EFFECT OF GAMMA RAYS RADIATION ON THE BACTERIOLOGICAL QUALITY OF ICE CREAM

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

EFFECT OF GAMMA RAYS RADIATION ON THE BACTERIOLOGICAL QUALITY

 OF ICE CREAM

EL-DOSOKY, H.F.A. ¹ and SHEREEN, S. MOSTAFA ²

¹ Food Hygiene Dept. Mansoura Provential Lab. Animal Health Research Institute

² Microbiololgy Dept. Mansoura Provential Lab. Animal Health Research Institute

Received: 26 December 2018;     Accepted: 31 January 2019

INTRODUCTION

Ice cream is a major dairy product of interest for large population. It is sold both in package (cups, cones and cartons); in open containers at the retail outlets, which is distributed manually in scoops, cones across counters. Due to its nutrient contents and long storage even though it is stored in a frozen state, the product can be a good source for microbial growth (Warke et al., 2000; Lee et al., 2009).

During processing of ice cream, there was a potential hazard due to addition of contaminated ingredients after the pasteurization step. Furthermore,   the  microbial  quality  of  ice  cream

Corresponding author: EL-DOSOKY, H.F.A.

E-mail address: rafat552008@yahoo.com

Present address: Food Hygiene Dept. Mansoura Provential Lab. Animal Health Research Institute

during retail marketing may affected by the post production handling of the product as well as efficiency and sanitary conditions during frozen storage. The lack of efficient frozen storage under warm tropical climatic conditions gives a chance for temperature changes during transportation and distribution of ice cream. Under such conditions, bacteria can proliferate leading to occasional food poisoning events (Champagne et al., 1994; Kanbakna et al., 2004). Radiation process has a positive effect in reducing the microbial counts and improving the safety and shelf-stability of food products without reducing their nutritional or sensory quality (Anon, 2003).

The present study was undertaken to investigate the efficacy of low-dose irradiation on 2 different flavors (vanilla and chocolate) ice cream to improve their microbial quality and getting safety.

MATERIALS AND METHODS

One hundred ice cream samples was collected individually from different supermarkets at Mansoura city Egypt, in the form of 2 groups (1^st group was 50 vanilla ice cream samples and 2^nd group was 50 chocolate ice cream samples). The samples were transferred to the laboratory in icebox and examined firstly before radiation for sensory and bacteriological examination. After that, the 2 groups were divided into 2 subgroups (each subgroup was 25 sample) The 1^st subgroup from the 2 groups was exposed to 2 KGy of gamma rays and the 2^nd subgroup from the 2 groups was exposed to 3 KGy of gamma rays. Each subgroup was packed in sterile polyethylene bag heat sealed then sent to the National Center for Radiation Research and Technology (NCRRT), Cairo, Egypt. The irradiation source was Cobalt 60 irradiation model ISS LEDDVATED. The dose rate was established using alanine transfer dosimeter (for measuring the dose rate) and variation in the absorption of irradiation dose was minimized by placing the samples within a uniform area of the irradiation field. After irradiation, 25 ml of each exposed samples after thawing was homogenized with 225 ml of 0.1% sterile peptone water in a stomacher for sample homogenization at 3000 rpm for 2.5 minutes followed by 10 folds 6 serial dilutions in 0.1% sterile peptone water. After that, the 2 groups (in the form of 4 subgroups) were examined as the following:

1- Sensory examination: experts in sensory evaluation evaluated changes in color, appearance, odor and texture and freshness quality.

2-Bacteriological examination:  

a) Aerobic plate count according to APHA (2001).

b) Staph. aureus count according to FDA (2002) using Baird-Parker agar plates, that were incubated at 35^o C for 48 hr. and the suspected Staph. aureus colonies were isolated and confirmed by catalase, coagulase, thermostable nuclease and Voges-Proskauer tests.

c) Bacillus cereus count according to the technique recommended by ISO 7932 (2004)

d) Coliforms count according to FDA (2005) using most probable number technique (MPN).

e) E. coli isolation according to FDA (2002) using sorbitol MacConkey agar medium (Oxoid, England).

f) Salmonella isolation according to the technique recommended by ISO 6579 (2002).

g) Listeria monocytogenesisolation according to the technique recommended by USDA; FSIS (1989) and FAO (1992).

h) Yersinia enterocoliticaisolation according toSchiemann (1982) using pre-enrichment culture on bile oxalate sorbose then culture on Cefuslodin Irgasan Novobiocin plate (CIN) according to Walker and Gilmour (1986).

