Available lysine content in human milk: stability during manipulation prior to ingestion

Available lysine content is an indicator of protein quality and nutritional value of milk. Many studies have examined the effects of extraction, treatment and storage of human milk upon its components, though no references are found regarding the possible changes in milk quality as defined by its content in essential amino acids such as lysine. The present study investigates the available lysine content in human milk and the variations in lysine resulting from milk manipulation as follows: (a) Cold storage (refrigeration at 4 degrees C for 48 hours, and frozen for 15 days at -20 degrees C); (b) Thermal treatment under conditions of low (Holder)(63 degrees C/30 minutes) and high pasteurization (75 degrees C/15 seconds). The results obtained show a decrease in milk lysine concentration after storage in both refrigerated and frozen samples. Pasteurization causes a highly significant loss of available lysine. The lysine losses were greater on applying low pasteurization versus the more gentle conditions of high pasteurization. CONCLUSIONS: While manipulation through cold storage or thermal treatment does not affect the protein content of human milk, its protein quality is modified. When human milk must be subjected to hygienization, it is preferable to apply high temperature treatment (75 degrees C, 15 seconds) than habitual pasteurization (63 degrees C, 30 minutes).     

Effect of chemical decontamination and refrigerated storage on the isolation of Mycobacterium avium subsp. paratuberculosis from heat-treated milk

AIMS: To assess the impact of chemical decontamination and refrigerated storage before culture on the recovery of Mycobacterium avium subsp. paratuberculosis from heat-treated milk. METHODS AND RESULTS: Five-millilitre samples of ultra heat-treated (UHT) milk spiked with Myco. paratuberculosis NCTC 8578, B4 or 806R (ca 10(6) CFU ml(-1)) were heated at 63 degrees C for 20 or 30 min by submersion in a water bath. Heat-treated milk (0.5 ml) was cultured immediately into BACTEC 12B medium or refrigerated at 4 degrees C for 48 h before culture. Milk samples that received a 20-min heat treatment were also subjected to decontamination with 0.75% cetylpyridinium chloride (CPC) for 5 h at room temperature before inoculation into BACTEC 12B medium when tested immediately and after 48 h at 4 degrees C. BACTEC vials were monitored for evidence of growth over an 18-week incubation period at 37 degrees C. CPC decontamination resulted in a significant reduction in the number of culture-positive milk samples recovered immediately after heating (P < 0.05) and after refrigerated storage for 48 h (P < 0.01). Refrigerated storage for 48 h before testing did not have any significant effect, beneficial or detrimental, on Myco. paratuberculosis recovery rates. CONCLUSIONS: CPC decontamination applied to milk immediately or 48 h after heating will adversely affect the recovery of viable Myco. paratuberculosis, possibly leading to nonrecovery of the organism although viable cells are present in the original milk sample. SIGNIFICANCE AND IMPACT OF THE STUDY: Published pasteurization studies in which milk samples were decontaminated before culture will have underestimated the survival capability of Myco. paratuberculosis after high-temperature, short-time pasteurization. CPC decontamination should not be applied to pasteurized milk in future studies.     

Effect of pasteurization and storage on some components of pooled human milk

Pooled human milk was subjected to Holder pasteurization and storage at −20°C up to 90 days and examined for its content of fat and

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-lactate and for lipid composition. This treatment reduced fats by 6% and

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-lactate by at least 7%. In addition, pasteurization and storage induced triglyceride hydrolysis. The absolute amount of free fatty acids (FFAs) which was 0.5% after collection, doubled after pasteurization and rose even more after storage. Different FFA compositions were found by several authors using the same analytical method even for milk samples subjected to the same treatment. More detailed information on procedures must be given to explain the different results.     

Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteurization

Milk from cows inoculated with Listeria monocytogenes was pooled for 2 to 4 days and then heated at 71.7 to 73.9 degrees C for 16.4 s or at 76.4 to 77.8 degrees C for 15.4 s in a high-temperature, short-time plate heat exchanger pasteurization unit. L. monocytogenes was isolated from milk after heat treatment in six of nine pasteurization trials done at 71.7 to 73.9 degrees C and in none of three trials done at 76.4 to 77.8 degrees C. An average of 1.5 to 9.2 L. monocytogenes cells was seen in each milk polymorphonuclear leukocyte before heat treatment in 11 of 12 pasteurization trials. Noticeable degradation of leukocytes with intracellular listeria was detected in unpasteurized milk after 3 days of storage at 4 degrees C, and by 4 days of storage leukocytes had deteriorated to cellular debris, suggesting that holding unpasteurized milk refrigerated for 4 or more days would eliminate a protective effect leukocytes may provide for increasing heat resistance of L. monocytogenes. Results indicate that under the conditions of this study, L. monocytogenes can survive the minimum high-temperature, short-time treatment (71.7 degrees C, 15 s) required by the U.S. Food and Drug Administration for pasteurizing milk.     

Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteurization.

Milk from cows inoculated with Listeria monocytogenes was pooled for 2 to 4 days and then heated at 71.7 to 73.9 degrees C for 16.4 s or at 76.4 to 77.8 degrees C for 15.4 s in a high-temperature, short-time plate heat exchanger pasteurization unit. L. monocytogenes was isolated from milk after heat treatment in six of nine pasteurization trials done at 71.7 to 73.9 degrees C and in none of three trials done at 76.4 to 77.8 degrees C. An average of 1.5 to 9.2 L. monocytogenes cells was seen in each milk polymorphonuclear leukocyte before heat treatment in 11 of 12 pasteurization trials. Noticeable degradation of leukocytes with intracellular listeria was detected in unpasteurized milk after 3 days of storage at 4 degrees C, and by 4 days of storage leukocytes had deteriorated to cellular debris, suggesting that holding unpasteurized milk refrigerated for 4 or more days would eliminate a protective effect leukocytes may provide for increasing heat resistance of L. monocytogenes. Results indicate that under the conditions of this study, L. monocytogenes can survive the minimum high-temperature, short-time treatment (71.7 degrees C, 15 s) required by the U.S. Food and Drug Administration for pasteurizing milk.     

Fate of Escherichia coli O157:H7 in unpasteurized milk stored at 8 degrees C

Escherichia coli O157:H7 and verocytotoxins were not found in any of 100 unpasteurized milk samples obtained from the bulk tanks of eight dairy farms located in the Puglia and Basilicata areas. Seven E. coli O157:H7 (EHEC) strains were inoculated separately into raw milk samples and then examined periodically to determine the fate of EHEC as influenced by the storage temperature (8 degrees C) and time. There was essentially no change in the viable population of three EHEC strains for up to 14 d. The remaining four strains showed an increase in population from < 2 log to 3 log cfu ml-1 in a time period of between 9 and 17 d. The results indicate good survival or even multiplication of E. coli O157:H7 in raw milk when stored at 8 degrees C and reaffirm the need for pasteurization and holding the milk at < or = 5 degrees C.     

Thermal inactivation of Yersinia enterocolitica in milk.

Three strains of Yersinia enterocolitica isolated from milk had D values at 62.8 degrees C from 0.24 to 0.96 min and z values of 5.11 to 5.78 degrees C. Since the pasteurization processes for dairy products recommended by the Food and Drug Administration are adequate to destroy large concentrations of these organisms, Y. enterocolitica in pasteurized milk probably results from substandard processing or recontamination after pasteurization.     

