EVALUATION OF THE HYGIENIC QUALITY OF RAW MILK BASED ON THE PRESENCE OF BIFIDOBACTERIA SPP. AS AN INDICATOR OF FAECAL CONTAMINATION IN ASSIUT CITY

EVALUATION OF THE HYGIENIC QUALITY OF RAW MILK BASED ON THE PRESENCE OF BIFIDOBACTERIA SPP. AS AN INDICATOR OF FAECAL CONTAMINATION IN ASSIUT CITY

MARWA M.N. EL-GENDI and LAMIAA M.T. ALI

Animal Health Research Institute, Assiut Lab.

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                                                  ABSTRACT __________________________________________________________________________________________

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Key words: Bifidobacteria, raw Buffalo's milk, raw Cow's milk.

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INTRODUCTION

The local dairy commodity chain, from farm to retail sites is informal and often escapes monitoring of quality. In addition, most stakeholders lack knowledge on hygiene and sanitary aspects of their production, which can result in poor production standards and contamination of local raw milk. Bifidobacteria are well known for their beneficial effects on health and are used as probiotics in certain food and pharmaceutical products. Since they constitute one of the most important bacteria group in human and animal digestive tract, they were recently considered as fecal contamination indicators in raw milk dairy products. Moreover, the Bifidobacteria spp. isolated in humans are different from that isolated from animals (Delcenserie, et al., 2005). Fecal contamination of raw milk in farms has been shown by the detection of the same and most frequent Bifidobacterium spp. in milk as in cow's dung. Raw milk can be assumed to be the first critical point in an HACCP analysis of the raw milk cheese industry, but a follow-up of contamination during the cheese-making process is also of interest. The standard in Europe for fecal contamination control of raw milk cheese is Escherichia coli (Beerens et al., 2000).

The general objective is to define a new standard, using the Bifidobacteria (genus Bifidobacterium) as indicator organisms in order to point out unsatisfactory hygienic conditions of raw material and food products. These new indicators will be applied on raw milk and cheese made from it, as the quality of which depends on the hygiene of milking, the farm environment and the hygiene along the cheese production chain. Also meat and meat products in which Bifidobacteria will indicate hygienic shortcomings during slaughter, cutting and debuting as well as retail trade. Good hygienic practices must lead to the elimination of all bacterial hazards such as Listeria, E. coli (Entero hemorrhagic E.coli (EHEC)) or Salmonella spp. The knowledge of the Bifidobacterium spp. that contaminate raw milk, cheese made with raw milk, meat and meat products will point out the sites from which the contamination derives the development of a fast, sensitive and generally applicable technique will enable industries to detect Bifidobacteria during processing and to take corrective actions immediately. Due to their intestinal ecology, milk is not the natural environment of Bifidobacteria. Their presence in milk is a sign of fecal contamination (Delcenserie et al., 2005) and can therefore serve as a marker of the level of hygiene in the production chain (Beerens et al., 2000). In addition, the species characterization and assessment of antibacterial potential could lead to its determination in milk, as a selection method of food safety strains. Moreover, an advantage in using Bifidobacteria instead of other fecal contamination indicators is the host specificity of Bifidobacterium spp in human or animal (Gavini and Beerens, 1999). Raw milk collected with proper hygiene should not contain Bifidobacteria.  In fact, the presence of Bifidobacteria in raw milk indicates fecal contamination and poor farm hygiene (Beerens et al., 2000; Beerens and Neut, 2005). This bacterium has been recently proposed as a fecal indicator in water (Lynch et al., 2002; Gilpin et al., 2003 and Nebra     et al., 2003) and in meat and raw milk samples (Beerens, 1998; Gavini and Beerens, 1999; Beerens   et al., 2000).

Bifdobacteria have been suggested as among the more promising alternative indicators of faecal pollution. They are present in faeces at levels 10:100 times greater than coliforms and, due to their anaerobic nature, fastidious growth requirements and inability to grow below 20°C, they are unlikely to multiply outside the intestinal tract (Mara and Oragui, 1983).

