Effects of breeding and hygienic practices on raw cow milk quality in Tadla area, Morocco

The main objective of this study was to evaluate the bacteriological quality of raw cow milk produced in two cooperatives in Tadla area in Morocco. During two periods (mid-June to mid-August, mid-November to mid-January), the milk samples (100) were collected at two milk collection centers (cooperatives) from cooperative 1 (semi-public farm) and cooperative 2 (Irrigated area). Samples were analysed for standard plate count (SPC), total coliform (TC) and feacal coliform (FC), lactic acid bacteria, spoilage microorganisms (proteolytic, lipolytic) and some of the pathogenic bacteria (staphylococci, clostridium and salmonella).

Our study in the region of Tadla showed the presence of heterogeneity in bacterial contamination among the cooperatives analysed. Thus, it appears that cooperative 2 is the heavily contaminated one. Whereas the less contaminated ones belong to the semi-public farm (i.e. cooperative 1). Procedures of milking, transportation, milking manner and pre-storage conditions could constitute the major factors involved in the contamination in cooperatives. Finally, these data represent the first investigation regarding the microbiological characteristics of raw cow milk produced in Tadla area from Morocco.

Introduction

In Morocco, the dairy industry is undergoing many problems related to the quality standards which are very hard to meet. This is due particularly to the heterogeneity of raw milk’s compositions. Many factors would contribute to this phenomenon such as the geographical area, breeding, climate conditions, and nutritional factors (Coulon 1994). Indeed, in Morocco, the milk collection centers still have many problems concerning hygienic quality of raw milk received (Srairi et al 2005; El Mouktafi 1988). This is due to the high temperature recorded in some regions of the country accompanied by a deficient milk refrigeration system. Furthermore, the non respect of the sanitation during milking and the handling would constitute an additional deteriorating factor (Aumaitre 1999). Therefore, it seems necessary to study the effects of such parameters on the quality of raw milk in order to improve and monitore an acceptable milk quality and through these studies, new breeding techniques can be described and applied in Morocco for better quality production.

To our knowledge, there was no breeding program in Morocco to include milk composition as well as microbiological characteristics (Hamama and El Mouktafi 1990). Only disparate official milk recording data exist but were not able to give a complete scheme. An exhaustive study has been reported by Mennane et al (2007) concerned particularly the effect of feeding from domestic waste on hygienic quality of raw cow’s milk in a determined region of Morocco. However, there is no detailed investigation concerning dairy production and its microbiological characteristics in other Moroccan regions. Until now, milk production and characterization are still at a premium and no data may exist to know really the situation. Thus, the main objective of this study is to investigate the microbiological characteristics of raw milk produced in two cooperatives of Tadla region (leading region in milk production in Morocco). These cooperatives follow differents manner of milking raw milk. All these findings have been evaluated during two different periods in the year, in attempt to assess whether temperature variations could influence significatly the microbiological composition of raw cow milk.

Material and methods

Characteristics of the dairy cattle cooperatives

The present investigation was carried out in two cooperatives representing the two cooperatives in the region of Tadla:

The raw cow milk samples were collected from milk collection centers (cooperatives) during the following periods: between the end of June and mid-August (period 1), between mid-November and mid-January (period 2). Samples were collected in the early morning. 250 ml samples of raw milk were taken starting from the cooled tanks, in sterile bottles and were immediately transported in an appropriated icebox to the laboratory. The test sample selection is carried out during two days of each week for the indicated periods. cooperative 1 (semi-public farm) applied a mechanically milking, and cooperative 2 who those adherents follow the traditional methods of milking raw milk.

Microbiological determinations

Ten ml of each sample were blended in 90 ml of sterile water (0.1%) in a disinfected mincer (blender) to prepare the initial dilution (1/10).

Standard Plate Count (SPC)

Appropriate serial dilutions (up to 10^-6) of the sample in distilled water were pour plated on standard plate count agar (PCA) (Difco Laboratory, USA). The plates were incubated at 30°C for 48 hours.

Staphylococci

Staphylococci counts were determined by the pour-plate method. Appropriate dilutions up to 10^-6 were plated on Mannitol Salt Agar (Difco, USA), the plates were incubated at 37 °C for 24 h. The small yellow colonies on the medium were counted and checked for Gram and catalase reactions. Catalase positive and gram positive colonies were spread cultured on trypticase soya agar slants for further characterization.

Coliforms

Coliforms were enumerated on Macconkey agar (Merck, Germany). The plates were incubated at 37°C for total coliforms (TC) and at 44°C for faecal coliforms (FC) for 24 hours. The appeared colonies on the medium were restreaked on the same medium for more purification. Isolated colonies were cultured on trypticase soya agar slants and incubated for 24 hours for further identification and cultures were stored at 4°C until identification.