Challenge testing of the lactoperoxidase system in pasteurized milk

AIMS: To determine the role of lactoperoxidase (LP) in inhibiting the growth of micro-organisms in pasteurised milk. METHODS AND RESULTS: Four micro-organisms of importance in the spoilage of pasteurized milk were challenged in lactoperoxidase (LP)-enriched ultra-heat treated (UHT) milk after subsequent pasteurization. Milk samples were stored at the optimum temperatures for growth of the individual bacteria. Pasteurization was carried out at 72 degrees C/15 s and 80 degrees C/15 s to determine the effect of the LP system on the micro-organisms. An active LP system was found to greatly increase the keeping quality (KQ) of milks inoculated with Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus thermophilus and pasteurized at 72 degrees C, but had little or no effect in milks heated at 80 degrees C, presumably due to virtual inactivation of LP at 80 degrees C. However, pasteurization temperature had no effect on the KQ of milks challenged with Bacillus cereus spores. CONCLUSIONS: This study suggests that the LP system, rather than heat-shocking of spores, is responsible for the greater KQ of milk pasteurized at 72 degrees C/15 s compared with 80 degrees C/15 s. SIGNIFICANCE AND IMPACT OF THE STUDY: The study emphasizes the care required in selecting pasteurization temperatures in commercial practice and to avoid the temptation to compensate for inferior quality of raw milk by increasing pasteurization temperature.     

Thermal inactivation of several Mycobacterium spp. in milk by pasteurization

I.R. GRANT, H.J. BALL AND M.T. ROWE. 1996. The thermal inactivation of Mycobacterium avium, Myco. bovis, Myco. fortuitum, Myco. intracellulare and Myco. kansaii in milk at 63.5°C was investigated. Survivors were enumerated after heating for 0, 5, 10, 15, 20 and 30 min and thermal death curves were constructed for each species. Mycobacterium bovis and Myco. fortuitum were found to exhibit linear thermal death curves and neither species demonstrated any survival after heating at 63.5°C for 30 min (equivalent to holder pasteurization). In contrast, Myco. avium, Myco. intracellulare and Myco. kansassi yielded thermal death curves which exhibited significant 'tailing' and all three strains survived holder pasteurization.     

Milk contamination and resistance to processing conditions determine the fate of Lactococcus lactis bacteriophages in dairies

Milk contamination by phages, the susceptibility of the phages to pasteurization, and the high levels of resistance to phage infection of starter strains condition the evolution dynamics of phage populations in dairy environments. Approximately 10% (83 of 900) of raw milk samples contained phages of the quasi-species c2 (72%), 936 (24%), and P335 (4%). However, 936 phages were isolated from 20 of 24 (85%) whey samples, while c2 was detected in only one (4%) of these samples. This switch may have been due to the higher susceptibility of c2 to pasteurization (936-like phages were found to be approximately 35 times more resistant than c2 strains to treatment of contaminated milk in a plate heat exchanger at 72 degrees C for 15 s). The restriction patterns of 936-like phages isolated from milk and whey were different, indicating that survival to pasteurization does not result in direct contamination of the dairy environment. The main alternative source of phages (commercial bacterial starters) does not appear to significantly contribute to phage contamination. Twenty-four strains isolated from nine starter formulations were generally resistant to phage infection, and very small progeny were generated upon induction of the lytic cycle of resident prophages. Thus, we postulate that a continuous supply of contaminated milk, followed by pasteurization, creates a factory environment rich in diverse 936 phage strains. This equilibrium would be broken if a particular starter strain turned out to be susceptible to infection by one of these 936-like phages, which, as a consequence, became prevalent.     

Effect of heat treatment on Listeria monocytogenes and Gram-negative bacteria in sheep, cow and goat milks

Sheep milk, compared with cow and goat milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65°C in a test-tube method. This effect was not solely due to fat content as cow milk artificially reconstituted to 10% homologous fat was not as protective. Listeria monocytogenes in whole sheep, cow and goat milks at an inoculum level of 1 times 10⁶ cfu ml^-1 was heated at 68°C for 15 s in the plate pasteurizer and survival was only detected in whole sheep milk after heating. Whole sheep, cow and goat milks containing high levels of L. monocytogenes (1 times 10⁶ cfu ml^-1) could not survive the current HTST plate pasteurization protocol.     

