Frozen Milk Samples Research Articles

Application of the Actiphage® Assay to Detect Viable Mycobacterium avium subsp. paratuberculosis Cells in Fresh Sheep and Goat Milk and Previously Frozen Milk and In-Line Milk Filters.

Mycobacterium avium subsp. paratuberculosis (MAP) is a well-known causative agent of paratuberculosis, a chronic infectious granulomatous enteritis of ruminants contributing to significant economic losses worldwide. Current conventional diagnostic tools are far from being sufficient to manage and control this disease. Therefore, increased attention has been paid to alternative approaches including phage-based assays employing lytic bacteriophage D29 to detect MAP cells. The aim of the present study was to assess the applicability and efficiency of the recently developed phage-based kit termed Actiphage® combined with IS900 real-time PCR (qPCR) for rapid detection and quantification of viable MAP in milk samples. We demonstrated that Actiphage® in combination with IS900 qPCR allows for rapid and sensitive detection and identification of viable MAP in milk samples with a limit of detection of 1 MAP per 50 ml milk. Using this method, the presence of viable MAP cells was successfully determined in 30.77% of fresh goat, sheep and cow milk samples originating from paratuberculosis-affected herds. We further used Actiphage assay to define the time-lapse aspect of testing naturally contaminated milk and milk filters frozen for various lengths of time by phage-based techniques. Viable MAP was detected in 13.04% of frozen milk samples and 28.57% of frozen milk filters using Actiphage-qPCR. The results suggest the ability to detect viable MAP in these samples following freezing for more than 1 year. The obtained results support the views of the beneficial role of this technology in the control or monitoring of paratuberculosis.

Holder pasteurization of donated human milk is effective in inactivating SARS-CoV-2.

Provision of pasteurized donor human milk, as a bridge to mother's own milk, is the standard of care for very low-birth-weight infants in hospital. The aim of this research was to confirm that Holder pasteurization (62.5°C for 30 min) would be sufficient to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in donated human milk samples. We spiked frozen milk samples from 10 donors to the Rogers Hixon Ontario Human Milk Bank with SARS-CoV-2 to achieve a final concentration of 1 × 107 TCID50/mL (50% of the tissue culture infectivity dose per mL). We pasteurized samples using the Holder method or held them at room temperature for 30 minutes and plated serial dilutions on Vero E6 cells for 5 days. We included comparative controls in the study using milk samples from the same donors without addition of virus (pasteurized and unpasteurized) as well as replicates of Vero E6 cells directly inoculated with SARS-CoV-2. We reported cytopathic effects as TCID50/mL. We detected no cytopathic activity in any of the SARS-CoV-2-spiked milk samples that had been pasteurized using the Holder method. In the SARS-CoV-2-spiked milk samples that were not pasteurized but were kept at room temperature for 30 minutes, we observed a reduction in infectious viral titre of about 1 log. Pasteurization of human milk by the Holder method (62.5°C for 30 min) inactivates SARS-CoV-2. Thus, in the event that donated human milk contains SARS-CoV-2 by transmission through the mammary gland or by contamination, this method of pasteurization renders milk safe for consumption and handling by care providers.

Comparison of the Potential Abilities of Three Spectroscopy Methods: Near-Infrared, Mid-Infrared, and Molecular Fluorescence, to Predict Carotenoid, Vitamin and Fatty Acid Contents in Cow Milk.

The objective of this work is to compare the ability of three spectroscopy techniques: molecular fluorescence, near-infrared (NIR), and mid-infrared with attenuated total reflectance (MIR-ATR) spectroscopy to predict the concentrations of 8 carotenoids, 6 vitamins and 22 fatty acids (FA) in cow’s milk. A dataset was built through the analysis of 242 frozen milk samples from different experiments. The milk compounds were analysed using reference methods and by NIR, MIR-ATR, and fluorescence to establish different predictive models. NIR spectroscopy allowed for better prediction of cis9-β-carotene, β-cryptoxanthin and the sum of carotenoids than the other techniques, with a coefficient of cross-validation in calibration (R2CV) > 0.60 and a coefficient of determination in validation (R2V) > 0.50. Their standard errors of prediction (SEP) were equal to 0.01, except for the sum of carotenoids (SEP = 0.15). However, MIR-ATR and fluorescence seem usable for the prediction of lutein and all-trans-β-carotene, respectively. These three spectroscopy methods did not allow us to predict (R2CV < 0.30) vitamin contents except, for vitamin A (the best R²CV = 0.65 with NIR and SEP = 0.15) and α-tocopherol (the best R²CV = 0.56 with MIR-ATR and SEP = 0.41), but all R²V were <0.30. NIR spectroscopy yielded the best prediction of the selected milk FA.

