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* Centre Especial de Recerca Planta de Tecnologia dels Aliments (CERPTA), CeRTA, XiT, Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
Unidad de Producción Animal, Universidad de las Palmas de Gran Canaria, Transmontaña s/n, 35416-Arucas, Spain
Instituto Canario de Investigaciones Agrarias, Apartado 60, La Laguna, Tenerife, Spain
1 Corresponding author: Toni.Trujillo{at}uab.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: heat and pressure treatment • microorganism • immunoglobulin G • caprine colostrums
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Although studies investigating the thermal destruction of human and bovine Ig in colostrum and milk have been reported, little information is available on caprine colostrum decontamination while maintaining IgG content (Argüello et al., 2003). On the other hand, heating experiments performed with IgG in bovine colostrum have shown higher IgG activity retention than that obtained with IgG in PBS buffer at similar pH (Domínguez et al., 2001). This difference suggests a protective effect of other milk components such as proteins, fat, and salts that cause a delay in denaturation and prevent aggregation of IgG during heat treatment. Goat milk and colostrum differ from their bovine counterparts in composition (CN, whey protein, fat, and salts), so research is required to verify the operating pasteurization conditions required to hygienize colostrum and retain IgG activity in goat colostrum.
Over the last decade, high-pressure (HP) treatment of foods and food components has gained increasing interest as a nonthermal method of modifying the structure and functional properties of food macromolecules, such as proteins, without affecting the nutritional value, flavor, color, and vitamin content. Effectiveness of HP in microbial inactivation has been reported for different foodborne pathogens and spoilage microorganisms (Smelt, 1998) with the purpose of enhancing product safety and improving the shelf life of the food. High pressure has been proposed as an alternative technology to thermal processing with various possible applications in the dairy industry (Trujillo et al., 2002) including sanitization of milk for making yogurt and fresh or ripened cheeses, fresh cheese sanitization, and cheese ripening acceleration.
The aim of this work was to determine the effect of heat (temperature and time) and high-pressure treatments on microbiological quality and immunoglobulin G stability of caprine colostrum.
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MATERIALS AND METHODS |
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For pressure treatments, colostrum samples in duplicate were packed into flexible tubes of 30 mL (Azlon, Bibby Sterilin Ltd., Stone, Staffordshire, UK), vacuum-sealed, and pressurized in a batch isostatic press (GEC Alsthom ACB, Nantes, France) at 400 and 500 MPa for 10 min (24 samples for analysis). Pressure was built up using a standard pressurization rate of about 200 MPa/min. During pressure buildup, temperature increase due to adiabatic heating was controlled with a heating/cooling system and was maintained at 20°C. After heat and pressure treatments, the samples were transferred to ice water for rapid cooling, and stored at 4°C until microbiological analyses were performed; samples for IgG determination were frozen at –20°C (<3 mo). In this study, we considered the global treatment including the initial phase of variable temperature or pressure from the experiment. Untreated colostrum samples in duplicate were used as a control (12 samples for analysis).
IgG Quantification
Colostrum IgG concentration was determined using an immunodiffusion method (Mancini et al., 1965). The standard curve was prepared in accordance with Catty and Raykundalia (1988) using pure goat IgG (Sigma-Aldrich, St. Louis, MO).
Microbiological Analyses
The microbiological quality of the colostrum samples was assessed by enumerating the following microorganisms. Total counts (TC) were enumerated on plate count agar medium (Oxoid Ltd., Basingstoke, UK) incubated for 72 h at 30°C. Enterobacteriaceae were enumerated on violet red bile glucose agar medium (Biokar Diagnostic, Bioser SA, Barcelona, Spain) incubated for 24 h at 37°C. Lactococci were enumerated on M17 agar medium (Oxoid Ltd.) incubated for 48 h at 30°C. Lactobacilli were enumerated on Rogosa agar medium (Biokar Diagnostics) incubated for 72 h at 30°C. Enterococci were enumerated on kanamycin esculin azide agar medium (Oxoid Ltd.) incubated for 48 h at 37°C. Coagulase-positive staphylococci were enumerated on Baird-Parker agar + rabbit plasma fibrinogen medium (bioMérieux S.A., Marcy-l’Etoile, France) incubated for 24 h at 37°C. Listeria monocytogenes were detected in a 2-stage enrichment procedure. Twenty-five grams of colostrum was preenriched in half Fraser broth (bioMérieux S.A.) incubated at 37°C for 24 h. One milliliter of the preenriched sample was then incubated in Fraser broth (bioMérieux S.A.) at 37°C for 24 h. Enrichment was then streaked onto Palcam agar medium (Oxoid Ltd.) and incubated at 37°C for 24 h. Salmonella spp. were also detected using a 2-stage enrichment procedure. Twenty-five grams of colostrum was preenriched in buffered peptone water (Oxoid Ltd.) incubated at 37°C for 24 h. One milliliter of the preenriched sample was then incubated in Muller Kauffman broth (bioMérieux S.A.) or 0.1 mL in Rappaport Vassiliadis broth (bioMérieux S.A.) at 37°C or 42°C for 24 h, respectively. Enrichments were then streaked onto XLD and SMID2 agar media (Oxoid Ltd.) and incubated at 37°C for 24 h.
