Dept. of Food Hygiene,

Fac. of Vet. Med., AssiutUniv. 

MICROBIOLOGICAL EVALUATION OF SOME INFANTS POWDERED MILK-BASED FOODS

(With 7 Tables)

By

Ahmed A-H. Ahmed; Enas El-Prince; Eman Korashy* and Marwa M.N. Al-Gendi*

* Assiut Animal Research Institute

(Received at 15/5/2008)

التقييم الميکروبيولوجي لبعض توليفات أغذية الأطفال الجافة

المحتوية علي اللبن

  أحمد عبد الحميد ، إيناس البرنس ، إيمان قرشي ، مروة الجندي

يتزايد الإقبال علي استعمال ألبان الأطفال الصناعية کبدائل للبن الأم نتيجة لعدم قدرة بعضهن علي اشباع أطفـالهن أو لظروف مرضية، لذلک کان لابد من البحث عن بدائل مناسبة للأعمار المختلفة تتوافر بها الشروط الصحية لضمان سلامة الأطفال. وتعد کل من الألبان الجافة والأغذية الجافة المحتوية علي اللبن من الأغذية واسعة التدوال لدي الصغار والکبـار، وتلوثها بالميکروبات الضارة من الأمور التي تستوجب الاهتمام. لذلک تضمنت هذه الدراسة فحص عدد 250 عينة عشوائية من أغذية الأطفال اللبنية الجافة بواقع 70 عينة من لبن البودرة للأطفال حديثي الولادة، 90 عينة من کل من أغذية الفطام الجافة المحتوية علي خلاصة الحبوب واللبن الجاف تم تجميعها من العديد من المحال التجارية والصيدليات في مدينة وقري أسيوط لمعرفة مدي تلوثها بالميکروبات المختلفة، وقد کانت صالحة للاستهلاک حيث تمتد فترة صلاحيتها لمدة لا تقل عن عام من تاريخ الإنتاج.  وقد دلت النتائج علي أن متوسط العدد الکلي للميکروبات الهوائية، المحبة للبرودة، الباسيلس سيريس و الخمائر والفطريات  في هذه العينات کان x 9.210 ، 6.1x10 و1.3 x ²10 ;  2.9 10x² ، x2.910 ³ و 2.8 x 10 ² ; 0.3 x 10 , 0.56 x 10, 7.2 x 10; 3 x 10، 8  x 10و 5.1 x 10/ جرام علي التوالي. من ناحية أخري فقد تم عزل ميکروبات  B.cereus,Enterococci  وanaerobs من العينات تحت الدراسة بنسب متفاوتة، بينما لم تتواجد ميکروبات coliforms، fecal coliforms وE.  coli نهائيا باستخدام طريقة MPN. بالإضافة إلى ذلک فقد أمکن عزل Enterobacteriaceae من العينات المفحوصة، بالنسبة لألبان الأطفال حديثي الولادة فقد تم عزل ميکروبات  Ent. cloaca، Serratia marcescens و K. oxytoca بنسب 24.9، 42.9 و 14.2 % علي التوالي. أما ميکروبات E.cloaca، Ent. sakazakii ، Serratia marcescens ، Serratia liquefaciens ،  Ent. aerogenes،  K. oxytoca،  Citrobacter freundii،  Hafnia alvei، spp. Proteus ، Salmonella spp. و Chryseomonas luteola فقد وجدت في أغذية الأطفال الجافة المحتوية علي اللبن بنسب  32.5، 15، 2.5، 5، 2.5، 10، 2.5، 12.5، 12.5، 2.5 و 2.5 % من العينات المفحوصة علي الترتيب. من ناحية أخري تبين أن 42.9، 7.1، 7.1، 21.4، 14.3 و 7.1 % من عينات اللبن الجاف کانت ملوثة بالميکروبات التالية علي التوالي، Ent. cloaca،   Ent. sakazakii، Serratia marcescens، Hafnia alvei، Yersinia pestis وYersinia pseudotuberculosis. ومن هذه النتائج أتضح أن أغذية الأطفال المحتوية علي اللبن کانت الأکثر تلوثا بميکروبات Enterobacteriaceae. وقد تم مناقشة الشروط الصحية الواجب اتخاذها لإنتاج ألبان وأغذية أطفال ذات جودة عالية وخالية من الميکروبات الممرضة.

