Use of ultraviolet radiation and hydrogen peroxide for improvement of microbial quality of tables eggs and assuring safety is under studied in India. Considering this fact in the present study, a total of 432 Table eggs were procured form local retail markets of Parbhani city and treated with UV radiation at different exposure time of 1 (UV-1), 2 (UV-2) and 4 (UV-4) minutes alone and with1.5 % H2O2 (HP) and analyzed for microbial quality of eggshell surface. Efficacy of ‘HP+UV-2’ & ‘HP+UV-4’ treatment was found to be significant (p<0.05) in reducing total viable count of eggshell surfaces whereas Staphylococcus spp. contamination was significantly (p<0.05) reduced up to 1.5 log10 cfu by use of ‘HP+UV-4’ treatment. Comparative effect of various treatments on yeast and mould counts indicated non-significant difference except for HP+UV-4. The study concluded that the combination of ‘HP+UV-4’ found to be superior than other treatment groups in enhancing microbial shell surface quality of Table eggs to safeguard the public health.
The poultry industry of India has changed dramatically over the last 50 years. Due to the organized efforts from integrators and farmers the poultry industry has grown and specialized in meat production and egg production. The Indian poultry sector has been one of the fastest growing sectors, with annual growth of about 8.5 % in eggs and 7.8 % in meat production (GOI, 2019). Egg is considered as proteinous food, which contain many vitamins and minerals except vitamin C and fully mixed egg contains about 65% water, 12% proteins and 11% fat (Jay et al., 2005). Many nutrient substances present in eggs create an excellent environment for the development of bacterial microflora, including pathogenic bacteria (Stepien, 2010). Egg spoilage is usually associated with freshly and poorly stored eggs owing to poor storage conditions, like relatively high humidity, which contributes to the high microbial growth. Microbes of egg many cause severe health problems like diarrhea, nausea and abdominal pain, since they are pathogenic (Ansah et al., 2009).
In order to achieve the objective of food safety in modern egg production and processing systems, sanitization of eggs with physical and chemical methods has become a common practice. Combination of physical and chemical methods should be explored for sanitization of eggshell surfaces mitigating the negative effects of heat on the functional properties of egg (Favier et al., 2001 and O’bryan et al., 2017). In order to preserve the microbial quality and reduce the potential for contamination of eggs with pathogenic microorganism sanitization of the eggshell surfaces during processing at egg production facilities is an important step. UV light may be lethal to most microorganisms found on eggshell surfaces (Sastry et al., 2000 and Wells et al., 2010). Similarly, research shows that lower concentrations of H2O2 can effectively reduce microbial levels from eggshell surfaces (Bayliss and Waites, 1982; Padron, 1995 and Wells et al., 2010). Morouj et al. (2016) reported that hydrogen peroxide (H2O2) and UV treatment resulted in the greatest reductions in eggshell aerobic plate counts compared to other treatments throughout egg storage. Therefore, keeping in view the relative advantages of ultraviolet radiation and hydrogen peroxide in consideration to safeguard the public health from various pathogens, assuring safety and quality of shell eggs, the present study was designed with the objective to study effect of hydrogen peroxide along with and without ultra violet radiation treatment on microbial quality of table eggs.
Materials and Methods
A total of 432 table eggs comprising of 144 samples (each sample contains 3 eggs) were randomly collected from different markets of Parbhani city and soon after collection the samples were transported to the laboratories within one hour under aseptic condition in sterile polybags.
Calculation of Surface Area of Eggs
Weighing of table egg was done for calculation of surface area for estimating Colony Forming Units of microbial load. A method of Bonnet et al. (1965) modified by Wall et al. (2008) was used for estimating the colony forming units (cfu) as cfu/cm2–
S = 4.68 P (2/3)
S = surface in cm2, P = egg weight in grams.