Effect of various storage conditions on the stability of quinolones in raw milk

ABSTRACTResearch on the storage stability of antibiotic residues in milk is important for method development or validation, milk quality control and risk assessment during screening, confirmation, qualitative or quantitative analysis. This study was conducted using UPLC-MS/MS to determine the stability of six quinolones – ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), sarafloxacin (SAR), difloxacin (DIF) and flumequine (FLU) – in raw milk stored under various conditions to investigate if quinolones degrade during storage of milk, and finally to determine optimal storage conditions for analysis and scientific risk assessment of quinolone residues in raw milk. The storage conditions included different temperatures and durations (4°C for 4, 8, 24 and 48 h; –20°C for 1, 7 and 30 days; –80°C for 1, 7 and 30 days), thawing temperatures (25, 40 and 60°C), freeze–thaw cycles (1–5), and the addition of different preservatives (sodium thiocyanate, sodium azide, potassium dichromate, bronopol and methanal). Most quinolones exhibited high stability at 4°C for up to 24 h, but began to degrade after 48 h. In addition, no degradation of quinolones was seen when milk samples were stored at –20°C for up to 7 days; however, 30 days of storage at –20°C resulted in a small amount of degradation (about 30%). Similar results were seen when samples were stored at –80°C. Moreover, no losses were observed when frozen milk samples were thawed at 25, 40 or 60°C. All the quinolones of interest, except sarafloxacin, were stable when milk samples were thawed at 40°C once and three times, but unstable after five freeze–thaw cycles. Preservatives affected the stability of quinolones, but the effects differed depending on the preservative and quinolone. The results of this study indicate optimum storage protocols for milk samples, so that residue levels reflect those at the time of initial sample analysis, and should improve surveillance programmes for quinolones in raw milk.

Analysis of the Storage Methods for Raw Human Milk from Mothers with Infants Admitted to a Neonatal Intensive Care Unit, According to Brazilian Regulations.

Milk safety is an important concern in neonatal units and human milk banks. Therefore, evidence-based recommendations regarding raw milk handling and storage are needed to safely promote supplying hospitalized infants with their mother's own milk. To evaluate raw human milk storage methods according to Brazilian milk management regulations by investigating the effects of refrigeration (5°C) for 12 hours and freezing (-20°C) for 15 days on the acidity and energy content in a large number of raw milk samples. Expressed milk samples from 100 distinct donors were collected in glass bottles. Each sample was separated into 3 equal portions that were analyzed at room temperature and after either 12 hours of refrigeration or 15 days of freezing. Milk acidity and energy content were determined by Dornic titration and creamatocrit technique, respectively. All samples showed Dornic acidity values within the established acceptable limit (≤ 8°D), as required by Brazilian regulations. In addition, energy content did not significantly differ among fresh, refrigerated and frozen milk samples (median of ~50 kcal/100 mL for each). Most samples tested (> 80%) were considered top quality milk (< 4°D) based on acidity values, and milk energy content was preserved after storage. We conclude that the storage methods required by Brazilian regulations are suitable to ensure milk safety and energy content of stored milk when supplied to neonates.

The relationship between fatty acid profiles in milk identified by Fourier transform infrared spectroscopy and onset of luteal activity in Norwegian dairy cattle

