Assiut University web-site: www.aun.edu.eg

MICROBIOLOGICAL EVALUATION OF SOME DRIED DAIRY DRINKS SOLD IN ASSIUT CITY

MARWA M.N. EL-GENDI and ZEINAB M. ABDEL-HAMEED

Animal Health Research Institute (Assiut Provincial Lab.) Food Hygiene Department

Received:  28 July 2016;       Accepted: 4 August  2016

INTRODUCTION

Nowadays, there are many dried dairy drinks that consumed as hot drinks in Egypt as salep, hot chocolate and coffee milk powder. The product salep is pronounced in Arabic dialect as Sahlab.

In the recent years, hot chocolate and coffee milk powder are generally prepared for immediate consumption and not stored in sealed bottles or cans. These drinks are consumed at home or through takeaway beverage shops, however, these drinks may have variable microbiological quality. Unhygienic handling of these ingredients may result in contamination and improper storage which allow microorganisms to multiply to a high level. Hot-served drinks are generally prepared with boiling hot water where microbiological hazards could be reduced by the high temperature; also, these drinks are more likely to be prepared upon ordering.

Salep was a popular beverage in the lands of the Ottoman Empire. Its consumption spread beyond

Corresponding author: Dr. ZEINAB M. ABDEL-HAMEED

E-mail address: Zenb@dr.com; moazahmednofel@yahoo.com

Present address: Animal Health Research Institute (Assiut Provincial Lab.) Food Hygiene Department

there to England and Germany before the rise of coffee and tea and it was later offered as an alternative beverage in coffee houses. In England, the drink was known as saloop, popular in the 17^th and 18^th centuries, its preparation required that the salep powder be added to water until thickened whereupon it would be sweetened, then flavored with orange flower or rose water. Substitution of British orchid roots, known as "dogstones", was acceptable in the 18^th century for the original Turkish variants (Davidson, 1987). 

Hot chocolate, also known as hot cocoa, is a heated beverage consisting of shaved chocolate, melted chocolate or cocoa powder, heated milk or water, and often sugarand considered a comfort food and is widely consumed in many parts of the world like USA (Sciscenti, 2015). Research has shown that the consumption of hot chocolate can be positive to one's health. A study conducted by Cornell University has shown that hot chocolate contains more antioxidants than wine and tea, therefore reducing the risk of heart disease (American ChemicalSociety, 2003).

Food safety control aims to safeguard public health and provide assurance on food safety. Microbiological analyses are useful ways to assess the safety and quality of food involved. Instant drinks are convenience products that require very little effort to reconstitute prior to consumption. Salep, ground cocoa based beverages (hot chocolate) and coffees milk powder are classified as instant foods that contain milk powder simply mixed with sugar or not. They are sources of high calorie intake and mineral nutrition (Shittu and Lawal, 2007).

Psychrotrophic bacteria are becoming increasingly dangerous to the dairy industry because they produce extracellular heat – resistant lipases and proteases (Sorhaug and Stepaniak, 1997). Moreover, the thermophilic bacilli could not only cause spoilage due to their production of acids and the rmostable enzyme excretion, but also cause food borne illness (Burgess et al., 2010).

Yeasts themselves are not commonly the cause of defect in dairy products unless they ferment lactose. In this case, they can grow rapidly and produce a characteristic yeasty or fruity flavor and obvious gas.. The presence of wild types of molds is undesirable as they may influence the organoleptic characteristics of the dairy products, they can produce mycotoxins and represent a potential health risk (Wouters et al., 2002). Spoilage of dairy products by molds is frequent and a matter of concern for human health.