Table 1: Design of experiment
|S. No.||Group Code||Treatment||Number of Samples|
|1.||C||Control without treatment||18|
|2.||HP||Hydrogen peroxide(H2O2) 1.5%||18|
|3.||UV-1||UV radiation – 1 min exposure||18|
|4.||UV-2||UV radiation – 2 min exposure||18|
|5.||UV-4||UV radiation – 4 min exposure||18|
|6.||HP+UV-1||UV radiation 1 min exposure + Hydrogen peroxide(H2O2) 1.5%||18|
|7.||HP+UV-2||UV radiation 2 min exposure + Hydrogen peroxide (H2O2) 1.5%||18|
|8.||HP+UV-4||UV radiation 4 min exposure + Hydrogen peroxide (H2O2) 1.5%||18|
|Total No. of samples*||144|
|Total number of eggs collected (144 x 3)||432|
Sanitization of eggs with Hydrogen Peroxide (HP) and Ultraviolet (UV) radiation
The table eggs were treated with 1.5 percent Hydrogen peroxide by using Hand sprayer purchased from local market whereas, for UV treatment kept in cabinet provides 250 nm UV rays where eggs were given exposure time of 1, 2 and 4 minutes. In UV cabinet eggs were placed in between two UV tubes so that all surfaces of eggs exposed with UV light. Details of control and treatment group is depicted in Table 1.
Microbial quality of eggshell surfaces of Table eggs with and without treatment with HP and UV radiation at different exposure time were assessed and evaluated. A method of Musgrove et al. (2005) was used for collection of eggshell rinsate sample with some modification. Three eggs were aseptically transferred to sterile polyethylene bag containing 300 ml peptone water. The surface of each egg was gently rubbed manually through the bag for 1 min. Egg was then removed and transferred to sterile bag separately. Rinsate was immediately used for estimation of TVC (BAM, 1998) and Differential Count in relation to Staphylococcus spp. (ISO-6888-2: 1999) and Yeast and Mould (ISO-21527-1). Isolation and identification of Salmonella spp. was done as per the ISO 6579: 1993, Reaffirmed 2005 method.
Results and Discussion
Average Eggshell Surface Area of Collected Samples
The average shell surface area in cm2 of collected egg samples was calculated and results are presented in Table 2. The average surface area of various treatment groups varied from 45.63±0.40 to 46.10±0.44 cm2 with mean average of 46.070±0.39 cm2. Data pertaining to the surface area of samples was further subjected to statistical analysis. Average eggshell surface area amongst the different groups of treatment did not differ significantly. The non-significant difference indicated that eggs from various treatment group and control were of similar size and did not have effect on microbial load.
Table 2: Details of average shell surface area of Table eggs samples collected under various groups.
|S. No.||Average ± SE (n=3)|
|Group code||Weight (grams)||Surface area (cm2)|
Mean average surface areas do not differ significantly *p<0.05
Effect of Sanitation on Microbial Quality of Eggshell Surfaces
The mean TVC, Staphylococcus spp. and Yeast and Mould counts of eggshell surfaces of Control (‘C’) and Treatment groups (‘HP’, ‘UV-1’, ‘UV-2’ ‘UV-4’, HP+UV-1’, ‘HP+UV-2’ and ‘HP+UV-4’ ) are given in Table 3.
Table 3: Comparative analysis of effect of H2O2, UV radiation and H2O2 with UV radiation on microbial quality of eggshell surface.
|Microbial count (log10 cfu/cm2)||Group code|
|Yeast and Mould||3.715±0.04a||3.384±0.08bc||3.427±0.09bc||3.505±0.09ab||3.191±0.07c||3.274±0.05c||3.189±0.05c||2.899±0.17d|
a, b, c, d, e means with different superscript in a row differ significantly * p < 0.05
Total Viable Count
Total viable counts (TVC) of eggshell surfaces given sanitary status at which the eggs are being handled in farm to retail process. The higher TVC counts indicated unhygienic handling and storage (Gole et al., 2013). A significantly (p<0.05) higher TVC count was observed in control group as compared to treatment groups. However, comparisons within UV treatment at various exposure time group indicate non-significant difference (p<0.05) but lower TVC count in ‘UV-4’ was observed. Significant reduction in TVC count of eggshell surfaces by UV radiation at 1 to 5 minutes exposure were reported by several research workers (Kuo et al., 1997; Chavez et al., 2002; Coufal et al., 2003; Patil, 2013 and Melo et al., 2019). UV radiation in the range of 250 to 260 nm was found to be lethal to various microorganisms including bacteria and Yeast and Mould (Bintsis et al., 2000; Patil, 2013). The observations in present study indicated that TVC of eggshell surfaces can be effectively reduced by exposing eggs to UV radiation for 4 minutes. Wells et al. (2010) also reported 1.5 log10 cfu reductions in TVC of eggshell surfaces after UV radiation (250 nm) for 4 minutes.