Since most bifidobacterial strains used in dairy products are common members of the human intestinal microbiota, they may behave as opportunistic pathogens, like other commensal bacteria (Saarela et al., 2002). Some commensal Bifidobacteria have been connected with certain dental infections, pulmonary infections, bacteremia, abscesses and bloodstream infections (Green, 1978; Gasser, 1994; Saarela et al., 2002). In infants aged 2 to 16 months, Brook (1996) described a prevalence of 57 Bifidobacterium isolates among 2033 specimens originating from chronic otitis media, abscesses, peritonitis, aspiration pneumonia and paronychia. In most cases, the non-probiotic B. dentium (formerly   B. eriksonii) was implicated, this is the only bifidobacterial species classified as a dental pathogen. A few cases also implicated B. adolescentis, which has been recovered purefrom otitis media (Brook, 1996). Despite these few cases, the recovery of Bifidobacteria in clinical infections is very rare in comparison to other commensals (Borriello et al., 2003). The most commonly used faecal indicator organisms, faecal coliforms, including E. coli, denote faecal pollution but not whether it is of human or animal origin. As a consequence, alternative indicators of faecal pollution better able to identify the faecal source have been suggested, including Bacteriodes spp., phages of Bacteroides fragilis,       F-RNA phage, Rhodococcus coprophilus, Bifdobacteria, and various chemical indicators (Sinton et al., 1998).

Bifidobacteria, previously known as Bacillus bifidus, are a group of microorganisms that were classified into 29 different species in the 9th Edition of Bergey’s Manual of Systematic Bacteriology (Scardovi, 1986). Nowadays, there are more than 33 species that were identified from the genus Bifidobacterium using recent molecular and biochemical methods of which 12 have been associated with the human gastrointestinal tract (Ventura et al., 2004).

Bifidobacteria are Gram-positive, non-motile, and non-spore-forming bacteria. They are part of normal intestinal microbiota in humans and animals, and are generally non-pathogenic. They were considered to be anaerobic (Simpson et al., 2004a).

Present study has been carried out to evaluate the hygienic quality of raw buffalo's and cow's milk sold in Assiut city based on the presence of Bifidobacterium spp. as indicators of faecal contamination. 

MATERIALS and METHODS

1- Raw milk samples: 70 samples of raw buffalo's and cow's milk (35 samples from each) were collected randomly from; some dairy farms, individual cases of dairy buffaloes and cows and dairy shops in Assiut city. Samples collected directly after milking and transported to the microbiological laboratory for examination. Each sample was kept in a sterile bag containing peptone water (0.1%) to which we added 0.25% L-cysteine.

2- Culture media: Numerous culture-based methods for Bifidobacteria detection, isolation and typing have been described. Culturing of Bifidobacterium and its evolution within gastrointestinal flora (human or animal) was carried out according to Martineau (1999); Rada and Petr (2000) and Petr and Rada (2001). MRS & BHIMup and two different culture media (CMup & Bifidobacterium media (BFM)) were used for isolation and identification of Bifidobacterium spp.

1-  Bifidobacteria isolation:

Two culture-based methods were used to detect Bifidobacteria spp. which comprised enrichment and isolation steps. The first method was done by inoculating 2 g of each sample of raw milk in 18 ml MRS broth as enrichment medium. 500 mg / l of L-cysteine hydrochloride has been added to MRS broth to reduce ox-reduction (ox-re) potential. The inoculated media were incubated anaerobically at 37C° for 48 hrs. In order to obtain single colonies, the cultured mMRS were streaked on the surface of Bifidobacterium media (BFM) and incubated anaerobically at 37C° for 72hrs. 

The second method was carried outaccording toisolation procedures described by Beerens (1998) and culture-based methods modified by Delcenserie et al. (2005). One millileter of each samples was transferred into 9 ml of enrichment medium BHMup [BHI, 37 g/l (oxoid, England), 5 ml/l of propionic acid, 0.5g/l Fe-citrate, 0.5 g/l cystein chlorhydrate, 5 g/l yeast extract and 2 g/l agar]. Mupirocin, 80 mg/lwas added before the useof the medium. The final pH was 5.0 and obtained with the addition of 1 mol/lNaOH solution. Tubes were incubated for 48 hrs at 37°C in Jars with anaerobic conditions. From each enrichment culture, 0.1 ml was spread on isolation medium CMup (Columbia blood agar, 0.5 g/l, Fe-citrate, 5 g/l glucose and 0.5 g/lcystein chlorhydrate). Mupirocin was added (50 mg/l) before the use of the medium. Plates were incubated anaerobically at 37°C for 72 hrs. Samples which have grown on both of the two selective media considered positive while samples which have grown on only one media, have been discarded. Colonies were rounded, blue and approximately 2mm in diameter a few minutes after removal from anaerobic condition. 