3-Detection of virulence genes: the isolated Staph. aureus and E. coli were examined by using PCR for detection of Staph. aureus enterotoxins genes and   E. coli (stx1 and stx2) genes; in which DNA extraction from the samples was performed using the QIA amp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56^O C for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit. Oligonucleotide Primers used were supplied from Metabion (Germany) were listed in Table 1.For multiplex PCR of each gene, primers were utilized in a 50- µl reaction containing 25 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 8 µl of water, and 7 µl of DNA template. The reaction was performed in an Applied biosystem 2720 thermal cycler. Analysis of the PCR Productsby electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 30 µl of the multiplex PCR products were loaded in each gel slot. Gelpilot 100 bp DNA ladder (Qiagen, Germany, GmbH) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.

Table 1: Primers sequences, target genes, amplicon sizes and cycling conditions for the bold genes of Staph. aureus and E. coli used in multiplex PCR.

RESULTS

Table 2: Statistical analytical results of the examined ice cream bold samples.

Table 3: Incidence of the isolated bacteria from the examined ice cream bold samples.

Fig. 1: Agarose gel electrophoresis of Staph. aureus PCR products using Staph. aureus enterotoxins primers for A, B, C, D and E enterotoxins

Lane Neg means negative control

Lane Pos means positive control

Lane L means 100 bp DNA ladder

Lane 1 means positive amplification of 102 bp for enterotoxin A for a chocolate ice cream sample 

Lane 2 means positive amplification of 209 bp for enterotoxin E for a vanilla ice cream sample

Lane 4 means positive amplification of 164 bp for enterotoxin B for a chocolate ice cream sample

Lane 3 & 5 means negative PCR products for vanilla and chocolate ice cream samples

Fig. 2: Agarose gel electrophoresis of E. coli PCR products using stx1 and stx2 primers

Lane 1 means DNA ladder

Lane 3 & 5 means negative PCR products for vanilla and chocolate ice cream samples   

Lane 2meanspositive amplification of 614 bp for stx1 gene in a vanilla ice cream sample

Lane 4 means positive amplification of 614 bp for stx1 gene and 779bp for stx2 gene in a vanilla ice cream sample

DISCUSSION

Ice cream considered as a delicious and tasty food, worldwide, its processing needs many steps and different food additives that may be bacteriologically contaminated. The obtained results of aerobic plate count (APC) for the examined ice cream samples before irradiation in Table 2 were nearly achieved  by Gunsen (2002) who mentioned that the mean levels of total mesophilic aerobic bacteria in unmixed, cocoa and total ice creams samples were 3.3x10⁵, 1.03x10⁴ and 1.33x10⁵ cfu/g, respectively; Aslantas (2002) found the number of viable aerobic bacteria ranged from 3.4x10³- 2.3x10⁶ cfu/g in the examined ice cream samples; Yucel and Ctak (2002) evaluated ice cream samples bacteriologically for total aerobic bacterialcounts which were 2.5x10² - 3.0x10⁴ cfu/ml. The difference in APC may be attributed to the sanitary status during processing or storage and the bacteriological state of additives. While the results of APC in Table 2 after gamma irradiation were nearly in accordance with those obtained by Kamat et al. (2001) who investigated vanilla and chocolate ice cream after exposure to 1 kGy where the counts were reduced by one log cycle; Kim et al. (2005) indicated that irradiation at 5 kGy or less was effective to ensure safety of ice cream and significantly reduced the level of APC; Badr (2013) showed that irradiation treatments significantly reduced the counts of microbial populations.

The achieved results of Staph. aureus count before irradiation in Table 2 & Fig 1 declared that the enterotoxigenic strains were found in 3 samples      (1 from vanilla ice cream and 2 from chocolate ice cream) for A, E & B enterotoxigenic genes, the results nearly as reported by Yucel and Ctak (2002) who found Staph. aureus count 1.0x10²-3.0x10³ cfu/ml in the examined ice cream samples also, Guner et al. (2004) found the average count of Staph. aureus was 1.2 -1.7x10³ cfu/g of examined ice cream samples and Gücükoğlu et al. (2012) found 10% of the examined ice cream contained enterotoxigenic Staph. aureus. Meanwhile, the mean results of Staph. aureus count in Table 2 after gamma irradiation with 2kGy and 3kGy were nearly in accordance with those obtained by Kamat et al. (2001) who investigated the efficacy of low-dose irradiation to improve the microbial safety of vanilla and chocolate ice cream samples which were exposed, at - 72⁰ C to irradiation at 1 kGy dose were effective in reducing Staph. aureus. Badr (2013) assured that Ice cream samples which were gamma irradiated in the frozen state at dose of 3 kGy completely inactivate the inoculated Staph. aureus. Ice cream samples that irradiated with 3kGy were acceptable for their sensory attributes during storage. The enterotoxigenic Staph. aureus could not be detected in the examined 4 subgroups after radiation.

