Incubation Of Milk Research Articles

Increasing the sensitivity of Listeria monocytogenes assays: evaluation using ELISA and amperometric detection.

An immunosensor for the detection of Listeria monocytogenes was developed. ELISA and amperometric studies were run in parallel to develop a more sensitive and rapid assay for the bacterium. Conditions for the immunosensor were primarily characterised using ELISA. A direct sandwich assay was employed and the affinities of two polyclonal (goat and rabbit) and one monoclonal (mouse) anti-L. monocytogenes antibodies were compared using this format. Owing to low sensitivity being obtained with all antibodies, biotin-avidin amplification and an indirect sandwich assay were employed. The system was then transferred to screen-printed electrodes (SPEs), the primary antibody being immobilised by cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the mode of detection being amperometric. Various parameters (limit of detection, working range, incubation time, cross-reactivity) of the systems were characterised. The effect of direct incubation in milk is also discussed. The final immunosensor had a working range of 1 x 10(6)-1 x 10(3) cells ml-1 and a detection limit of 9 x 10(2) cells ml-1. The assay took about 3.5 h to complete.

The Major Lactococcal Cell Wall Autolysin AcmA Does Not Determine the Rate of Autolysis of Lactococcus lactis subsp. cremoris 2250 in Cheddar Cheese

Abstract A range in the levels of autolysis of 17 commercial Lactococcus lactis cheese starters was found after growth and incubation in milk for 72 h at 30°C. Autolysis levels in milk usually (but not always) correlated with autolysis in 0.1 m Na phosphate buffer. One strain, Lc. lactis subsp. cremoris strain 2250, was highly autolytic. The major lactococcal autolysin (N-acetyl muramidase; AcmA) was cloned from 2250 and sequenced. Comparison of the predicted amino acid sequence with AcmA from Lc. lactis MG1363 showed the presence of ten mostly conserved substitutions, equivalent to 98% amino acid homology. Three substitutions (all conserved) were in the active site domain, but none of these involve the acidic amino acids expected to be required for muramidase activity. This concurs with earlier results (Riepe et al. (1997) Applied and Environmental Microbiology 63 , 3757–3763) showing that there is no significant difference in enzymatic activities of the two AcmAs. A 2250 acmA deletion mutant (2250Δ acmA ), completely lacking any AcmA activity, was used to assess the contribution of AcmA to the autolysis of 2250 in milk and Cheddar cheese. In milk, AcmA contributed significantly to high autolysis of 2250. Nevertheless, autolysis of 2250Δ acmA in milk was higher than most of 16 other Lc. lactis strains, all of which have AcmA present. By contrast, in cheese, 2250Δ acmA autolysed almost to the same extent as 2250 wild-type. Factors other than AcmA must therefore cause the high levels of autolysis seen in Lc. lactis subsp. cremoris 2250 in cheese. These factors may be important in causing high autolysis of other autolytic Lc. lactis strains.

Manufacture of cheese made from CO2-treated milk

Cheeses were manufactured from pasteurised milk (control), pasteurised milk acidified to pH 6.0 with CO2, and milk acidified to pH 6.0 with CO2 prior to pasteurisation. Production of cheese from CO2-treated milk at pH 6.0 reduced the amount of rennet necessary for coagulation by about 75%. Although acidification reduces the amount of lactic acid produced by starter during incubation of milk, no significant differences in lactic acid content were detected between cheeses manufactured from non-acidified or CO2-acidified milks. Cheeses produced from CO2-treated milk showed less proteolysis than control cheeses, but no significant differences in sensory characteristics between cheeses were detected.