Bacillus cereus is a common contaminant frequently isolated from foods and dairy products and the fact that the bacterium has a remarkable ability to survive different environmental stresses makes it difficult to be removed from dairy products. The spores survive pasteurization and transfer from milk to pasteurized milk products. The psychrotrophic strains often limit the keeping quality of pasteurized milk and its products (Giffel et al., 1996). Cases of B. cereus food poisoning in milk-based products have been reported (Meer et al., 1991; Andersson et al., 1995; Schoeni and Wang, 2005) and as many as 69-85% psychrotrophic B. cereus isolates from milk and milk products have been found to be enterotoxigenic (Griffiths, 1990). B. cereus produces two types of toxins – emetic (vomiting) and diarrhoeal – causing two types of illness. The emetic syndrome is caused by emetic toxin produced by the bacteria during the growth phase in the food. The diarrhoeal syndrome is caused by diarrhoeal toxins produced during growth of the bacteria in the small intestine (Ehling-Schulz et al., 2006). B. cereus is responsible for several outbreaks of foodborne diseases due to its emetic toxin and enterotoxin. Enterotoxins, cytotoxin K (CytK), nonhemolytic enterotoxin (Nhe) and hemolysin BL (Hbl), have been recorded in several diarrheal cases due to food poisoning from B. cereus (Zhang et al., 2016).

One of the objectives of this study was to use multiplex PCR to detect enterotoxin-producing B. cereus in some dried dairy drinks. The specificity of primers of two enterotoxins; cytK and hblC of B. cereus was verified by inclusivity and exclusivity tests using single PCR.

The aim of the present study was to investigate the microbiological quality of some dried dairy drinks that consumed hot with special focus on the incidence of enterotoxigenic B. cereus in the examined products.

MATERIAL AND METHODS

A) Sampling:

A total of 75 random samples of salep, hot chocolate and coffee milk powder (25 each) were purchased from different dairy shops and supermarkets in Assiut city, Egypt; in the period from May to June 2015. All the samples were still valid for consumption. Cartons and packets of the samples were cleaned, thoroughly mixed and aseptically opened. Tenfold serial dilution was carried out according to A.P.H.A. (1992). Microbiological examination was done for the samples before preparation (powdered form) and after preparation (reconstituted form). Preparation of the samples was proceed according to the manufacturing methods by using sterile distilled water.

B) Total psychrotrophic count: was done according to Gilliland et al. (1976).

C) Enumeration of total thermophiles:

One ml of the sample and its decimal dilutions was plated using milk plate count agar (MPCA) and incubated at 55^ᵒ C for 48 h as described by Frank and Yousef (2004).

D) Enumeration of total yeasts and molds:was done according to Harrigan and MacCance (1976) by using malt extract agar (containing 500 mg each of chlortetracycline and HCL chloramphenicol).

E) Total Bacillus spp. count:

One milliliter from each dilution was mixed with molten (45^ᵒ C) mannitol–egg yolk–polymyxin (MYP) agar (FDA, 1998) and incubated at 37^ᵒ C for 24 to 48 h. The colonies that appeared were counted and identified.

F) Isolation of B. cereus: was done according to Varadaraj (1993)

B. cereus was isolated using surface plating technique on polymyxin-pyruvate-eggyolk-mannitol-bromothymol blue agar (PEMBA) (Oxoid). B. cereus was appeared as peacock blue coloured colonies surrounded by a zone of precipitation of egg yolk. The isolates of typical colonies were picked for further identification.

G) Identification of B. cereus isolates:

The isolated   organisms   were   identified   microscopically   and biochemically according to Koneman et al. (1992):

1. Microscopical identification:

1.1. Staining

1.2. Motility test

2. Biochemical identification:

2.1. Catalase test

2.2. Egg yolk lecithinase

2.3. Nitrate reduction test

2.4. Modified VogusProskauer test

2.5. Citrate utilization test

2.6. Gelatin hydrolysis

2.7. Starch hydrolysis

2.8. Indole production test

2.9. Sugar fermentation

3. Serological identification: was done according  to TeGiffel et al. (2000)

H) Polymerase Chain Reaction (PCR) technique:

1.1. Material used for PCR for detection of B. cereus enterotoxins genes:

Protocol applied forDNA Extraction using QIAampkit was done according to Shah et al. (2009).