To investigate the feasibility of milk fatty acids as predictors of onset of luteal activity (OLA), 87 lactations taken from 73 healthy Norwegian Red cattle were surveyed over 2 winter housing seasons. The feasibility of using frozen milk samples for dry-film Fourier transform infrared (FTIR) determination of milk samples was also tested. Morning milk samples were collected thrice weekly (Monday, Wednesday, Friday) for the first 10 wk in milk (WIM). These samples had bronopol (2-bromo-2-nitropropane-1,3-diol) added to them before being frozen at −20°C, thawed, and analyzed by ELISA to determine progesterone concentration and the concentrations of the milk fatty acids C4:0, C14:0, C16:0, C18:0, and cis-9 C18:1 as a proportion of total milk fatty acid content using dry-film FTIR, and averaged by WIM. Onset of luteal activity was defined as the first day that milk progesterone concentrations were >3 ng/mL for 2 successive measurements; the study population was categorized as early (n=47) or late (n=40) OLA, using the median value of 21 DIM as the cutoff. Further milk samples were collected 6 times weekly, from morning and afternoon milkings, these were pooled by WIM, and one proportional sample was analyzed fresh for fat, protein, and lactose content by the dairy company Tine SA, using traditional FTIR spectrography in the wet phase of milk. Daily energy-balance calculations were performed in 42 lactations and averaged by WIM. Animals experiencing late OLA had a more negative energy balance in WIM 1, 3, 4, and 5, with the greatest differences been seen in WIM 3 and 4. A higher proportion of the fatty acids were medium chained, C14:0 and C16:0, in the early than in the late OLA group from WIM 1. In WIM 4, the proportion of total fatty acid content that was C16:0 predicted late OLA, with 74% sensitivity and 80% specificity. The long-chain proportion of the fatty acids C18:0 and cis-9 C18:1 were lower in the early than in the late OLA group. Differences were greatest in WIM 4 and 5. Differences in concentrations of cis-9 C18:1 were seen between the groups from WIM 1. No relationship was seen between OLA and milk concentrations of either protein or fat, or between OLA and the milk fat:protein ratio. The differences in milk fatty acid proportions between the 2 groups are most likely related to differences in energy balance. The study shows that frozen milk samples can be tested for fatty acids by FTIR spectroscopy and that FTIR spectroscopy of milk can be used to provide real-time information about cow reproductive function.

Evaluation of the effect of freezing milk samples on the refractive index for copper(II) serum determination

&lt;p&gt;This work aimed to evaluate the effect of freezing of raw and whole UHT milk samples on the refractive index for copper(II) serum (RICS) determination. Eighteen samples were used in this study (9 raw and 9 whole UHT milk samples). RICS values were determined before and after freezing on different days (1, 5, 8, 15, 22, 29 and 57 days). The RICS values from frozen samples were lower than from fresh samples. However, the reduction in RICS was considered acceptable because this value was below to the expanded uncertainty of the method. Freezing milk is adequate to preserve samples for RICS determination.&lt;/p&gt;&lt;p&gt;&amp;nbsp;&lt;/p&gt;&lt;p&gt;DOI: 10.14685/rebrapa.v6i1.142&lt;/p&gt;

Inactivation of Escherichia coli in milk by high pressure processing at low and subzero temperatures

Inactivation of Escherichia coli strain ATCC 25922 in frozen and unfrozen milk by high pressure processing at low or subzero temperatures was investigated. Frozen (−21 °C) and unfrozen (3 °C) milk samples were exposed to a pressure of 300 MPa for 0.5–15 min at varying temperatures between −5 °C and 5 °C. Reduction in the number of E. coli in unfrozen milk was enhanced by pressure treatment at subzero temperatures, whereas no survival was detected in frozen milk samples after a 10 min pressure treatment at −3 °C, corresponding to a minimum of 6.8 log cycle reduction in the number of E. coli. A substantial reduction of 3.8 log reduction in number of E. coli was observed after a 30 s pressure treatment at −3 °C. Results suggested that the mechanical forces could play a role in enhanced inactivation of E. coli in frozen milk samples.