THE ASSESSMENT OF THE BACTERIOLOGICAL CONDITION OF MILK BOTTLES

SUMMARY: A study of the relative values of a number of bacteriological tests for assessing the condition of milk bottles indicated that the colony count of the bottle rinse solution on yeastrel milk agar incubated for 4 days at 30°, combined with a clot-on-boiling test applied to 1 ml. of rinse in 9 ml. of sterile milk after incubation for 72 hr. at 19–20°, gave the most useful results.
The mean of the ratios of colony counts at 30° to those at 37° was 15·1, while it was as high as 22·9 for rinses with 37° of over 600 for an unsatisfactory bottle should be retained when the test is done at 30°. The thermoduric colony count of rinses of milk bottles, even when laboratory pasteurized in milk, did not provide any additional information to that given by the colony count at 30° made without pasteurization. A high proportion of the organisms in bottle rinses survived laboratory pasteurization in milk, the survival rate being highest in efficiently treated bottles.
The clot-on-boiling test gave results in general agreement with colony counts and served to indicate the potential influence of badly contaminated bottles on the keeping quality of milk placed in them. A substantial proportion of rinses with satisfactory colony counts reduced methylene blue within 48 hr. at 19–20°.
Colony counts at 37° were on the average much lower for bottles treated with steam than for bottles submitted to detergent treatment in various types of bottle washing machines. Treatment of bottles by steam or hypochlorite was more efficiently done on the farms than at the dairies.     

Milk Contamination and Resistance to Processing Conditions Determine the Fate of Lactococcus lactis Bacteriophages in Dairies

Milk contamination by phages, the susceptibility of the phages to pasteurization, and the high levels of resistance to phage infection of starter strains condition the evolution dynamics of phage populations in dairy environments. Approximately 10% (83 of 900) of raw milk samples contained phages of the quasi-species c2 (72%), 936 (24%), and P335 (4%). However, 936 phages were isolated from 20 of 24 (85%) whey samples, while c2 was detected in only one (4%) of these samples. This switch may have been due to the higher susceptibility of c2 to pasteurization (936-like phages were found to be approximately 35 times more resistant than c2 strains to treatment of contaminated milk in a plate heat exchanger at 72°C for 15 s). The restriction patterns of 936-like phages isolated from milk and whey were different, indicating that survival to pasteurization does not result in direct contamination of the dairy environment. The main alternative source of phages (commercial bacterial starters) does not appear to significantly contribute to phage contamination. Twenty-four strains isolated from nine starter formulations were generally resistant to phage infection, and very small progeny were generated upon induction of the lytic cycle of resident prophages. Thus, we postulate that a continuous supply of contaminated milk, followed by pasteurization, creates a factory environment rich in diverse 936 phage strains. This equilibrium would be broken if a particular starter strain turned out to be susceptible to infection by one of these 936-like phages, which, as a consequence, became prevalent.     

Impact of pasteurization on the self-assembly of human milk lipids during digestion

Human milk is critical for the survival and development of infants. This source of nutrition contains components that protect against infections while stimulating immune maturation. In cases where the mother's own milk is unavailable, pasteurized donor milk is the preferred option. Although pasteurization has been shown to have minimal impact on the lipid and FA composition before digestion, no correlation has been made between the impact of pasteurization on the FFA composition and the self-assembly of lipids during digestion, which could act as delivery mechanisms for poorly water-soluble components. Pooled nonpasteurized and pasteurized human milk from a single donor was used in this study. The evolving FFA composition during digestion was determined using GC coupled to a flame ionization detector. In vitro digestion coupled to small-angle X-ray scattering was utilized to investigate the influence of different calcium levels, fat content, and the presence of bile salts on the extent of digestion and structural behavior of human milk lipids. Almost complete digestion was achieved when bile salts were added to the systems containing high calcium to milk fat ratio, with similar structural behavior of lipids during digestion of both types of human milk being apparent. In contrast, differences in the colloidal structures were formed during digestion in the absence of bile salt because of a greater amount of FFAs being released from the nonpasteurized than pasteurized milks. This difference in FFAs released from both types of human milk could result in varying nutritional implications for infants.     

Survival of Enterobacter cloacae and Pseudomonas paucimobilis in yoghurts manufactured from cow's milk and soymilk during storage at different temperatures.