The results of Bacillus cereus count before gamma irradiation in Table 2 were nearly in accordance with Abdel-Haleem (2005) who isolated Bacillus cereus from the examined ice cream samples with count ranged from 3 cfu/g. The mean values of Bacillus cereus count in Table 2 after gamma irradiation with 2kGy and 3kGy were in accordance with Kamat et al. (2001) who mentioned that low-dose irradiation improve the microbial safety of vanilla and chocolate ice cream and resulted in reduction of microbial population by one log cycle.

The obtained results in Table 2 for coliforms count in the examined ice cream samples were before irradiation nearly similar to Warke et al. (2000) where they found coliforms count was 3.0x10²-5.8x10⁴ cfu/ml in the examined ice cream samples. Presence of coliforms in the examined ice cream samples with higher count indicates poor hygienic practices during manufacturing, post processing contamination and unsatisfactory transportation. Meanwhile, coliforms in Table 2 after irradiation with 3kGy could not be detected in the 4 subgroups. Kim et al. (2005) mentioned that Gamma irradiation significantly reduced the level of coliforms population in ice cream. Badr (2013) mentioned that Enterobacteriaceae were completely inactivated in ice cream samples irradiated at 2 kGy.

Results in Table 3 declared that the incidence results of E. coli in the examined samples before irradiation were 12% and 10% in 1^st and 2^nd group of vanilla and chocolate ice cream respectively. There were 2 isolates of E. coli from vanilla ice cream were positive for stx1 (Fig 2). While, in chocolate ice cream the isolated E. coli were negative for stx1 and stx2. The results after irradiation in Table 3 for the 4 subgroups declared that E. coli could not be detected in the examined samples.

The incidence results of Salmonellaspp. in Table 3 declared that Salmonellaspp. could not be detected by traditional methods or by PCR in all the examined groups and subgroups of vanilla and chocolate ice cream before and after irradiation. These results were in accordance with Warke et al. (2000); Windrantz and Arias (2001); Bostan and Akn (2002); Aslantas (2002) and Gunsen (2002) whom mentioned that Salmonella spp. was not isolated in any of the examined ice cream samples.

The incidence results of L. monocytogenes before irradiation in Table 3 were 10% and 8% in 1^stand 2^nd group of vanilla and chocolate ice cream respectively. These results were in accordance with Warke et al. (2000) who mentioned that                  L. monocytogenes was detected in only one sample of the opened examined ice cream samples; Cordano and Rocourt (2001) found L. monocytogenes in 3.5% of the examined ice cream samples and Windrantz and Arias (2001) showed that presence of L. monocytogenes in ice cream samples were 12.3%. While, higher results recorded by Molla et al. (2005) they stated that Listeria species were isolated from 43.5% of the examined ice cream samples and        L. monocytogenes were isolated mainly from 19.6% of ice cream samples in vice with Ambily and Beena (2012) mentioned that Listeria spp. was not isolated from any of the examined samples and Marouf et al. (2014) failed to detect L. monocytogenes in all examined samples. While, after irradiation the obtained results assured that L. monocytogenes failed to be detected. the results of subgroups were in agree with Kamat et al. (2001) who investigated the efficacy of low-dose irradiation to improve the microbial safety of ice cream where vanilla and chocolate ice cream were exposed to irradiation at 1 kGy resulted in elimination of L. monocytogenes and Badr (2013) stated that Enterobacteriaceae were completely inactivated in samples irradiated at 2kGy. Furthermore, irradiation at 3kGy completely inactivate the inoculated L. monocytogenes.

Regarding the incidence results of Y. enterocoliticain the examined ice cream samples before irradiation were 6% and 2% for 1^stand 2^nd groups of vanilla and chocolate ice cream respectively in Table 3. These results were in accordance with El-Prince and Hussein (2001) they could detect Y. enterocolitica in 1% of the examined ice cream samples and Erdogrul (2002) found that, out of 71 examined ice cream samples, 2 were Y. enterocolitica positive. Meanwhile, Y. enterocolitica could not be detected after irradiation which were nearly similar to those achieved by Kamat et al. (2001) who investigated the efficacy of low-dose irradiation to improve the microbial safety of ice cream where vanilla and chocolate ice cream were exposed to 1 kGy resulted in reduction of microbial population and Y. enterocolitica was eliminated.

CONCLUSION

From the achieved results in the present study, it could be concluded that gamma irradiation can be applied at dose of 3 kGy to improve the microbial safety of frozen ice cream products without adverse effects on their sensory acceptability.

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