Salmonella

25 g of the sample were added to 125 ml of sterile buffered peptone water and incubated for 18 hours at 37°C. Tree tubes of tetrathionate broth and 3 tubes of selenite-cystein broth (Difco, USA) were inoculated with 1 ml from cultures of buffered peptone water and incubated for 24 hours at 37°C. Positve tubes of both media were streaked on Hektoen agar (Difco, USA). The method described by Poelma et al (1984) was used for the identification.

Spore forming bacteria

The initial dilution was heat activated at 80°C for 10 min and immediately cooled in iced water. Anaerobic sulfite reducing Clostridium were groew on SPS medium (Merck, Germany) in tubes which were then inoculated with 2, 1 and 0.5 ml of the heat activated dilution and incubated at 30°C for 24 hours.

Spoilage microorganisms

Proteolytics

The appropriate dilutions up to 10⁴ were plated on the medium milk contain. The plates were incubated at 30 °C for 48 to 72 h. After solidification, a clear halo around the colonies will be counted.

Lipolytics

Dilutions up 10^-6 were plated on Tween 80 medium. The plates were incubated at 30 °C for 48 to 72 h. Colonies of the lipolytic bacteria were surrounded by an opaque halo due to the release of free fatty acids.

Lactic acid bacteria

The lactic acid bacteria were counted on MRS (De Man Rogosa and Sharpe) medium incubated at 30 °C for 48 h.

Statistical analysis

Data obtained from all microbological analysis were analysed by analysis of variance (ANOVA) and by the least significant difference (LSD) (SAS, proc glm).

Results

To assess the diversity and influence of conditions of production on raw cow milk microflora, the microbial composition of 100 raw cow milk samples was determined. The results obtained were depicted in tables 1 and 2, which summarize the mean count of the seven bacterial groups analysed of locally produced raw milk collected from two cooperatives presenting different breeding and milking ways during period 1 (mid-June to mid-August) and period 2 (mid-November to mid-January) respectively.

Table 1. Mean counts for all microbial groups in period 1
Coop	FC (10³)	T C (10⁵)	Staph (10³)	SPC (10⁶)	LAB (10⁶)	Proteo (10³)	Lipo (10⁵)
1	2.4^b	0.4^b	1.3^b	1.6^b	0.9^b	2.4^a	2.1^b
2	36.4^a	21.3^a	8^a	21.3^a	177.6^a	0.8^b	12.1^a
Coop: cooperative; FC: faecal coliforms; TC: total coliforms; Staph: staphylococci; SPC: standard plate counts; LAB: lactic acid bacteria; Proteo: proteolytic; Lipo: lipolytic. ^a-b each distinct letter indicates a different group as obtained by the analysis variance. Means within columns without a common superscript are significantly different (P < 0.05).

Table 2. Mean counts for all microbial groups in period 2
Coop	FC (10³)	T C (10⁵)	Staph (10³)	SPC (10⁶)	LAB (10⁶)	Proteo (10³)	Lipo (10⁵)
1	0.1^b	0.02^b	0.5^a	1,6^b	0.2^b	2.5^a	0.5^b
2	44.8^a	12.5^a	1.5^a	0.9^a	2.7^a	1.5^a	10.2^a
Coop: cooperative; FC: faecal coliforms; TC: total coliforms; Staph: staphylococci; SPC: standard plate counts; LAB: lactic acid bacteria; Proteo: proteolytic; Lipo: lipolytic. ^a-b each distinct letter indicates a different group as obtained by the analysis variance. Means within columns without a common superscript are significantly different (P < 0.05).

When the breeding and milking ways were considered, the variance analysis of our results of all microbial contamination showed that there is a significant difference between cooperatives 1 and 2. The least significant difference (LSD) mentioned the presence of two groups during periods 1 and 2 of cooperatives 1 and 2. Comparaison of results obtained in the two cooperatives, are presented in figures 1-7. As depicted by these figures, it appears clearly that cooperative 1 had the lowest levels of all bacteria. Considering FC, the significative difference (P < 0.05) is evidenced for the two periods analyzed (Figure 1).

Thus, the mean values of FC were 2.48x10³ and 4.63x10⁴fcu/ml in period 1 respectively of cooperatives 1 and 2 (Table 1) , whereas in period 2 the mean values were 1.98x10²and 5.91x10⁴fcu/ml (Table 2) respectively. Similarly, when the TC are considered, the average values were limited between 4x10⁴and 3.17x10⁶fcu/ml in period 1 but in period 2, the average values were ranged between 1.98x10³ and 1.6x10⁶ fcu/ml, respectively for cooperatives 1 and 2 (Figure 2).