1.2. Primer sequences of B. cereus used for PCR identification system: Application of PCR for identification of heamolysin BL (hblC) and cytotoxic K (cytK) genes of B. cereus was performed essentially by using Primers (Pharmacia Biotech) as shown in the following table:

1.3. Amplification reaction of B. cereus (Ngamwongsatit et al., 2008):

The amplification was performed on a Thermal Cycler (Master cycler, Eppendorf, Hamburg, Germany). The multiplex PCR amplification was performed in a final volume of 20 ul containing 5 μl of DNA templates with final concentration 1X PCR buffer (10mM Tris-HCl pH 8.3 and 50 mMKCl), 1.5mM MgCl2, 200 μM of each dNTP, 5U Taq DNA polymerase and 0.4μMhlbC primer and 0.2 μMcytK primer. Reactions were carried out with the following cycling conditions: initial denaturation at 95°C for 5 min, followed by 30 cycles of 94°C for 45 sec, annealing at 54°C and 56°C for 1 min in case of hblC and at 58°C in case of cytK, elongation at 72°C for 2 min and final extension at 72°C for 5 min. Amplified products were analyzed by 1.5% of agarose gel electrophoresis (Applichem, Germany, GmbH) in 1x TBE buffer stained with ethidium bromide and captured as well as visualized on UV transilluminator. A 100 bp plus DNA Ladder (Qiagen, Germany,GmbH) was used to determine the fragment sizes.

RESULTS

Table 1: Statistical analytical result of total psychrotrophic count of the examined samples (n=25 of each).

*˂ 10 means no colonies were detected in the plates.

Table 2: Statistical analytical results of total the rmophilic count of the examined samples (n=25 of each).

*˂ 10 means no colonies were detected in the plates.

Table 3: Statistical analytical results of total yeasts and molds count of the examined samples (n=25 of each).

*˂ 10 means no colonies were detected in the plates.

Table 4: Statistical analytical results of total Bacillus count of the examined samples: (n=25 of each).

*˂ 10 means no colonies were detected in the plates.

Table 5: Incidence of Bacillus spp. isolated from the examined samples: (n=25).

Figure 1a: Agarose gel electrophoresis of multiplex PCR of hblC (695bp) and cytK (565 bp) virulent genes for characterization of B. cereus.

Lane M: 100 bp ladder as molecular size DNA marker.

Lane 1: Control positive B. cereus for hblC and cytK genes.                   

Lane 2: Control negative.

Lanes 3, 7, 10, 12 & 13: Positive B. cereus strains for hblC gene. 

Lanes 11 & 15: Positive B. cereus strains for cytK gene.

Lanes 4, 5, 6, 8, 9, 14, 16 & 17: Positive B. cereus strains for both hblC and cytK genes.

Figure 2 b: Agarose gel electrophoresis of multiplex PCR of hblC (695bp) and cytK (565 bp) virulent genes for characterization of B. cereus.

Lane M: 100 bp ladder as molecular size DNA marker.

Lanes 18, 19, 21, 23, 24, 26 & 29: Positive B. cereus strains for hblC gene. 

Lanes 20, 27 & 28: Positive B. cereus strains for cytK gene.

Lanes 22, 25 & 30: Positive B. cereus strains for both hblC and cytK genes.

DISCUSSION

The microbiological results of total psychrotrophic count of the tested dried dairy drinks are presented in Table 1.Out of 25 samples of salep, 8 samples in a percentage of 32% were positive for counting of total psychrotrophes in the powdered form. The maximum, minimum and average counts were 7x10³, <10 and 1.2x10³cfu/g, respectively. While, 5.8x10⁴,<10 and 1.25x10⁴  were the maximum, minimum and average counts of the salep samples as the reconstituted form, respectively. The number of positive samples increased after reconstitution of the salep samples to become 13 samples in a percentage of 52%.  9x10³ and 1.4x10⁵ were the maximum counts of the hot chocolate examined samples of the powdered and reconstituted forms, respectively in percentages of 4 and 36% as shown in Table 1.4 and 8% of the examined samples of coffee milk powder were positive for total psychrotrophic count in the powdered and reconstituted forms, respectively with maximum numbers of 2x10³ and 1.3x10⁵cfu/g.

