, PR5, PR10 and R cheeses than in P cheese. Commercial graders awarded the highest flavour scores to 4-month-old PR1 cheeses and the lowest to P or R cheese. An expert panel of sensory assessors awarded increasingly higher scores for fruity/sweet and pungent aroma as the level of raw milk increased. The trend for aroma intensity and perceived maturity was R>PR10>PP5>PR1>P. The NSLAB from raw milk appeared to influence the ripening and quality of Cheddar cheese.