Toxicokinetics of Methyl Parathion in Lactating Goats

Lactating goats were exposed by oral administration to a subtoxic dose of methyl parathion (MPT) (5 mg/kg/day ; 3 days). Neither MPT nor its metabolite methylparaoxon (MPO) was detected in plasma, urine, or milk. MPT and MPO were unaffected by incubation in milk for 12 h. Conversion of MPT to aminomethyl parathion by rumen microflora limited bioavailability. Absorption did occur because 7.4% of the total dose of MPT was excreted in urine as dimethyl phosphate (DMP) and dimethyl thiophosphate (DMTP), and serum cholinesterase (ChE) activity was depressed to 58% of control. In lactating goats given an intravenous bolus of MPT at a dose (5 mg/kg) that was slightly toxic (salivation and nervousness), the elimination half-life was 0.81 ± 0.17 (mean ± SE) h. Although MPO, DMP, and DMTP were not detected in plasma, 67.8% of the intravenous dose was excreted in urine as DMP and DMTP. ChE activity was depressed to 52% of control. It was concluded that dosages of MPT not causing overt signs of toxicity are not associated with excretion of MPT or its toxic metabolite MPO in milk.

Assessment of mammary gland metabolism in the sow: I. Development of methods for the measurement of cellular metabolites in milk and colostrum

We have modified deproteinization methods and a number of spectrophotometric and bioluminescent methods in order to measure the concentrations of cellular metabolites in small volumes (< 0.3 ml) of sows' colostrum and milk. For the majority of the assays, recoveries ranged from 92 to 105%. The binding of ATP and UTP to a calcium phosphate-citrate-caseinate complex in milk, and the decrease in ATP (92%/h) and UTP (18.1%/h) concentrations during in vitro incubation of the whey fraction suggested that it was unlikely ATP and UTP (both < 1 microM) could exist free in sows' milk. The mean concentrations (range) of cellular metabolites in milk (6-11 d post partum) were: glucose, 669 microM (220-1367); glucose 6-phosphate, 63.0 microM (27.6-101.4); glucose 1-phosphate, 18.3 microM (13.1-24.8); UDPglucose, 296 microM (170-494); UDPgalactose, 635 microM (230-945); lactose, 162 mM (124-187); UDP, 105 microM (85-130); UMP, 1760 microM (1326-2587); inorganic phosphate, 13.5 mM (1.4-29.3); ATP, < 0.5 microM; ADP, 53.6 microM (10.5-171.25); AMP, 215 microM (61.6-491.6); cAMP, 22.3 microM (3.5-61.6); galactose, 198 microM (118-474) and fructose, 226 microM (172-283). Differences in the concentrations of glucose, glucose 6-phosphate, glucose 1-phosphate, UDPgalactose and cAMP between fore and hind milk samples indicated postsecretory changes in the concentrations of certain metabolites. Changes in the concentrations of metabolites during in vitro incubation of milk and of colostrum suggested that these postsecretory changes were probably due to the actions of enzymes present in mammary secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of polymer‐binding on the specificity of lipases

Abstract The flavour of a food system is strongly related to the production of free fatty acids (FFA). The controlled lipolysis and liberation of desired fatty acids are therefore of special interest in the food industry. Lipases that meet the food safety regulations, often do not necessarily possess the desired specificity. It was observed that the profile of FFA can be altered by a pre‐treatment of the lipase with synthetic, as well as natural, polymers. The profile of fatty acids released during incubation in milk was strongly correlated to the polarity of polymer used. The higher the polarity of the polymer, the more unsaturated fatty acids were liberated. After an incubation of the untreated lipase in milk, the oleic‐ versus stearic acid was found to be 3:1. However, using a lipase modified with a dimethyl polysiloxane (Mc Reynolds constant is 44), the ratio was found to be 1.5:1 and a modification with cyanoallyl polysiloxane (Mc Reynolds constant: 844) increased the ratio to 5:1. When applied for l...