The efficiency in reducing the total bacterial population of treatments was calculated as percentage of reduction related to raw milk counts [100 – (f x 100)/i], where f = bacterial population after treatment, and i = bacterial population before treatment.
Statistical Analysis
The 6 batches of colostrum were divided in 10 aliquots each. Two random aliquots from each batch were assignment to 1 of 5 different treatments (raw, 56°C/60 min, 63°C/30 min, 400 MPa, and 500 MPa).
The statistical analysis used to evaluate the effects of different treatments on colostral IgG and microbiological parameters was a GLM procedure using duplicates as separate results, and a posthoc Tukey analysis was performed using SPSS (SPSS Inc., Chicago, IL).
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RESULTS AND DISCUSSION |
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In this study the main groups of microorganisms that comprise the microbiota commonly found in raw milk included total counts as a useful indicator for monitoring the bacteriological quality of raw colostrums; Enterobacteriaceae as hygiene indicators (their absence can be useful in assessing heat treatment efficiency); enterococci, in spite of the fact that these microorganisms are highly resistant to conditions of freezing, drying, and heat treatments as in standard pasteurization; lactococci and lactobacilli as typical lactic acid bacteria of milk; and coagulase-positive staphylococci, Listeria monocytogenes, and Salmonella spp., because of their interest as pathogens and, in the case of coagulase-positive staphylococci, by their relative resistance to hygienizing treatments.
Table 1
shows bacterial log counts of microorganisms and IgG in raw caprine colostrums. The IgG concentrations at the first milking were 21.51 to 36.71 mg/mL. Results agree with previous experiments in Majorera goats (Argüello et al., 2003, 2006) and are consistent with those reported by Ubellarte et al. (1987) in Saanen and Camosciata goats, and by Levieux et al. (2002) in Saanen and Alpine goats.
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An elevated number of Enterobacteriaceae in milk or colostrums indicates deficient handling during milking, collection, or manipulation. In the present study, Enterobacteriaceae were a constant component of the microbiota, with counts that ranged from 1.5 x 102 to 1.15 x 103 cfu/mL. Enterococci were found at a high level with notable variations between samples (from 4.0 x 103 to 4.35 x 104 cfu/mL). Lactococci and lactobacilli were 2 permanent groups of the colostrum microflora but with significantly higher counts for lactococci (1.25 x 105 to 7.2 x 105 cfu/mL) compared with lactobacilli (1.0 x 102 to 9.5 x 102 cfu/mL).
Coagulase-positive staphylococci were detected in 4 of the 6 samples analyzed, with counts that varied from 1.0 x 101 to 1.25 x 102 cfu/mL, and only one sample exceeded the threshold value for the number of bacteria (m = 102 cfu/mL) but without prejudice to compliance with the limits established in Europe (European Union, 1992) for this group in raw goat milk: n = 5, C = 2, m = 102 cfu/mL, M = 5 x 102 cfu/mL, where n = number of sample units comprising the sample; m = threshold value for the number of bacteria, and the result is considered satisfactory if the number of bacteria in all sample units does not exceed "m"; M = maximum value for the number of bacteria, and the result is considered unsatisfactory if the number of bacteria in one or more sample units is "M" or more; c = number of sample units where the bacteria count may be between "m" and "M", the sample being considered acceptable if the bacteria count of the other sample units is "m" or less.
Neither Salmonella spp. nor Listeria monocytogenes were detected in any colostrum sample using the protocols described above.
The results obtained for caprine colostrums confirm the studies made by Poulsen et al. (2002) in bovine colostrums, who reported that 82% of samples analyzed presented poor microbiological quality, and more of these colostrum samples had close to or even greater than 106 cfu/mL with a high content of coliforms. However, these authors also found some pathogens such as Escherichia coli and Salmonella spp. According to Stewart et al. (2005), bacterial counts are very low (<105 cfu/mL) in bovine colostrum samples collected directly from the udder if disinfection of cow teats before milking and postmilking is carried out. However, counts significantly increase in relation to the colostrum production method, harvest equipment, and storage processes used on-farm.