2-       Identification of isolates:

a-       Phenotypic characterization: Each strain isolated was cultured on MRS agar (MRS agar to which we added, 0.25% L-cysteine). Inoculated plates were incubated anaerobically at 37°C for 24 to 48h. Isolates observed were stained with methylene blue and examined microscopically (1000 x). Only strains with the “Y” phenotypic form were selected for the identification (Rasic and  Kurman, 1983).

All strains were initially submitted to Gram staining, catalase and spore formation test. Colonies and cells morphology characteristics on TPY containing 0.2% cysteine-HCl, MRS and M17 agar were also examined. Gas production from glucose was determined in MRS broth containing inverted Durham (Grill et al., 2000). Citrate utilization, in the presence of carbohydrates, was performed on the media of Kihal et al. (1996).

b- Carbohydrate fermentation

The carbohydrates fermentation was determined on TPY broth containing bromocresol purple (0.04 g/l) as a pH indicator, and supplemented with 1% of the following carbohydrates: lactose, sucrose, xylose, arabinose, sorbitol, fructose, galactose, mannose, cellobiose, raffinose, melizitose and melibiose. To ensure anaerobic conditions, each tube was supplemented with two drops of sterile liquid paraffin after inoculation (Samelis et al., 1994; Saidi et al., 2002). The results obtained for morphological, physiological and biochemical tests were compared with those in standard texts for identification (Scardovi, 1986; Miyake et al., 1998, Ingrassia et al., 2001) and the isolates assigned to appropriate species. The isolates belonging to the genus Bifidobacterium were identified to species level using sugar fermentation and the profiles were compared with the reference strains in standard texts for identification.

RESULTS

Bifidobacterium spp. was identified in 65.71 % (23samples) and 51.42% (18 samples) of raw buffalo's and cow's milk, respectively (Table, 1). Isolates were identified and findings revealed that raw cow's milk samples harbored B.dentium in 61.11% while B. suis was identified in 27.77% of examined samples(Table, 2). B. bifidum were isolated from 2 samples (11.11%). B. dentium, B. suis and B. bifidum were found contaminating 47.82, 39.13 and 13.04% of examined raw buffalo's milk (Table, 2).

Table 1: Percentage of positive raw Cow’s and Buffalo's milk samples that contain Bifidobacteria spp.

Table 2: Bifidobacteria spp. isolated from the examined samples of raw cow's and buffalo's milk.

DISCUSSION

Presence of B. bifidum revealed that raw cow's and buffalo's milk may be contaminated with feces of the human (adult and infants). However, isolation of      B. suiscould be attributed to pollution with feces of piglets. Also the detection of B. dentium in examined samples could be attributed also to contamination with human discharges coming from; mouth suffering dental caries, human abscesses, vaginal discharges and feces. These organisms considered potentially pathogenic and have hazards effect on human health.

Commensal Bifidobacteria (B. dentium) have been connected with certain dental and other infections (Leo et al., 2008). Lynch et al. (2002), Nebra et al. (2003) and Bonjoch et al. (2004) recorded that         B. adolescentisor B. dentiumare indicators of fecal pollution. As these species are dominant in human feces, they will indicate a contamination of human origin (Scardovi, 1986).

Kouamé-Sina et al. (2011) found that isolates belong to five different species of Bifidobacterium were present in 9% of milk samples. Most of the Bifidobacterium isolated were B. minimum (53%) and B. pseudolongum sub spp. globosum (24.4%). The other strains are composed of one strain of                B. thermophilum, B. thermacidophilum sub spp. Suis and B. magnum.