Summary

A total of two hundred and fifty random samples of infants milk formulae (IMF) for babies after birth (70 samples), milk-based cereal weaning food and dried milk powder (90 samples each) were purchased from different shops and pharmacies in Assiut city and villages around the city. These samples were transferred to the laboratory in their packages to be examined microbiologically to evaluate their quality. The average values of aerobic plate count (APC), psychrotrophic, B. cereus and total yeasts and molds counts were 9.2x10, 6.1x10 and 1.3x10²; 2.9x10²,2.9x10³ and 2.8x10²; 0.3x10, 0.56x10 and 7.2x10 and 3.0x10, 8.0x10 and 5.1x10 cfu/g of examined samples, respectively. Moreover, B. cereus, enterococci and anaerobes could be isolated in various percentages from the examined samples. Furthermore, Ent. cloaca, Serratia marcescens and Klebsiella oxytoca were isolated from IMF in percentages of 42.9, 42.9 and 14.2%, respectively. Concerning milk-based cereal baby food, Ent. cloaca, Ent. sakazakii; Serratia marcescens; Serratia liquefaciens; Ent. aerogenes; K. oxytoca; Citrobacter freundii; Hafnia alvei; Proteus spp.; Salmonella spp. and Chryseomonas luteola were found in 13, 6, 1, 2, 1, 4, 1, 5, 5, 1, 1 and 1 of tested samples, respectively. While, Ent. cloaca; Ent. sakazakii; Serratia marcescens; Hafnia alvei; Y. pestis and Y. pseudotuberculosis were existed in dried milk powder samples in  incidences of 42.9, 7.1, 7.1, 21.4, 14.3 and 7.1 %, respectively. Recommendations were suggested to safeguard the existence of such microorganisms in infants' milk food and to avoid their undesirable changes resulted in economic losses as well as public health hazards.

Key words: Microbiological evaluation, Infants milk powder, Milk-based cereal weaning food, Dried milk powder.

Introduction

In recent years, there has been a growing use of IMF and baby foods with dairy base as replacers of fresh milk or in addition to it. Milk-based cereal weaning food constitutes a major category, which are fed to babies above 3 months old. Uses of IMF have been decreasing in industrial countries for over forty years as a result of antenatal education, increased understanding of their risks, and social activism. Most major medical and health organizations strongly advocate breastfeeding over their use except in unusual circumstances (WHO, 2001). No other breast milk substitute is as safe as commercial IMF, when produced according to International Standards. Because IMFis not a sterileproduct, it is anexcellent medium to supportbacterial growth. Bovine milkis an essential ingredientof IMF and apotential source of bacteriathat are pathogenic tohumans (Breeuwer et al., 2003). All available data indicate the increased infection risk arising from multiplication of potentially pathogenic bacteria in reconstituted formula kept at room or warmer temperatures for prolonged periods of time (Codex Committee on Food Hygiene, 2004) primarily in hospital neonatal intensive care units (ICU). 

Nowadays, the growing use of baby foods has made its microbial quality of primary concern, due to the high susceptibility of children to food-borne diseases. Enterotoxigenic Staph. aureus, Enterococci, Proteus spp. and Clostridial organisms were isolated from baby foods by Becker et al. (l984) and Saudi et al. (1984). Enterobacter spp. were reported as being the fifth and third most common among those recovered from the urinary and respiratory tracts (as nosocomially acquired infections), respectively, of patients in ICU (Jarvis and Martone, 1992 and Borderon et al., 1996). Also, other organisms, as E. coli, Ent. agglomerans, Ent. cloacae, Ent. sakazakii, K. pneumoniae, K. oxytoca, and Citrobacter freundii, were detected in powdered IMF (Iversen et al., 2004). Moreover, dried milk products are known to be frequently contaminated with B. cereus, principally with its spores. Viable spores may germinate and the vegetative cells can proliferate and produce toxin; which could potentially even occur at refrigeration temperatures (Becker et al., 1994 and Jaquette and Beuchat, 1998). Furthermore, enterococci existence in samples indicated fecal contamination and unsanitary conditions during handling and production. So, the public health significance can not be denied, specially, when the organisms found in a tremendous number in the product as they have been implicated in several food poisoning outbreaks (ICMSF, 1978). Food-borne diseases also may occur in infants as a result of anaerobes (Bouer-Hertzberger, 1982) or yeasts and molds contaminated products which is inactive of unhygienic production.