The total the rmophilic counts ranged from 2.6x10⁴ to <10cfu/g with an average 2.68x10³ and ranged from 6x10³ to <10cfu/g with an average 6.8x10² for the powdered and reconstituted forms of the salep samples, respectively (Table 2).

On other hand, only one positive sample of the hot chocolate has the rmophilic bacteria in a percentage of 4% for both powdered and reconstituted forms; 40 cfu/g was the average count of both powdered and reconstituted forms of the hot chocolate samples with a maximum count of 1x10³cfu/g for both. The results of coffee milk powder samples for total the rmophillic count could be detected only in the powdered form in one sample (4%) with a count ranged from 1x10³ to˂10cfu/g with an average count of 40 cfu/g.

Table 3 revealed that the average count of total yeasts and molds of the powdered form of  the salep samples was 1.72X10³cfu/g with counts ranged from 1X10⁴ to <10cfu/g , while in the reconstituted form, the average count was 9.6X10² and the counts ranged from 7X10³ to <10cfu/g.12 and 32% were the percentages of the positive examined samples of the powdered and reconstituted forms of the hot chocolate, respectively, with the maximum counts 2X10³ and 1X10⁴ cfu/g and the average counts 1.6X10² and 9.2X10²cfu/g. The maximum, minimum and average counts  of the powdered and reconstituted forms of coffee milk powder samples were 1.1X10⁴&2.1X10⁴ and <10&˂10 and  1.01X10³ &2.6X10³cfu/g, respectively.

The results documented in Table  4 showed that the average counts of total Bacillus count of the powdered form of the examined samples of salep, hot chocolate and coffee milk powder were 4x10², 3.2x10² and 40 cfu/g, with maximum and minimum counts ranged from 5x10³ to<10,2x10³ to <10 and 1x10³ to <10cfu/g, respectively. The percentages of the positive samples of the reconstituted form of the same products respectively were 28, 40 and 8% where the maximum counts of the salep, hot chocolate and coffee milk powder samples recorded 1.8x10⁴, 1.8x10⁴ and 4x10³cfu/g, respectively.

The manufacture of salep is based on traditional method without any regard to the quality of raw material used and/ or the hygienic quality of the products. Under such conditions, many microorganisms can find access to the milk products (Soomro et al., 2002) leading to a low shelf life of the base products. Most of these products are sold in the market without proper packaging and unduly exposing them to atmospheric contamination and has become an important public health issue and a great concern to everybody. In developing countries, fruit juices, drinks, meals and snacks sold by street-food vendors are widely consumed by millions of people (Tambekar et al., 2011).

From Table 5, B. megaterium, B. subtilis, B. circulans, B. licheniformis and B. maceranswere detected in percentages 8, 8, 4, 8 and 4% in the examined samples of salep in the powdered form while in the reconstituted form, B. subtilis, B. licheniformis, B. sphaericus, B. coagglans, B. stearothermophilus and B. polymyxawere isolated and identified in percentages of 4, 4, 4, 8, 4 and 4%, respectively. Other Bacillus spp. could be found in the examined samples of hot chocolate in the powdered form as B. megaterium, B. circulans, B. licheniformis, B. macerans and B. stearothermophilus in percentages of 8, 4, 4, 4and 4%, respectively. After preparation of the hot chocolate samples according to manufacturing procedures, B. megaterium, B. subtilis, B. circulans B. coagglans and B. stearothermophilus were detected in 2, 3, 1, 1 and 1 of the examined samples, respectively. B. licheniformis, B. sphaericus and B. coagglans were isolated from the powdered form of coffee milk powder samples in percentages of 4, 4 and 4%, respectively. B. megaterium, B. subtilis, B. licheniformis, B. sphaericus, B. stearothermophilus and B. polymyxa were present in the examined samples of coffee milk powder in the reconstituted form.