Phagocytosis of mastitis isolates of Staphylococcus aureus and expression of type 5 capsular polysaccharide are influenced by growth in the presence of milk

Phagocytosis by bovine polymorphonuclear granulocytes of seven capsular polysaccharide type 5 Staphylococcus aureus strains isolated from mastitis [corrected] was investigated by means of luminol-dependent chemiluminescence. Bacteria were grown on four different agar media (brain heart infusion, Columbia broth, modified staphylococcus medium 110, and skim milk) and were opsonized by normal bovine serum. When compared to growth on brain heart infusion agar, Columbia agar, and modified staphylococcus medium 110 agar, growth on skim milk agar rendered five of the strains more resistant to opsonization. The other two strains were resistant in all culture media used. Short periods of incubation in milk after growth on brain heart infusion agar did not augment resistance to phagocytosis, indicating that mere adsorption of milk components on bacteria was not responsible. The variability of the chemiluminescence response of polymorphonuclear leukocytes was pronounced among strains with each growth medium except milk. Growth on modified staphylococcus medium 110 and on milk agar favored the masking of teichoic acid, as shown by inagglutinability with rabbit antiserum. Interestingly, agglutination by a monoclonal antibody to capsular polysaccharide type 5 was optimal when bacteria were grown on skim milk agar. This suggests that capsular polysaccharide participated in the masking effect. These findings indicate that masking of the bacterial target of most of the naturally acquired opsonins present in normal bovine serum occurred when bacteria grew in the presence of milk, resulting in an increased resistance to phagocytosis by polymorphonuclear leukocytes.

Production of extracellular lipases and proteinases during prolonged growth of strains of psychrotrophic bacteria in whole milk.

Three strains of psychrotrophic Pseudomonas spp., each with different lipolytic and proteolytic phenotypes (including a proteinase-deficient mutant), were cultured separately in whole milk at 7 degrees C. Growth rates were the same during logarithmic growth phase, but during early stationary phase the cell densities were related to the activities of lipase and proteinase in the cultures. Only one strain underwent pronounced death phase. Proteinase activity was not detected in the culture of the proteinase-deficient mutant, but in those of the other strains it increased to a plateau, or continued to increase linearly. Lipase activity of the culture of each strain reached a peak in early stationary phase; in late stationary phase activity was highest for the proteinase-deficient mutant strain where degradation of lipase by bacterial proteinase would have been least. The ability of psychrotrophic bacteria both to survive for long periods and to produce high levels of proteinases and lipases on prolonged incubation in milk emphasizes the spoilage potential arising from psychrotrophic bacteria in inadequately cleaned dairy equipment.

Effect of preservation media on proliferation and collagen biosynthesis of periodontal ligament fibroblasts.

Abstract The viability of periodontal ligament (PDL) cells, which are responsible for the production of ligament proteins, is essential for clinical healing of replanted exarticulated teeth. Cultured human PDL fibroblast‐like cells were used to study the effects of various transport media on cell proliferation, collagen synthesis and total protein synthesis. The PDL cells were obtained from intact premolars of a healthy young male patient by trypsinization and scraping the PDL tissue. Proliferation of the cells, the activity of prolyl 4‐hydroxylase, total protein and collagen synthesis were studied after 60 min incubation in Dulbecco's Modified Eagle's Medium (DMEM), milk, saliva and tap water. Proliferation capacity was also investigated after prolonged incubation in milk. The results indicated that milk and saliva were superior to water in maintaining these cell functions, but as the saliva used in this study does not resemble that in the clinical situation, we recommend milk as a temporary storage medium for exarticulated teeth before replantation.

The occurrence and growth of yeasts in dairy products

Yeasts were isolated, identified and enumerated from 161 samples of retail dairy products. Highest yeast populations (up to 10 6–10 7 cells/g) were found in yogurt and cheese samples while lower counts occurred in samples of pasteurized milk, cream, butter and ice cream. Candida famata, Kluyveromyces marxianus, Candida diffluens and Rhodotorula glutinis were the most frequency isolated species. The growth of these and other species was demonstrated during the refrigerated storage of cream, butter and cheese samples and by their inoculation and incubation in milk and yogurt samples. The predominance and growth species was probably related to their production of protein and fat hydrolysing enzymes and the ability to grow at 5°C.