Short communication: Influence of storage and preservation on microbiological quality of silo ovine milk

This study was designed to analyze the effects of the storage and preservation conditions on counts of mesophilic, thermoduric, psychotrophic, coliform, Escherichia coli, Streptococcus agalactiae, and Staphylococcus aureus organisms in silo ovine milk. A total of 910 analytical determinations were conducted from aliquots of 10 silo ovine milks. The conditions tested were unpreserved and azidiol-preserved milk stored at 4°C, and unpreserved milk stored at −20°C. Milk aged 2, 24, 48, 72, and 96h post-collection for refrigerated aliquots, and 7, 15, and 30 d post-collection for frozen aliquots. The factors silo and storage conditions significantly contributed to variation of all microbiological variables, although milk age effect within storage was only significant for mesophilic, psychrotrophic, and coliform bacteria counts. In refrigerated raw milk, mesophile, psychrotroph, and coliform counts significantly increased over 96h post-collection, whereas the other groups and bacteria species tested maintained their initial concentration. In all cases, azidiol preservation maintained the initial bacterial concentration in raw sheep milk under refrigeration throughout 96h. Thus, azidiol was a suitable preservative for microbiological studies in sheep milk. Smallest counts were registered for frozen samples, particularly for coliforms, E. coli, Strep. agalactiae and Staph. aureus. Estimates of mesophilic, thermoduric and psychrotrophic organisms showed similar values on both azidiol-preserved and frozen milk samples. Coliforms and E. coli counts significantly decrease over time after freezing. Consequently, freezing at −20°C could also be appropriate for analysis of mesophilic, thermoduric, and psychrotrophic bacterial groups, but not for coliforms or mammary pathogens.

Diagnosis of intramammary infection in samples yielding negative results or minor pathogens in conventional bacterial culturing

Up to half of quarter milk samples submitted for mastitis diagnosis are culture-negative results or lead to identification of coagulase-negative staphylococci or Corynebacterium bovis in conventional culturing, the so-called minor pathogens. The interpretation and usefulness of these results in terms of udder and animal health management is limited, even though the amount of resources spent is relatively high. This work aimed to test two methods of analysis of milk samples with the goal of increasing detection of intramammary pathogens. In the first study, 783 milk samples were processed in duplicate: before and after freezing at -20°C for 24 h, using standard bacteriological techniques. There was a significant difference between the two methods with samples frozen for 24 h yielding significantly fewer Gram-positive catalase-positive cocci, Gram-negative bacilli, Gram-positive bacilli and significantly more samples leading to no growth, than samples before freezing. The number of samples yielding Gram-positive catalase-negative cocci was not significantly affected by freezing. In the second study, a real-time PCR-based test was performed on milk samples with an individual quarter somatic cell count above 500,000 cells/ml that were either negative (n=51 samples) or that led to the isolation of minor pathogens in culturing: Corynebacterium bovis (n=79 samples) or non-aureus staphylococci (NAS, n=32). A mastitis pathogen, beyond the result obtained with standard bacteriology, was detected on 47% of the no-growth samples, on 35% of the samples from which C. bovis had been isolated and on 25% of the samples from which NAS had been isolated. The most commonly detected major pathogen was Escherichia coli, followed by Streptococcus uberis, Arcanobacterium pyogenes/Peptoniphilus indolicus and Streptococcus dysgalactiae. These results suggest that simply freezing milk samples for 24 h does not increase the detection of intramammary bacteria in milk samples and therefore should not be recommended. However, use of the real-time PCR-based test may be useful in diagnosing intramammary infections when milk samples with high somatic cell counts are culture-negative or when culturing results in the detection of minor pathogens.

Viability of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in goat milk samples stored under different conditions

Control programs for contagious agalactia (CA) involve monitoring milk samples to detect this disease. This study was designed to establish the effects of the preservatives generally used in dairy laboratories and storage temperature on the viability of Mycoplasma ( M.) agalactiae ( Ma) and M. mycoides subsp. capri ( Mmc) in goat milk samples. In total, 1440 determinations were conducted for each mycoplasma species in milk samples subjected to different storage temperatures (refrigeration at 4 °C or freezing at −20 °C), preservation strategies (no preservative, NP; azidiol, AZ; or bronopol, BR) and storage times at each temperature (0, 2, 4, 6, 8, 10 and 24 h at 4 °C and 48 h, 1 week, 2 weeks and 4 weeks at −20 °C). Our findings reveal the similar viability of Mmc in milk samples stored at 4 °C for 24 h under the three preservation conditions examined. In contrast, the isolation of Ma in refrigerated milk samples was compromised by the presence of BR, and in smaller measure by the treatments AZ and NP. Freezing milk samples considerably reduced the viability of both mycoplasmas. Given the different sensitivity of the two mycoplasma species to BR, refrigerated milk samples treated with AZ could be used to detect infections caused by both species through culture-based methods.

