Introduction
Consumers are increasingly aware of the link between food and health, and this has spurred the development of specialty health-promoting foods (Clydesdale 2004), typically called functional foods (FF). The dairy industry is at the forefront of the development of FF, particularly by using probiotic cultures (Leatherhead Food International 2006). Probiotics can be defined as "Live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host" (Araya et al. 2002). They have been associated to positive effects on gut physiology, diarrhoea, immune functions, and cancer (Ouwehand et al. 2003). Fermented milks, such as yogurt, are the main commercial probiotic-containing products, but non-fermented milks enriched with the health-promoting bacteria now appear on the market. In these products, the addition of probiotics does not affect taste, and the product is therefore sensitive to off-flavours, which could develop during storage.
The shelf life of pasteurized milk is partially associated to the bacterial microbiota it contains as it leaves the dairy plant. The bacterial population of pasteurized milk is comprised of thermoresistant bacteria and cells resulting from post-pasteurization contamination from the lines, pumps, and packaging equipment. In plants with high sanitary standards, this post-pasteurization contamination does not contribute the majority of microorganisms in the pasteurized milk, and total counts are principally due to the thermoresistant microbiota from the raw milk (Boor and Murphy 2002). Therefore, the determination of the microbial population of a pasteurized milk product can be a useful tool to evaluate plant sanitation as well as to help predict shelf life.
In pasteurized milks, the total plate counts are generally below [10.sup.3] CFU/mL (Boor and Murphy 2002), but the probiotic populations of the products recently marketed are typically of [10.sup.7] CFU/mL. This means that the contaminating bacteria only represent 0.01% of the total population of the probiotic-enriched products. Although the contaminating microbiota are thus at low initial levels, the psychrotrophic fraction could potentially generate off-flavours during storage, and was of concern. It is thus a challenge to selectively enumerate the non-probiotic contaminating microbiota in such products. For this purpose, a means to inhibit the growth of the probiotic bacteria must be carried out without affecting the colony development of the contaminating microbiota.
Many studies have been carried out on the selective enumeration of lactobacilli and bifidobacteria in fermented yogurts. In these assays, a number of strategies to promote, differentiate, or repress growth of selected species in plating media have been suggested: pH (Rybka and Kailasapathy 1996; Dave and Shah 1996; Roy et al. 1997), chromogenic indicators (Kneifel and Pacher 1993; Bracquart 1981), antibiotics (Dave and Shah 1996), bile (Dave and Shah 1996), and inorganic salts (Dave and Shah 1996; Arroyo et al. 1995; Roy et al. 1997), which include phosphates (Wright and Klaenhammer 1984). In addition to medium composition, temperature (Champagne et al. 1997) and oxygen (Shah 2000) can serve to selectively enumerate the cultures. In most cases, specialized media, such as de Man--Rogosa--Sharpe (MRS), M17, or Reinforced Clostridium Medium (Rybka and Kailasapathy 1996; Dave and Shah 1996; Onggo and Fleet 1993; McCann et al. 1996), were used, and no study was found using the basic plate count agar (PCA) as a base medium for selective enumeration of microbiota in probiotic-containing milk products. Furthermore, in all assays where selective enumerations were examined, the aim was to differentiate probiotics from each other or from the starter cultures in the foods. Although the analysis for contaminating pathogens is routinely carried out on commercial probiotic cultures, there is no report on the selective enumeration of total contaminating bacteria in a food product containing high probiotic populations.
Petrifilm[TM] aerobic count (AC) plates have been used for the total enumeration of lactic cultures (Champagne et al. 1994), and including MRS as a diluent proved useful in this respect (Nero et al. 2006). Furthermore, selective enumeration of lactic cultures in foods with Petrifilm[TM] AC was achieved by replacing the MRS diluent by KF or KFS media (Ortolani et al. 2007). However, no attempt has been made to prevent the development of probiotics in Petrifilm[TM] AC plates while allowing the development of the non-probiotic contaminating microbiota.
The aim of this study was to develop a medium, which would prevent the growth of Bifidobacterium lactis BB-12 and Lactobacillus rhamnosus Lb-Immuni-T, as well as enable the assessment of the non-probiotic standard plate count (SPC) of contaminating bacteria in pasteurized milk enriched with the probiotic bacteria. We report on the effect of phosphate addition to SPC agar or plate count broth (PCB), as well as the use of Petrifilm[TM] AC plates for this goal.
Materials and methods
Probiotic bacteria
Lactobacillus rhamnosus Lb-Immuni-T[TM] cell suspensions were prepared at the Agropur Research and Development Centre (Granby, Quebec) from a commercial freeze-dried product of a confidential source. Bifidobacterium animalis subsp. lactis BB-12[R] (B. animalis subsp. lactis BB-12) was purchased from Chr. Hansen (Denmark). The bifidobacteria were packaged in frozen pellets and kept at -40[degrees]C.
Probiotic-enriched milks
Commercial 2% fat pasteurized milk was obtained from Agropur Natrel (St. Bruno, Quebec). Nine different production lots were analyzed directly, and 6 were stored at 7[degrees]C for 1 week prior to use. The microbial analyses were carried out prior to and after addition of the probiotic bacteria to clearly ascertain the effect of probiotic supplementation on the results. Addition of the bifidobacteria was carried out by mixing 5 g of frozen pellets with 15 mL of cold UHT milk (Grand-Pre, Ste-Claire, Quebec) or 0.1% sterile peptone (BD, Becton Dickinson, Franklin Lakes, New Jersey), and adding the appropriate amount of this cell suspension into pasteurized milk to obtain a population of approximately 2 x [10.sup.7] CFU/mL of milk. The lactobacilli-supplemented milks were prepared by adding 4 g of the freeze-dried stock culture of L. rhamnosus Lb-Immuni-T to 100 mL of sterile UHT milk (Grand-Pre) or 0.1% sterile peptone water, allowing a 15 min rehydration period at 22[degrees]C and then adding the appropriate amount of the cell suspension to …
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