Free Fatty Acids in the Development of Breast Milk Jaundice

The incubation of milk, at 40C, from mothers of infants with breast milk jaundice (BMJ) is reported to result in significantly higher levels of free fatty acids (FFA; compared with milk from controls. Single milk samples collected under standard conditions were obtained from four mothers of infants with BMJ and 14 control donors matched for stage of lactation. Milk samples were analyzed for the concentrations of FFA, using thin‐layer gas chromatographic techniques. In addition, serum total fatty acids were measured in mothers and infants. The concentrations of FFA increased after storage of the milk from both the jaundiced and control groups. No differences were observed in the composition of milk FFA before or after incubation, when respective values were compared between these two groups. Similarly, no differences were detected in serum total fatty acids in either infants or mothers. The observation that increased levels of FFA in milk are associated with BMJ was not confirmed.

Post-secretory aggregation of caseins.

Particles as large as several micrometer diam. have been observed occasionally in normal milk and commonly in prepartum and postpartum colostrum. These particles can be dissociated by EDTA and their appearance closely resembles that of normal casein micelles. However, they are often too large to have been completely formed within the Golgi vesicles of mammary epithelium and hence some degree of post-secretory aggregation of caseins is thought to occur. Two possible mechanisms of post-secretory aggregation of caseins are: (1) a continuation of the normal processes of micelle assembly in the alveolus and (2) aggregation as a result of limited proteolysis of the caseins during the time the milk is in the mammary gland. Incubation of milk with fibrinolysin, however, failed to produce aggregation of normal micelles.

Electron microscopic observations of the interaction of casein micelles and milk fat globules with bovine polymorphonuclear leucocytes during the phagocytosis of staphylococci in milk

Polymorphonuclear leucocytes (PMN) isolated from bovine blood or milk were examined in the electron microscope before and after incubation with staphylococci in either milk or a synthetic salts medium supplemented with serum. Milk PMN on isolation contained prominent milk fat globules and a few small vacuoles containing granules resembling casein micelles. Neither of these were seen in blood PMN. Milk PMN contained fewer lysosomes than blood PMN, and it is suggested that phagocytosis of fat globules and casein micelles in vivo, with consequent degranulation, accounts for their previously observed deficient phagocytosis of staphylococci. On incubation in milk in vitro with staphylococci, both blood PMN and milk PMN ingested apparently large quantities of granules resembling casein micelles, forming phagocytic vacuoles separate from those containing staphylococci. The ingested staphylococci appeared largely intact, despite the loss of lysosomes from the PMN cytoplasm. In contrast, the staphylococci ingested by blood PMN in synthetic medium appeared to be dead after 2 h. These observations support previous findings that casein inhibits the bactericidal activities of PMN, and suggest possible mechanisms for this inhibition.

Digestion and Absorption of Bovine Milk Xanthine Oxidase and its Role as an Aldehyde Oxidase

The effects of acidic and intestinal proteolytic environments on bovine milk xanthine oxidase (XO) activity were determined in order to evaluate the extent to which this enzyme was absorbed in biologically active form. The inhibition of XO by folic acid and the relative affinities of XO for the oxidation of palmitaldehyde, stearaldehyde, and xanthine were compared. The effects of acid and gastric juice on XO activity were measured by incubating purified enzyme, and non-purified enzyme (milk), in buffers ranging in pH from 2 to 9. Fresh gastric juice was also incubated with milk. Increasing amounts of the enzyme were inactivated as the pH of the incubation mixture was reduced below pH 6.5. Below pH 3.5, the enzyme was completely inactivated. Gastric juice, pH juice incubated with milk. Milk XO activity was reduced 36% when mild was incubated with an equal volume of gastric juice. Homogenized milk had 59% less XO activity compared with raw molk. Fresh raw milk XO, homogenized milk XO, and purified XO were equally susceptible to inactivation by acid or gastric juice. After incubation of milk with gastric juice, or gastric juice followed by pancreatin, XO activity was associated with a macromolecule of 300,000 daltons molecular weight and subunits containg activity were not found. It was estimated that 0.00008% of the XO in the intestine was absorbed. Both folic acid and allopurinol inhibited XO activity in vitro. Allopurinol was 3.5 times more potent an inhibitor than folic acid. A large excess of dietary folic acid did not reduce rat liver or intestinal XO activity in vivo. XO had a much greater affinity for xanthine than for palmitaldehyde or stearaldehyde substrates. It was estimated that of 100 mg of XO in fresh raw milk, 41 mg remained after homogenization, 27 mg entered the intestine and only 20 ng were absorbed as intact enzyme.