Monitoramento da qualidade do leite humano ordenhado e distribuído em banco de leite de referência

O leite humano (LH) coletado, processado e distribuído em Bancos de Leite humano (BLH), deve configurar-se como uma alternativa inócua e nutricionalmente satisfatória aos recém-nascidos que o utilizem. Com o propósito de monitorar a qualidade do LH em um BLH de referência, foram avaliadas 20 amostras de LH quanto aos parâmetros microbiológicos, físico-químicos, bem como as condições higiênico-sanitárias do BLH em relação à qualidade do ar ambiental, recipientes de armazenamento do leite e mãos dos manipuladores. As amostras de leite analisadas apresentaram quantidade de contaminantes deterioradores, que foi reduzida com a pasteurização, contudo os coliformes não foram eliminados. Não foi detectada a presença de Staphylococcus coagulase positiva. Na composição centesimal, foram verificadas grandes perdas do valor energético total, 23,28% nas amostras de leite congelado (LC) e 23,86% das amostras após a pasteurização (LAP), em decorrência da grande perda lipídica. Quanto aos parâmetros de pH, acidez em graus Dornic e umidade, verificaram-se alterações significativas (p &lt; 0,05) entre as amostras de leites antes e depois do congelamento e LAP. Pode-se inferir que o monitoramento e os processos de conservação melhoraram a qualidade microbiológica do leite, todavia os processos de pasteurização e de congelamento influenciaram negativamente na composição físico-química e no valor nutricional.

Effects of storage methods on the recovery of Mycoplasma species from milk samples

Mycoplasma species are fastidious microorganisms causing mastitis in dairy cows. Storage by freezing milk samples affects their viability. The purpose of this study was to compare the effect of alternative storage methods on their recoverability. In Experiment I, mycoplasma counts from fresh milk samples were compared to those same samples stored for 1, 3, and 5 days at refrigerated (5 °C) temperatures. Experiment II was done to compare the mycoplasma counts of fresh milk samples with those stored frozen (−20 °C) with addition of 0%, 10%, 30% and 50% glycerol (v/v). Two strains of each of 5 species: M. bovis, M. californicum, M. bovigenitalium, M. canadense and M. alkalescens, were selected and inoculated into bulk tank milk free of this pathogen. Compared to those in fresh milk samples, counts were approximately reduced by: 0.3 log 10 CFU/ml in 5 day refrigerated milk ( P < 0.05) and by 1.0 log 10 CFU/ml in milk frozen without glycerol ( P < 0.05). Addition of glycerol (10% and 30%, v/v) to milk samples increased the number of recovered Mycoplasma by up to 0.4 log 10 CFU/ml in frozen milk samples ( P < 0.05). No significant interactions were detected between either Mycoplasma species or starting concentration and survival as effected by storage method. Refrigerating milk samples for 5 days and freezing milk samples lowers the number of recovered Mycoplasma species . The addition of glycerol to achieve 10% and 30% v/v solutions improves the recovery of Mycoplasma species from frozen milk samples. To maximize detection of this pathogen, fresh milk samples should be cultured without storage.

Bacterial growth in human milk during continuous feeding.

The present study was undertaken to determine both the time at which exponential growth of bacteria ensued in human milk during a continuous feeding and whether the fat content of the milk changed during a continuous feeding. Breast milk samples were obtained from 19 healthy lactating women between the 7th and 28th postpartum day. Nine samples were refrigerated and utilized in a simulated continuous feeding within 24 hours of collection. The remaining 10 samples were frozen for four to six weeks until use in the simulated feeding study. Bacterial colony counts and fat content (by creamatocrit) were then measured at the beginning of the simulated feeding and at hourly intervals from five to eight hours after the initiation of the study. Results indicated that logarithmic growth of the contaminating bacteria was not observed during the study time. No significant increase in total colony count was observed over the course of study in the frozen milk samples, whereas a significantly increased colony count was apparent by six hours for the fresh milk samples. Fat content of the milk decreased significantly within six hours for the frozen samples and within eight hours for the fresh milk. These data would suggest that when continuous drip feeding methods are employed for providing human milk to newborn infants, it may be desirable to change the entire feeding apparatus (syringe, tubing, and milk) at four hour intervals in order to optimize caloric intake and minimize the risk of bacterial overgrowth.