The survival of two microbial contaminants, Enterobacter cloacae and Pseudomonas paucimobilis, in yoghurts manufactured from cow's milk and soymilk was investigated during storage for 45 days at 4 and 12 degrees C. Sensory panel tests performed before microbiological investigation, showed that the flavor of soy-yoghurts made with cocoa powder or malt added did not have the beany taste of soy beans. Both contaminants were significantly resistant to low pH values during storage for 32 days at 4 degrees C. The survival at 4 degrees C was remarkable in both plain and flavored yoghurts and a population close to 10(2) C.F.U./ml was observed after 38 days of storage. Experiments performed with soymilk yoghurts showed an enhanced survival of P. paucimobilis at 4 degrees C compared to the storage in cow's milk yoghurts; microbial values were close to 7-8 x 10(6) C.F.U./ml after 16 days. Soymilk exhibited a protective effect on L. delbrueckii subsp. bulgaricus and S. thermophilus at 12 degrees C and, compared to the survival in cow's milk yoghurts, a larger number of viable cells of both probiotic microorganisms (10(6) and 10(8) C.F.U./ml, respectively) were observed after 36 days of storage.     

A NOTE ON THE THERMODURIC BACTERIAL CONTENT OF FARM WATER SUPPLIES

SUMMARY: Significantly higher thermoduric colony counts were obtained when 5 ml. of water were laboratory pasteurized in 5 ml. of sterile milk or 2 ml. of water were pasteurized in 8 ml. of sterile milk, than when 10 ml. of water were directly pasteurized; 86% of the water samples had higher thermoduric counts after pasteurization in milk. Large differences were not common, only 19% of the ratios being over 5 and 5% over 10. Aerobic sporing rods were dominant in the thermoduric microflora irrespective of the method of pasteurization.     

Inactivation of Mycobacterium paratuberculosis in cows' milk at pasteurization temperatures.

The thermal inactivation of 11 strains of Mycobacterium paratuberculosis at pasteurization temperatures was investigated. Cows' milk inoculated with M. paratuberculosis at two levels (10(7) and 10(4) CFU/ml) was pasteurized in the laboratory by (i) a standard holder method (63.5 degrees C for 30 min) and (ii) a high-temperature, short-time (HTST) method (71.7 degrees C for 15 s). Additional heating times of 5, 10, 15, 20, and 40 min at 63.5 degrees C were included to enable the construction of a thermal death curve for the organism. Viability after pasteurization was assessed by culture on Herrold's egg yolk medium containing mycobactin J (HEYM) and in BACTEC Middlebrook 12B radiometric medium supplemented with mycobactin J and sterile egg yolk emulsion. Confirmation of acid-fast survivors of pasteurization as viable M. paratuberculosis cells was achieved by subculture on HEYM to indicate viability coupled with PCR using M. paratuberculosis-specific 1S900 primers. When milk was initially inoculated with 10(6) to 10(7) CFU of M. paratuberculosis per ml, M. paratuberculosis cells were isolated from 27 of 28 (96%) and 29 of 34 (85%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. Correspondingly, when 10(3) to 10(4) CFU of M. paratuberculosis per ml of milk were present before heat treatment, M. paratuberculosis cells were isolated from 14 of 28 (50%) and 19 of 33 (58%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. The thermal death curve for M. paratuberculosis was concave in shape, exhibiting a rapid initial death rate followed by significant "tailing." Results indicate that when large numbers of M. paratuberculosis cells are present in milk, the organism may not be completely inactivated by heat treatments simulating holder and HTST pasteurization under laboratory conditions.     

Antimicrobial effect and shelf-life extension by combined thermal and pulsed electric field treatment of milk