Effect of Heat (Temperature and Time) and Pressure Treatments on Microbiological Quality and IgG Concentration of Colostrums
Table 2 shows bacterial log counts of microorganisms and IgG in raw, heat-, and pressure-treated colostrums. Heat and HP treatments significantly reduced TC (1.5 to 1.32 log) and there were no statistical differences among the treatments assayed. Overall, HP treatments were as efficient in reducing the total bacterial population as were heat pasteurization treatments: reductions of 95.50 and 96.93% for pressure treatments of 400 and 500 MPa, and 91.61 and 97.59% for heat treatments of 56°C for 60 min and 63°C for 30 min, respectively. These results are similar to those reported by Buffa et al. (2001) studying the effect of pasteurization (72°C, 15 s) and HP (500 MPa, 15 min, 20 °C) treatments on TC in caprine milk.
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Reductions in lactococci counts by the technological treatments were similar to those described for TC (1.45 log), and enterococci were significantly reduced in either pasteurized or HP-treated colostrums (2.47 log).
Coagulase-positive staphylococci were not detected in heat or pressure-treated samples. Gervilla et al. (1999) studying HP inactivation of Staphylococcus aureus 534 CECT in ewe’s milk at different temperatures for 10 min and pressure treatments from 200 to 500 MPa found that this microorganism is very resistant to pressure treatment, although at 500 MPa and 25 °C, reductions between 1.9 to 2.4 log were achieved.
Because no Salmonella spp. nor Listeria monocytogenes were detected in any raw colostrums, we cannot draw conclusions about the efficacy of heat and HP treatments on the destruction of these pathogenic microorganisms. However, there are numerous studies in the literature about the destruction of foodborne pathogens by both heat and pressure treatments. Effectiveness of HP in microbial inactivation has been reported using E. coli, L. monocytogenes, Salmonella spp., Staphylococcus aureus, and Bacillus cereus in milk (Patterson and Kilpatrick, 1998; Gervilla et al., 1999; McClements et al., 2001; Wuytack et al., 2003).
The IgG values in the colostrums after heat and pressure treatments are shown in Table 2
. A reduction in colostrum IgG concentration was observed in all treatments assayed: 
14 and 15% for heat treatments of 56°C for 60 min and 63°C for 30 min, and 20 and 38% for pressure treatments of 400 and 500 MPa, respectively, but statistical differences were only observed between raw colostrums and those HP-treated at 500 MPa. Overall, treatments of 56°C for 60 min and 63°C for 30 min would be considered equivalent heat treatments because they diminished bacterial counts to the same degree, and no significant differences in IgG contents were observed between treatments.
The reduction observed on the IgG concentration after heat treatments was similar to that observed in bovine colostrum by Meylan et al. (1996) and Tyler et al. (2000) at 63°C for 30 min (12% IgG reduction) and McMartin et al. (2006) at 63°C for 120 min (34% IgG reduction), but was lower than that reported by Argüello et al. (2003) in the same goat breed. These authors applied heat treatments of 57°C for 10 min to the colostrums and then transferred them to a thermos bottle preheated with boiling water for 1 h, obtaining an IgG reduction of 38%. By contrast, Steinbach et al. (1981) found no reduction in bovine colostrum IgG concentration after a heat treatment of 55°C for 30 min.
The reduction in IgG concentration observed after pressure treatments agrees with the results obtained by Felipe et al. (1997), who did not observe differences in the levels of Ig in goat milk (determined from their loss of solubility at pH 4.6 by gel permeation fast protein liquid chromatography and SDS-PAGE) with pressure treatments up to 300 MPa, but some aggregation occurred between 300 and 500 MPa. According to Howlett et al. (1992), bovine IgG does not undergo conformational changes below 210 MPa, but when pressure is increased to 820 MPa, some conformational changes and aggregation appear to occur, the rate of change being faster between 210 and 460 MPa.
Although rheological measurements were not conducted on raw and treated colostrums, some samples of HP-treated colostrums, especially those treated at 500 MPa, presented higher viscosity (visually) compared with their raw and heat-treated homologues. This observation could be explained by the denaturation of ß-LG produced by HP treatment (Felipe et al., 1997), which could produce large protein aggregates increasing colostrum viscosity.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Although these results are promising because they suggest the possibility of hygienizing goat colostrum by heat or pressure (400 MPa) treatments while maintaining Ig content, the findings are preliminary and should be interpreted with caution. The study has been performed with 6 batches of goat colostrum using small volumes (20 to 30 mL) and under laboratory conditions to simulate heat or pressure pasteurization conditions. Further research is needed to study the use of commercial on-farm batch pasteurization equipment to treat larger volumes of caprine colostrum, as would be the situation under farm conditions. On the other hand, the results have shown that HP processing at 400 MPa could be an alternative to heat treatment of colostrums. High-pressure processing, in a batch or semi-continuous process, is gaining commercial acceptance, and some medium capacity (500 kg/h) industrial applications are currently in operation. The process can result in excellent product quality but remains relatively expensive and so is restricted to high added-value products.
Received for publication February 24, 2006. Accepted for publication September 16, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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-lactalbumin and serum albumin in colostrums and milk during the early post partum period. J. Dairy Res. 69:391–399.[Medline]
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