B. stearothermophilus are extremely heat resistant, up to 20 times more resistant than Clostridiumbotulinum spores (Iciek et al., 2008). B. stearothermophilus spores typically survive canningand sterilization procedures of food products and may cause spoilage problems especially where foods must be stored atelevated temperatures for a long time. Growth of B. stearothermophilus spores results in flat sour spoilage because acid is produced but with little or no gasgenerated (Brackett, 2001).

Regarding, the incidence of B. cereus isolated from the powdered and reconstituted forms of salep samples were 24 and 36%, respectively. Also, Table 5 showed that the incidence of B. cereus in both the powdered and reconstituted forms of hot chocolate samples was 16 %.B. cereus was isolated from 8 and 12 % of the powdered and reconstituted forms of coffee milk powder examined samples, respectively. A microbiological risk assessment defined the risk of B. cereus as gastroenteritis associated with consumption of vended hot chocolate. High level of B. cereus isolation does not indicate an immediate risk to public health; however, it may indicate a sub-optimal hygienic conditions and further improvement on the hygienic measures is required. The production of powdered hot chocolate drinks can involve the use of a variety of powdered products as; whey powder, powdered milk, powdered milk protein and cocoa powder which found to have the potentially contaminated B. cereus and also has been identified in hot drinks vended from automatic vending machines. Chung- Wang et al. (1993) couldidentify B. cereus in vended hot chocolate and cocoa, while Nelms et al. (1997) could isolate B. cereus within a vended hot chocolate drink, in and around the mixing bowl of the vending machine.

Figures 1a & b referred to the enterotoxigenic B. cereus that could be detected by multiplex PCR. Two types of virulent genes for characterization of B. cereus were used; hblC (695bp) and cytK (565 bp). All the 28 B. cereusisolates were tested for the presence of both enterotoxin genes. The figures cleared that out of 28 strains of B. cereus, 12 were harbored hblC gene. However, the cytotoxic (cytK) gene was found in 5 strains which lack hblC gene. Elsewhere, 11 strains carried both types of genes were noticed. So this study revealed that all the B. cereus isolates from the examined dried dairy products were enter otoxigenic.

Food manufacturer may need to observe good personal and hygienic practices on handling and storage of ingredients for preparation of salep; prevent packaging contamination and avoid prolonged storage under high temperatures.

Chung-Wang et al. (1993) examined 4 varieties of hot drinks obtained from automatic vending machines in Taiwan. A number of specific organisms were also isolated from the drinks and drinks powders by biochemical means. Coffee had a microbial load of 1.5 log cfu/ml. Apowder of chocolate mix was free from B. cereus. The absence of B. cereus from cocoa powders may call into question the sensitivity of the test procedures used, as it is a common contaminant and wasfound within the vended drink. A variety of other Bacilli were identified within the powder including B. subtilis and licheniformis. B. cereus that found contaminating the drink was confirmed positive for emetic toxin production. Levels of B. cereus above 10⁵ /g are thought to be required to produce sufficient toxin to cause illness (Granum, 1997).

Contamination of liquid milk with B. cereus and its spores can also lead to the presence of the organism within any powdered food product containing powdered milk. The contamination of powdered milk and other products derived from powdered milk with B. cereus has been widely reported (Wijnands et al., 2006).B. cereus has also been found in other dried food products such as potato (Turner et al., 2006), pepper (Chooet al., 2007) and importantly, cocoa and hot chocolate powder (Gabis et al., 1970, TeGiffel et al., 1996; Ardhana and Fleet, 2003).

The examined salep samples were found to have hygienic problems in the present study. Hygienic conditions of the ingredients used may be a contributing factor to the microbiological quality of this product.

Although B. cereus has been recognized as a cause of food poisoning for more than 30 years, it is still difficult to control, especially in the dairy industry. Morphological, biochernical, serological and toxigenic characteristics are commonly used to identify and classify B. cereus (Tallen and Kotewicz, 2012).

Further research should focus on the identification and characterization of the microorganisms found within the dried dairy drinks to provide information on the source of contamination and the population dynamic within the products.

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