Effect of Copper on Microorganisms in the Manufacture of Swiss Cheese1,2

Commercial strains of Lactobacillus bulgaricus, Streptococcus thermophilus, lactic streptococci, and propionibacteria were grown in the presence of 0, 2, 4, 8, and 16ppm copper to simulate conditions that might exist in Swiss cheese manufacture. In general, loss of viability and inhibition of acid production was hastened by an increase in copper concentration although genus, species, and strains differed. During the first 3h of incubation in milk, mixed-strain lactic cultures were not affected by the addition of copper up to 16ppm. Later, inhibition and increased death rates occurred depending upon copper concentration. S. thermophilus strains exhibited a wide variety of sensitivities to copper. However, they seemed to overcome partly their sensitivity to copper after about 16h incubation. Two of three strains of L. bulgaricus showed slight reduction in cell numbers and acid production with increasing copper concentration. The third strain more closely resembled the lactic streptococci in its greater sensitivity to copper. Two of four strains of propionibacteria were more sensitive to copper than were the other two. Carbon dioxide production in broth at pH 7.0 differed among the strains. Incubation periods up to 20 days, however, tended to overcome copper inhibition. At an initial broth pH of 5.4, carbon dioxide production was delayed markedly by 16ppm copper, but, with prolonged incubation, two of the four strains attained carbon dioxide production equal to copper-free controls.

İNEK SÜTÜ VE YOĞURDU ÜZERİNDE AMİNO ASİT SPEKTRUMU, TOTAL PROTEİN VE LAKTOZ YÖNÜNDEN ARAŞTIRMALAR

Suınrnary: The purpose of this study was to investigate the possiblc changes in amino acid speetrum, protein and lactose contents of milk during fermentation. In this study, 25 milk and 25 yoghurt samplcs were used. The yoghurt was made in our laboratory by the incubation of milk which was fermented with 2 % yoghurt yeast at 4O°C for 12 hours. The amino acid determination was carried out using an automatic amino acid analyzer. The protein content was determined by KjeWdal method and the polarimetric method was used for the determination of lactose. The mean values of the amino acids as g fi 00 g of protein in mil k and yoghurt were as follows, rcspectively:

Study on the Phospholipase in Cow's Milk

The phospholipids in cows milk are known to be high in unsaturated fatty acids content. Taking of this point, the authors considered that if these unsaturated fatty acids were lilerated by hydrolysis, they developed the off-flavor of milk by autoxidation. However, no study was reported concerning the presence of phospholipase in milk. The present study deals with the occurrence and the origin of the phospholipase.(1) The presence of phospholipase was suggested by the increase of lysophosphatidylethanolamine (LPE) during the incubation of milk.(2) Addition of toluene to milk as a preservative resulted in repressing the production of LPE. But free fatty acids and LPE were increased by the incubation of milk-bacteria with milk-phospholipids. These results indicated that the phospholipase was not present originally in milk but was produced by multiplied bacteria.(3) During the incubation of milk, the increase of lysophosphatidylcholine was not observed. Therefore, this enzyme seemed to have specific activity against phosphatidylethanolamine.