Effects of Freezing Milk Samples on the Recovery of Alimentary Pathogens and Indicator Microorganisms

The objective of the present study was to determine the effects of freezing and subsequent storage on quantitative results of bacteriologic culturing of selected alimentary pathogens and indicator microorganisms in milk. Two model experiments were carried out. In the first experiment, raw milk samples were frozen and stored at -20 °C for 72 hours, 7 days or 21 days. After thawing, the following counts of indicator microorganisms were assessed: total bacteria count and counts of coliform and psychrotrophic microorganisms. The counts of these microorganisms determined before freezing served as control. In the second experiment, milk samples were inoculated with strains of shigatoxigenic Escherichia coli, enterotoxigenic Staphylococcus aureus and bacteria Listeria monocytogenes and frozen. After storage for the above times, recovery was performed and colony-forming unit counts per millilitre were assessed. It was documented that freezing had a considerable adverse effect on the recovery of shigatoxigenic E. coli (P &lt; 0.01) after 7 days of storage. A decline in counts of coliform bacteria (P &lt; 0.01) and psychrotrophic microorganisms (P &lt; 0.01) was detected as soon as after 72 hours storage. The decline in counts of E. coli and psychrotrophic microorganisms continued with the time of storage - 72 hours and 7 days (P &lt; 0.05); 72 hours and 21 days (P &lt; 0.01). In contrast, no effect was recorded for L. monocytogenes and total bacterial count. Freezing caused a slight increase (P &lt; 0.05) in S. aureus counts in milk samples after 72 hours and 7 days of storage. The results of this study indicate that the freezing of milk samples is unsuitable for sample storage before the assessment of hygienic quality because potential risk of misdiagnosis may be high.

Inter-laboratory studies for the evaluation of ELISA kits for the detection of chloramphenicol residues in milk and muscle

The use of chloramphenicol in veterinary medicine was banned in the EU in 1994. As the Community Reference Laboratory for antibiotic residues in food of animal origin, one of our functions is to organize inter-laboratory studies. A first inter-laboratory study for the analysis of chloramphenicol (CAP) in milk by ELISA kits was organized in 2001 and a second one for the detection of CAP in pig muscle by ELISA kits in 2002. These studies were intended to allow participants to control their CAP ELISA methods when used routinely and also to compare the performances of various ELISA kits for the detection of chloramphenicol in milk (commercial or in-house kits). In 2001, 15 participants received ten randomly coded frozen milk samples (four blank samples and six spiked milk samples from 0.5 to 5.0 μg/l). In 2002, 20 participants received eight randomly coded frozen muscle samples (two blank samples and six incurred muscle samples from 2.1 to 6.5 μg/kg). They were asked to analyse each sample in triplicate with the ELISA kit of their choice. Different kits from different suppliers were used and compared in the two studies. The results of the two inter-laboratory studies on ELISA kits were satisfactory regarding qualitative results. The global rates of false compliant results of 2.2% for milk and 0.0% for pig muscle samples were lower than 5% whichever kit was used. The global rates of false non-compliant results (16.7% and 10% for milk and muscle respectively) were also satisfactory. The distribution of false non-compliant results depends on the kind of kit used and on the detection limit for milk as well as for muscle. Moreover the sample preparation was very important to avoid false non-compliant results. Finally, this study demonstrates that ELISA kits for chloramphenicol in milk and muscle globally show good repeatability and accuracy. So these kits could be considered as suitable tests for screening purposes.

Effect of freezing goat milk samples on recovery of intramammary bacterial pathogens

With the aim of evaluating the effect of freezing goat milk samples on recovery of intramammary pathogens, 1200 milk samples from udder halves with subclinical intramammary infection were studied. Samples (20 ml) were frozen at −20 and at −80 °C. Thawing was carried out at room temperature at 7, 14, 21, 28, 58, 118, 178, 236 and 730 days after collection and bacteriological analyses were carried out to determine the number of colony forming units/ml (CFU/ml). Mixed model statistical analysis showed that bacterial group, temperature of storage, interaction of bacterial group and temperature of storage and the interaction of bacterial group, time and temperature of storage were statistically significant effects. For coagulase negative staphylococci (CNS), least squares means of log CFU/ml recovered at −20 and −80 °C were not different. Nevertheless, for Gram negative bacilli (GNB) a significant decrease was detected in samples frozen at −20 vs. −80 °C. At both temperatures and at different times of storage, significant increases were detected between log CFU/ml of CNS and values on day zero. At −20 °C, a significant decrease in GNB recovery was detected between freezing days zero and 730. This difference was not detected when goat milk samples infected by GNB were frozen at −80 °C. The results show that frozen milk samples can be useful in goat subclinical mastitis control programs.