The typical habitats of Bifidobacteria are human, warm-blooded animal and honeybee intestinal tract (Scardovi, 1986). Members of genus Bifidobacterium are among the most common microorganisms in the human gut, comprising up to 3% of the total faecal microflora of adults (Sghir et al., 2000). They are more numerous in the infant gut, where they form up to 91% of the total microflora in breast-fed babies being supported by bifidogenic factors presented in human milk and up to 75% in formula-fed infants (Harmsen et al., 2000). Using classical culturing methods it has been found that B. adolescentis and B. longumare major bifidobacterial species in theintestine of adults (Biavati et al., 1986; Mutai and Tanaka, 1987; Gavini et al., 2001). B.infantis and B. breveare predominant species in the intestinal tract of human infants (Benno et al., 1984; Biavati et al., 1984; Mutai and Tanaka, 1987). In addition,             B. bifidum, B. catenulatum, B. pseudocatenulatum,   B. angulatum, B. gallicum, and B. dentium have also been reported to be human intestinal bifidobacteria (Scardovi, 1986). Matsuki et al. (1999) who used for the detection of bifidobacteria in human gut species-specific polymerase chain reaction (PCR) reported, that the most common species in the breast-fed infants are B. breve, B. infantis, B.longum, and         B. bifidum. In adult intestinal tracts, the                     B. catenulatum group was the most common taxon, followed by B. longum and B. adolescentis.

In the case of raw milk samples, an enrichment step was necessary because of the possible relatively low levels of bifidobacteria (10 to 10⁶/ ml -1) compared to those in human or animal feces (10⁷ to 10¹⁰/ g-1). Beerens (1998) recommended the using at the enrichment step the BHI medium with addition of propionic acid, yeast extract, iron citrate, and at the isolation step, paromomycin as selective agent. However, the high number of lactobacilli that were not inhibited by paromomycin hides bifidobacteria at low dilutions. Rada and Petr (2000) showed that bifidobacteria were resistant to mupirocin when lactobacilli were susceptible. Mupirocin (pseudo-monic acid A) was originally isolated from Pseudo-monasfluorescens and used as a topical antibiotic (Sutherland et al., 1985). In raw milk samples, addition of mupirocin at the enrichment step can eliminate most of the lactobacilli strains present that could hide Bifidobacteria in raw milk. If some lactobacilli strains were still present after the enrichment step, one might suppose that they would be eliminated during the isolation step by mupirocin, when present. Grand et al. (2003) also used mupirocin as selective agent for detection of Bifidobacteria in probiotic milk products, as did Mikkelsen et al. (2003) in gastrointestinal samples from piglets and Simpson et al. (2004b) in probiotic as animal feed.

Moreover, Rhodes and Kator (1999) enumerated sorbitol-fermenting Bifidobacteria to define human fecal pollution in estuarine watersheds. However, in raw milk cheese, the principal contamination was shown to be of animal origin (Beerens et al., 2000), most likely by cow dung onfarm, since the same species, B. pseudolongum, was isolated from both kinds of samples. Therefore, in food industries, it seems important to define the human or animal origin of the contamination.

The milk contamination by Bifidobacteria is also an indicator of fecal contamination (Beerens et al., 2000) and is due to the lack of hygiene in farms. Its presence on cow’s udder reflects environmental contamination with feces of other animals living in farms or in the vicinity. Our results indicated insignificant variation in the prevalence of Bifidobacterium along the dairy production chain. Contamination by Bifidobacterium was present at all stages of milk production. These results indicated fecal contamination at all stages of the production chain, from production to selling point of raw milk. This suggests a lack of sanitary education of farmers and milk vendors. This sanitary education of farmers is important especially on good hygiene practices through simple actions like washing and disinfecting of hands, cow’s udders and milking utensils (Bonfoh et al., 2003).

Finally it could be concluded that the hygienic quality of local raw milk was assessed as poor, based on the detection of the Bifidobaterium in raw milk (Therefore the Bifidobaterium isolated in this study are rather an indication of poor hygiene). In order to improve the microbiological quality of local raw milk, fermentation as well as heating is needed to inhibit the growth of contamination germs. The safety strategy should be developed, with good hygiene practices related to cleanliness of animals and their environment as well as sanitation of the milking process (milker’s hands, milking utensils).

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