For the standard plate count (SPC) the difference was also significant in the two periods as the difference between the mean values of cooperatives 1 and 2 was higher in period 1 (figure 3): 1.63x10⁶and 2.13x10⁷ fcu/ml and in period 1.63x10⁶ and 9.68x10⁶fcu/ml respectively of cooperatives 1 and 2.

The bacterial loads differ also from the hottest period (period 1) to the cold one (period 2) for staphylococcus and lactic acid bacteria. Thus, in period 1 and 2, the results for staphylococcus aureus (Figure 4) evidenced the presence of a significant difference between the cooperatives 1 and 2.

The mean values for staphylococcus aureus in period 1 were 1.06x10³and 1.13x10⁴fcu/ml (P < 0.05) and in period 2 : 6.57x10² and 1.93x10³fcu/ml (P < 0.05) respectively of cooperatives 1 and 2. Concerning lactic acid bacteria (LAB), tables 1 and 2 show that a significant higher (P < 0.05) LAB count has been found in cooperative 2 when compared to cooperative 1. This difference is evidenced in the two periods analysed (Figure 5).

For proteolytic (Figure 6) and lipolytic microorganisms (Figure 7), the analysis of variance shows that the difference is significant (P < 0.05) only for lipolytic microorganisms in two periods (Tables 1-2).

Figure 6. Presence of spoilage microorganisms (proteolytic) in raw cow milk. Note that in contrast to the other bacterial groups, the mean count of this group is significantly higher in cooperative 1 than in cooperative 2, both in period 1 and period 2

Figure 7. Spoilage microoraganisms (lipolytic microorganisms) mean count in 100 raw cow milk samples taken from two different cooperatives located in Tadla area. Note that the load is higher in cooperative 2 compared to cooperative 1 for each period

Of note, salmonella and clostridium were not detected in raw milk samples collected from two cooperatives.

Discussion

Taken together, the results showed a variability in raw cow’s milk quality between two cooperatives (1 and 2) during the periods of investigation (one cold and the other hot to very hot period). Concerning FC and TC levels in our study were lesser than the results reported in Gharb region (North of Morocco) by Ounine et al (2004) and by Riahi (1981). The existence of coliform bacteria may not necessarily indicate a direct fecal contamination of milk, but more precisely as an indicator of poor hygiene and sanitay practices during milking and further handling. This could explain clearly the significant difference evidenced between the two cooperatives. Indeed, in cooperative 1, milking is made mechanically and the occurrence of fast refrigeration in this cooperative contributes further to the presence of lesser levels.

The standard plate count (SPC) is taken as an indicator concerning the hygienic conditions of the raw milk in collecting centers. In this sense, we note that the heavy contamination was found during the period 1 (i.e the hottest period). Furthermore, the cooperative 1 contain the lowest levels of SPC compared to the cooperative 2. More contamination of raw milk in cooperative 2 is due to the low microbiological quality of the water used for cleaning utensils and animals, as well as milk storage conditions which would lead to a poor quality of milk in this cooperative (Chatelin and Richard 1981; Piton and Richard 1982). Our results in this study are in agreement with reports of Srairi et al (2005), Amhouri et al (1998) and Bonfoh et al (2003). Although diffrences in stphylococci loads were evidenced between cooperatives 1 and 2, they are still lower compared to the levels founded by Hamama (2002) in Rabat region. Staphylococci count reached an average of 10³ cfu/ml in fresh milk samples. These levels are still insufficient to induce a risk of intoxication by staphylococci toxin production (Bergdoll 1970) and are still lower than the limits accepted in USA and EU for the raw cow milk. The fact that the smallholder adopt a traditional milking imply the presence of higher staphylococci loads. The presence of lactic acid bacteria in high numbers are undesirable in fresh cow milk because of the acid production and coagulation which is a default in fresh milk. In our study, the levels of lactic acid bacteria were higher in cooperative 2 during the priod 1, corresponding to the hot months, so the raw milk quality under these condition favorise the increase of lactic acid bacteria load in raw milk.

Some lactic acid bacteria can also be sheltered to the udder surface according to Desmasures et al (1997) or generally, on the body of the cows (Salama et al 1995). Therefore a minimal hygiene around the udder would preserve this type of flora (Bacic et al 1968).

The results of proteolytic and lipolytic microorganisms showed that there is a significant difference in two period only for lipolytic microorganisms. The acidification of milk is related to the relative proportions of technological flora and spoilage flora of milks which are proteolytic and lipolytic microorganisms. In period 1, the significant difference was showed in cooperative 2 concerning lipolytic microorganism and was characterized by high level than cooperative 1, the same situation was observed in period 2. Taken together, the results obtained in the present data evidenced clearly the determined role of breeding way and practices in the quality of raw milk. This is of high importance, considering the fact that the major part of this milk is destined to be processed industrially as a pasteurized milk.

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