Aims:  The impact of a combined hurdle treatment of heat and pulsed electric fields (PEF) was studied on native microbiota used for the inoculation of low-fat ultra-high temperature (UHT) milk and whole raw milk. Microbiological shelf-life of the latter following hurdle treatment or thermal pasteurization was also investigated.
Methods and Results:  UHT milk was preheated to 30°C, 40°C or 50°C over a 60-s period, pulsed for 50  μ s or 60  μ s at a field strength of 40 kV cm⁻¹ or for 33  μ s at 50 kV cm⁻¹. Heat and PEF reduced the microbial count by a maximum of 6·4 log in UHT milk (50°C; 50 kV cm⁻¹, 33  μ s) compared to 6·0 log ( P  ≥ 0·05) obtained by thermal pasteurization (26 s, 72°C). When raw milk was treated with a combination of hurdles (50°C; 40 kV cm⁻¹, 60  μ s) a 6·0 log inactivation of microbiota was achieved and microbiological milk shelf-life was extended to 21 days under refrigeration (4°C) vs 14 days in thermally pasteurized milk. Native microbiota was decreased by 6·7 log following conventional pasteurization.
Conclusions:  The findings suggest that heat and PEF achieved similar inactivation of native microbiota in milk and longer stabilization of microbiological shelf-life than thermal pasteurization.
Significance and Impact of the Study:  A hurdle approach of heat and PEF could represent a valid milk processing alternative to conventional pasteurization. Hurdle treatment might also preserve native milk quality better due to less thermal exposure.     

Influence of oligosaccharides on the viability and membrane properties of Lactobacillus reuteri TMW1.106 during freeze-drying

Freeze-drying is a process commonly used in starter culture preparation. To improve the survival rate of bacteria during the process, cryoprotectives are usually added before freezing. This study investigated the influence of the addition of sucrose, fructo-oligosaccharides (FOS), inulin and skim milk on the viability and membrane integrity of Lactobacillus reuteri TMW1.106 during freezing, freeze-drying and storage. The effect of drying adjuncts on survival was correlated to their interaction with bacterial membrane by determination of the parameters membrane fluidity and membrane lateral pressure. Sucrose, FOS and skim milk significantly enhanced survival of exponential-phase cells of L. reuteri during freeze-drying. Cellular viability during storage of exponential-phase cells remained highest for cells dried in the presence of skim milk and inulin. Membranes of these cells were completely permeabilized after freeze-drying. The application of FOS significantly improved survival of stationary phase cells of L. reuteri TMW1.106 after freeze-drying and storage. This increased viability of L. reuteri TMW1.106 in the presence of FOS correlated to improved membrane integrity. Fructo-oligosaccharides and fructans, but not gluco-oligosaccharides interacted with membrane vesicles prepared from L. reuteri TMW1.106 as indicated by increased membrane lateral pressure in the presence of FOS and fructans. Increased membrane integrity of stationary phase L. reuteri TMW1.106 was attributed to direct interactions between FOS and the membrane which leads to increased membrane fluidity and thus improved stability of the membrane during and rehydration.     

Effect of Different Heat Treatments and Disinfectants on the Survival of <Emphasis Type="Italic">Prototheca zopfii</Emphasis>

Bovine mastitis caused by the yeast-like alga Prototheca zopfii represents a serious veterinary problem and may result in heavy economic losses to particular dairy farms. The purpose of this study was to evaluate the survival of 50 isolates of P. zopfii in milk subjected to different heat treatments and the survival of further 106 P. zopfii isolates after exposure to three classes of teat disinfectants: iodine (Dipal), quaternary ammonium compounds (Teat), and dodecylbenzenesulphonic acid (Blu-gard). Of the 50 isolates tested for thermal tolerance, 29 (58%) survived heat treatment at 62°C for 30 s and 13 (26% of all isolates) of those survived after heat treatment at 72°C for 15 s. None of the 106 isolates were able to withstand the in-use concentrations of the three disinfectants tested. The highest disinfectant concentrations that permitted survival of at least one isolate were dilutions: 1:1,000 for Dipal (survival rate of 52.8–57.5%), 1:100 for Teat (88.7–90.6%), and 1:10 for Blu-gard (100%). No differences in the survival rates of P. zopfii were observed with respect to the duration of exposure to disinfectant. The results of this study support the previous findings that P. zopfii may resist high-temperature treatments, including that applied in the high-temperature, short-time (HTST) pasteurization process. The obtained data also demonstrate the efficacy of the three classes of teat disinfectants against P. zopfii, with the efficacy of iodine being most pronounced. The study emphasizes the necessity of using higher temperatures in the pasteurization of raw milk to kill the Prototheca algae, as well as the particular suitability of the iodine for the control procedures of protothecal mastitis.