Enterobacteriaceae are common in food processing environment and their numbers may change as a result of novel contamination, changes in sanitation measures and conditions of growth but they remain prominent and it is hardly possibly to eliminate them (Cox et al., 1988). In heat-processed foods as well as in ready-to-eat foods, the presence of species of this family should have public health significance (Iversen and Forsythe, 2004). Due to the use of baby foods as substitute of/or with mother milk so, the evaluation of their microbial quality is of great concern.

Materials and Methods

Collection and preparation of samples:

A total of two hundred and fifty random samples of IMF (70 samples), milk-based cereal weaning food and dried milk powder (90 samples each) were collected from different shops and pharmacies in Assiut city and villages around the city. These samples were still valid for consumption as their shelf life is at least to be more than one year from production time and were transferred to the laboratory in their packages to be examined microbiologically to evaluate their quality. Cartons and cans of samples were cleaned, thoroughly mixed and aseptically opened. 11 g of the prepared samples were mixed with 99 ml of sterile 0.1 % peptone water and thoroughly mixed to give a dilution of 1/10 and then ten fold serial dilutions were prepared (A.P.H.A., 1992).

Experimental techniques included:

1) Aerobic plate count (APC) using Standard plate count agar (A.P.H.A., 1992).

2) Psychrotrophic count using Crystal Violet Tetrazolium agar medium (Gilliland et al., 1976).

3) Enumeration and isolation of B. cereus using Brain-Heart infusion broth and KG agar (Kim and Goepfert, 1971).

4) Total yeasts and molds count using malt extract agar (containing 500 mg each of chlortetracycline and HCL chloramphenicol) (Harrigan and MacCance, 1976).

5) Enterococci count using KF streptococcal agar (Deibel and Hartman, 1982) and isolation using KF broth and KF agar (Morrifon et al., 1997).

6) Detection of anaerobic spore formers: “Stormy fermentation test” (Crückshank et al., 1969).

7) Total coliforms, fecal coliforms and Escherichia coli count using MPN/ ml (A.O.A.C., 1975).

8) Isolation and identification of Enterobacteriaceae (FDA, 2002) using violet red bile agar (VRBL). Isolates were identified using biochemical tests including Triple Sugar Iron (TSI), Urease test, Sugar fermentation tests, IMViC tests, catalase test then oxidase test.

Results

The obtained results were recorded in Tables 1- 7.

Table 1: Statistical analytical results of aerobic plate count in the examined samples of powdered milk-based foods.

* Colonies could not be detected on the plates.

Table 2: Statistical analytical results of psychrotrophic count in the examined samples of powdered milk-based foods.

* Colonies could not be detected on the plates.

Table 3: Statistical analytical results of Bacillus cereus count in the examined samples of powdered milk-based foods.

* Colonies could not be detected on the plates.

Table 4: Statistical analytical results of total yeasts and molds count in the examined samples of powdered milk-based foods.

* Colonies could not be detected on the plates.

 Table 5: Incidence of different microorganisms in the examined  samples of powdered milk-based foods.

                                  No.^* Number of positive samples

* Colonies could not be detected on the plates, but could be isolated (<100 / g)

Table 6: Incidence of Enterobacteriaceae in the examined samples of powdered milk-based foods.