Effects of storage on the physicochemical and antibacterial properties of human milk

Storage of human milk for limited periods of time is unavoidable in neonatal units and also in the home where increasing numbers of mothers go back to work soon after delivery. Many mothers, convinced of the importance of prolonged exclusive or complementary breast-feeding, often express and store human milk for use during the period of separation. This study examines the effects of different storage methods on the pH and some antibacterial activities of human milk. Portions of milk and colostrum samples from healthy lactating mothers were stored at 4°C and -20°C for periods ranging from one day to 12 weeks. The stored milk samples were analysed for pH, bactericidal and bacteria sequestration activities against a serum-sensitive Escherichia. coli, and compared with freshly collected samples, with and without EDTA. Milk became progressively more acidic during storage. While the bactericidal activities of refrigerated samples diminished rapidly, up to two-thirds of the original activity level was maintained by freezing for up to three months. The ability of milk fat globule membrane to adhere to suspended bacteria was gradually lost in frozen milk samples, while it was greatly enhanced during the first few days in refrigerated samples, before declining sharply. This study shows that loss of bactericidal activity in refrigerated milk is well compensated for by enhanced bacteria sequestration activity, and allays any fears that might arise concerning the suitability of stored human milk for infant consumption.

Manufacture of yoghurt from stored frozen sheep’s milk

The effects of long-term deep-frozen storage of sheep’s milk on its physicochemical, microbiological and physical stability characteristics, as well as on the corresponding characteristics and the sensory attributes of the yoghurt manufactured from the stored frozen milk after thawing were examined. Bulk whole sheep’s milk, after clarification and pasteurisation (72° C for 15 s), was sampled for analysis and then poured into plastic bags, frozen in a moving-air freezer at −25° C in thin blocks (21 cm long, 21 cm high and 2.7 cm thick) weighing about 1.2 kg, and stored at −20° C for up to 6 months. At 2, 4 and 6 months of storage, samples were removed, thawed and analysed. Moreover, set type yoghurts were manufactured from these samples and examined. The results indicated that there were no significant ( P>0.05) differences between fresh (control) and stored frozen milk samples in pH, acidity, acid degree value, peroxide value, protein sediment, apparent viscosity or coliform counts. The frozen samples had significantly ( P<0.05) lower total bacterial counts than the fresh milk. No significant ( P>0.05) differences in pH, acidity, lactose, appearance and colour, body and texture, flavour, overall acceptability, consistency, apparent viscosity or syneresis were observed between the control milk yoghurt and the yoghurts made from milk stored frozen for up to 6 months.

Recovery of bacteria from goats' milk following freezing and the prevalence of bacterial infection in milk from goats with an elevated somatic cell count

Aims. To establish the prevalence of bacterial infection of goats with high somatic cell counts and to determine the effect of freezing milk samples on the probability of culturing the same pathogen again. Methods. Milk samples were collected from both halves of 99 goats with a somatic cell count > 650,000 cells/ml. The milk samples were split into four aliquots with one of the aliquots being cultured immediately. The other three aliquots were frozen at —20 °C, and successive aliquots of 39 samples from which pathogens were isolated at Day 0 were thawed and cultured 28, 56 and 84 days later. Results. Coagulase-negative staphylococci were the most common isolates. Freezing and thawing did not significantly reduce either the number of isolates cultured or the number of colony forming units from these successive aliquots. However, three of 39 selected pathogens were not cultured at one or more of the successive thawings. Goats from herds that were housed had a higher probability of having a pathogen isolated from milk than goats from herds kept on pasture. Conclusions. Freezing of milk samples may be used for subsequent culture of bacteria from goats' milk.