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Assessment of the bacterial diversity of treated and untreated milk during cold storage by T-RFLP and PCR-DGGE methods

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Dairy Science & Technology

Abstract

The evaluation of the effect of treatments on the dynamics of milk microbiota by appropriate and reliable methods is critical for a better control of the microbiological quality of milk. The aim of this study was to compare the diversity and the alterations of the microbiota in treated and untreated milk samples during cold storage at 4 °C or 8 °C up to 7 days. Three treatments were applied to raw milk, carbon dioxide addition (CO2), thermization (TH), and microfiltration (MF), and untreated raw milk (UT) was used as a control. Two molecular fingerprinting methods, terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) were used. According to the T-RFLP and polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) profiles, the dominant bacteria in milk were presumably affiliated with the psychrotolerant genera Streptococcus, Staphylococcus, Pseudomonas, and Aerococcus. At day 7, phylotypes related to Pseudomonas spp., Trichococcus floculiformis and Prevotella spp. were dominant in UT and CO2 milk samples but not in TH milk samples while Staphylococcus spp. and Stenotrophomonas spp. were specifically dominant in TH and MF milk samples, respectively. Principal component analysis of T-RFLP profiles revealed the strong effect of treatment on the dynamics of specific bacteria. PCR-DGGE profiles revealed the dominant bacteria associated with the effect of treatments (TH, CO2, and MF) on milk microbiota, which can then be used as markers for the quality of treated milk. The T-RFLP method had a higher resolution than PCR-DGGE in this study and can thus be used as an accurate tool for analysis of the bacterial community in milk.

采用 T-RFLP 和 PCR-DGGE方 法评价处理乳和非处理乳在冷藏过程中细菌多样性

摘要 采用恰当,可靠的方法评价处理过程对乳微生物动力学的影响,对更好地控制乳微生物质量是非常重要。本研究比较了处理乳与非处理乳在冷藏条件下 (4 °C 或 8 °C下 7 天) 微生物的多样性及其变化。分别采用充二氧化碳, 热杀菌 (TH) 和微过滤 (MF) 方法对原料乳进行处理。两种分子指纹图谱方法, 即末端限制片段长度多态性 (Terminal restriction fragment length polymorphism,T-RFLP) 和变性梯度凝胶电泳 (Denaturing gradient gel electrophoresis, DGGE) 用于本研究中。根据 T-RFLP 和 PCR-DGGE 的图谱, 推测乳中主要微生物为耐寒性微生物 Streptococcus, Staphylococcus, Pseudomonas 和 Aerococcus 属。冷藏 7 天后, 在 UT 和 CO2 处理的乳样中, 以 Pseudomonas spp., Trichococcus floculiformis, Prevotella spp. 为主导菌群, TH 处理乳中无上述菌群, 但以 Staphylococcus spp. 为主, MF 处理乳样中则以 Stenotrophomonas spp. 为主导菌群。T-RFLP 的主成分分析揭示了处理方法对特定微生物有强烈的影响。PCR-DGEE 图谱揭示了主导菌群与处理方法 (TH、CO2 和 MF) 对乳样微生物菌群的作用是密切联系, 这种方法可以作为评价处理乳质量的标记。T-RFLP 方法与 PCR-DGGE 方法相比, 就有更高的分辨率, 因此可以作为乳中微生物群落分析的精确工具。

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Acknowledgments

This study was supported by a grant from Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT), Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du Québec (MAPAQ), Novalait, and Agriculture and Agri-Food Canada award to G. LaPointe, D. Roy, and D. St-Gelais and by the NSERC Canada Research Chair awarded to D. Roy. The authors thank D. St-Gelais, S. Turcot, S. Labrie, and L. Lagacé for their scientific advice and technical assistance.

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  1. Institut des Nutraceutiques et des Aliments Fonctionnels (INAF), Université Laval, 2440 Hochelaga Blvd, Québec, QC, Canada, G1V 0A6

    Eric Andriamahery Rasolofo, Gisele LaPointe & Denis Roy

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  1. Eric Andriamahery Rasolofo
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  2. Gisele LaPointe
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  3. Denis Roy
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Correspondence to Denis Roy.

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Rasolofo, E.A., LaPointe, G. & Roy, D. Assessment of the bacterial diversity of treated and untreated milk during cold storage by T-RFLP and PCR-DGGE methods. Dairy Science & Technol. 91, 573–597 (2011). https://doi.org/10.1007/s13594-011-0027-4

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  • DOI: https://doi.org/10.1007/s13594-011-0027-4

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