 Table 7: Frequency distribution of Enterobacteriaceae isolates recovered from positive samples of powdered milk-based foods

Discussion

The results presented in Table 1 pinpoint that the average values of APC/g were 9.2x10; 6.1x10 and 1.3x10² of examined IMF, milk-based cereal weaning food and dried milk powder samples, respectively. In case of IMF, relatively similar findings were obtained by Jarchovská et al. (1980); Saudi et al. (1984); Bhatt et al. (1992); Al-Ashmawy et al. (1993); El-Shinawy et al. (1995) and El-Prince and Korashy (2003). However, lower count was estimated by Schwab et al. (1982) (52/g), while Moustafa et al. (1984) and Sabreen (1986) recorded higher counts. Carneiro et al. (2003) detected unacceptable colony counts for the majority of the IMF samples and the contamination rates were related to inadequate handling. According to the limits proposed by A.P.H.A. (1992), Egyptian Standards (2001) and U. S. Dairy Exports Council (1996-2002) of dried milks that, APC must not exceed 5 x 10⁴/g, therefore, all of examined samples are considered satisfactory. Moustafa et al. (1984); Sabreen (1986); El-Prince and Korashy (2003) and Sayed (2004) recorded higher counts of APC in milk-based cereal baby foods and according to the standards of ICMSF (1974), it is evident that all examined baby food samples were within the range of accepted quality (5 x10⁴- 5 x 10⁵ APC/g). Higher counts were recorded by El-Prince and Korashy (2003) in milk powder. Many authors recommended the APC as an index of hygienic measures, organoleptic quality, safety and utility of infant foods.

The psychrotrophic count ranged from <100 to 1.2x10⁴ with an average of 2.9x10²/ g of IMF samples which are higher than that showed by El-Prince and Korashy (2003). In case of milk-based cereal weaning food, the counts ranged from <100 to 7.8x10³ with an average count of 2.9x10³ cfu/g. Higher count was detected by Sayed (2004) however, psychrotrophs were not demonstrated by El-Prince and Korashy (2003).While, in dried milk powder the count lie in between <100 to 1x10⁴ and an average of 2.8x10² cfu/g (El-Prince and Korashy, 2003 detected lower finding) (Table 2). In the absence of psychrotrophs or presence of large thermodurics, certain thermoduric psychrotrophs can grow and induce spoilage of the product (Richter et al., 1992 and Meer et al., 1993).

The average counts of B. cereus in the concerning samples were 0.3x10; 0.56x10 and 7.2x10 cfu/g, respectively as shown in Table 3. Moreover, they could be isolated in incidences of 54.3; 18.9 and 38.9 % from the examined samples, respectively (Table 5). Variant counts and percentages were demonstrated by El-Prince and Korashy (2003). Dried milk products, such as milk powder, milk substitute and IMF, contaminated with B. cereus, even at low levels should be considered as potential vehicles for food-borne B. cereus disease. As these products contain an elevated level of carbohydrates (starch, sucrose or lactose) and minerals, they can promote proliferation and enterotoxin production when they are reconstituted and held at ambient temperature for extended periods, potentially even at refrigeration temperature (Jaquette and Beuchat, 1998).

Regarding total yeasts and molds count (Table 4), the results revealed that 77.1; 60 and 83.3% of the examined samples were contaminated in average counts of 3.0x10; 8.0x10 and 5.1x10 cfu/g. Nearly similar incidence was obtained by Jesenská and Hardinová (1981). Sabreen (1986); El-Shinawy et al. (1995) and El-Prince and Korashy (2003) recorded higher counts in samples of IMF. It is evident that most of positive samples did not comply with the Egyptian Standards (2001) where yeasts and molds must not exceed 10 /g. The obtained finding of milk-based cereal baby food was coincident with that reported by Bhatt et al. (1992) (66.67%). However, higher incidences were postulated by Moustafa et al. (1984) (93.33 %) and Aboul-Khier et al. (1985) while, lower percentages were detected by El-Prince and Korashy (2003) and Sayed (2004). The percentage of yeasts and molds in milk powder agree to a certain extent with that estimated by Bhatt et al. (1992) and Ismail and Saad (1995). While, higher counts and lower percentages (60 %) were detected by El-Prince and Korashy (2003). Yeasts and molds may grow over a wide range of temperature and gain entrance to milk powder either from the milk used, air contamination or utensils. So, their presence is indicative of unsatisfactory sanitation during processing and handling of the product. The high level of these microorganisms may be due to post heat treatment contamination.

The incidences of enterococci were 20, 1.1 and 13.3 % and their numbers were less than 10²/g (could not be detected on the plates) in all the positive examined samples, respectively (Table 5). However, the prevalence of enterococci in dried milk products has been reported by various investigators as Jarchovská et al. (1980); El-Bassiony and Aboul-Khier (1983); Saudi et al. (1984); Sabreen (1986) and Sayed (2004). The prevalence of enterococci in dairy products has long been considered as a result of unhygienic conditions during their production and processing. However, their presence has often been shown to be unrelated with direct faecal contamination (Franz et al., 1999 and Gelsomino et al., 2001).

About the anaerobes (Table 5), they were detected in a percentage of 44.3% for IMF (lower incidences were detected by Sabreen, 1986 and El-Prince and Korashy, 2003); 54.4% for milk-based cereal baby food (Sabreen, 1986; El-Prince and Korashy, 2003 and Sayed 2004 detected higher counts) and 38.9 % for dried milk powder. It is worth to mention that, the probability of food-borne illness may occur to children due to consumption of contaminated products with anaerobes which is indicative of careless methods of production (Bouer-Hertzberger, 1982).

In addition, coliforms, fecal coliforms and E. coli failed to be detected. Similar findings were obtained by Jarchovská et al. (1980); El-Shinawy et al. (1995) and El-Prince and Korashy (2003), while Sayed (2004) could identify only one contaminated sample (3.3%) in a level of 7.3 MPN/ g. On the other hand, Schwab et al. (1982); Moustafa et al. (1984); Saudi et al. (1984); Sabreen (1986); Bhatt et al. (1992); Al-Ashmawy et al. (1993) and Carneiro et al. (2003) detected coliforms in most of tested IMF. Our results were in accordance with A.P.H.A. (1992); Egyptian Standards (2001) and U.S. Dairy Exports Council (1996-2002) that coliforms must be less than 10/g and E. coli was absent. The absence of coliforms with failure to detect fecal coliforms and E. coli can be considered as an index of satisfactory sanitation.

Furthermore, Enterobacteriaceae existed in the examined samples at percentages of 10, 44.4 and 15.6 %, respectively (Table 6). Ent. cloaca, Serratia marcescens and Klebsiella oxytoca were isolated from IMF in percentages of 42.9, 42.9 and 14.2%, respectively (Table 7). Higher result was recorded by Saudi et al. (1984) who detected Enterobacteriaceae in 52.5% of IMF. Carneiro et al. (2003) identified K. pneumoniae, Citrobacter freundii, Cedacea davisae, K. planticola and Ent. cloacae. While, Iversen and Forsythe (2004); Estuningsih et al. (2006); Shaker et al. (2007) and Townsend et al. (2007) succeeded to isolate Enterobacteriaceae from IMF. Concerning milk-based cereal baby food, Ent. cloaca, Ent. sakazakii; Serratia marcescens; Serratia liquefaciens; Ent. aerogenes; K. oxytoca; Citrobacter freundii; Hafnia alvei; Proteus spp.; Salmonella spp. and Chryseomonas luteola were found in 13, 6, 1, 2, 1, 4, 1, 5, 5, 1, 1 and 1 of the examined samples, respectively (Table 7). Iversen and Forsythe (2004) and El-Prince et al. (2007) could distinguish 14 and 26 isolates related to family Enterobacteriaceae, respectively; however, higher incidences were obtained by Sabreen (1986). Moreover, Ent. cloaca; Ent.sakazakii; Serratia marcescens; Hafnia alvei; Y. pestis and Y. pseudotuberculosis were demonstrated in dried milk powder samples in incidences of 42.9, 7.1, 7.1, 21.4, 14.3 and 7.1 %, respectively. Iversen and Forsythe (2004) isolated 36 strains of Enterobacteriaceae from examined 72 samples of milk powder, however, El-Prince et al. (2007) could not isolate any strain of this family.

The above achieved results declared that IMF, milk-based cereal baby food and dried milk powder are liable to contamination by some pathogenic microorganisms constitute public health hazard. Therefore, sailing of these products should be controlled with health authorities to eliminate potentially occurring hazards arising from microbial pollution.

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