Significantly (p<0.05) lower TVC count was observed in hydrogen peroxide (HP) alone group in comparison with Control (‘C’) group. Effectiveness of hydrogen peroxide in eliminating eggshell surface microflora was reported by Sheldon and Brake (1991). Similarly, Wells et al. (2010) reported effectiveness of 1.5 percent hydrogen peroxide in reduction of bacterial count from eggshell surfaces. In present study effects were on similar lines in relation with control of shell surface microflora. The comparison of mean TVC count of groups (‘HP+UV-1’, ‘HP+UV-2’ and ‘HP+UV-4’) indicates significantly (p<0.05) lower count in ‘HP+UV-4’ group. The results are in agreement with earlier work carried out by Morouj et al. (2016). The present findings are also in accordance with Woodring (2011) and Wells et al. (2010) where reduction in total viable counts of eggshell surfaces were recorded 2 to 3 log10 cfu/egg after treating samples with hydrogen peroxide and UV radiation. In comparison among all groups of experiment (‘HP’ , ‘UV-1’, ‘UV-2’ ‘UV-4’, HP+UV-1’, ‘HP+UV-2’ and ‘HP+UV-4’ ) the efficacy of ‘HP+UV-2’ & ‘HP+UV-4’ treatment was found to be significant (P<0.05) than other treatments in controlling shell surface microflora. It was also observed that combination of Hydrogen peroxide and UV radiation with exposure time of 2 and 4 minutes were equally effective in reducing total viable count of eggshell surfaces.
Staphylococcus spp. is one of the predominant Gram-positive pathogens found on eggshell surface (Ansah et al., 2009). Significantly (p<0.05) lower count of Staphylococcus spp. was seen on UV treatment group compared to control group; however non-significant difference was observed within UV treatment groups. Observations indicated that Staphylococcus spp. contamination can be reduced by exposing the eggshell surfaces to UV radiation for 1 to 4 minutes. The results of present study are not in agreement with study carried out by Kuo et al. (1997). They did not observe significant reduction in Staphylococcus spp. count after exposing eggs to UV radiation for 15 minutes. The results are corroboration with earlier studies carried out by Reu et al. (2006) and Patil (2013). The comparison of ‘C’ and ‘HP’ indicated that eggshell surface count of Staphylococcus spp. was significantly (p<0.05) lower in ‘HP’ group. Comparatively higher reduction of Staphylococcus spp. was reported by Maktabi et al. (2018) by using 0.5 percent Hydrogen peroxide. French et al. (2004) also reported bactericidal effect of hydrogen peroxide on Staphylococcus aureus isolated from poultry environment. The comparison of mean Staphylococcus spp. count of all groups (‘C’, ‘HP+UV-1’, ‘HP+UV-2’ and ‘HP+4’) revealed significantly (p<0.05) lower count in ‘HP+UV-4’ group. Current results are in agreement with earlier studies carried out using Hydrogen peroxide or UV radiation individually or in combination (Reu et al., 2006; Wells et al., 2010; Gottselig, 2011 and Patil, 2013).
In comparison among all groups of experiment (‘HP’ , ‘UV-1’, ‘UV-2’ ‘UV-4’, HP+UV-1’, ‘HP+UV-2’ and ‘HP+UV-4’ ) the efficacy of ‘HP+UV-4’ treatment was found to be significant (P<0.05) than other treatments which indicated that Staphylococcus spp. contamination could be significantly (p<0.05) reduced up to 1.5 log10 cfu. It was also observed that ‘UV-4’ and ‘HP+UV-2’ treatment was at par with each other in control of Staphylococcus spp. contamination.
Yeast and Mould
Yeast and Mould are ubiquitous in nature. Eggs were contaminated because of improper environmental and storage conditions (Arthur and Somuah, 2001). The yeast and mould count was significantly (p<0.05) higher in ‘C’ group as compared to other groups of experiment (‘HP’ , ‘UV-1’, ‘UV-2’ ‘UV-4’, HP+UV-1’, ‘HP+UV-2’ and ‘HP+UV-4’). Within the UV treatment groups ‘UV-4’ group had significantly (p<0.05) lower count than ‘UV-1’ and ‘UV-2’ group. Results are suggestive of high efficacy of ‘UV-4’ radiation method in reduction of yeast and mould contamination. Findings of the present study are on similar lines with earlier work carried out by Kuo et al. (1997) and Patil (2013). Several other workers also used UV radiation to control yeast and mould growth on food products (Coufal et al., 2003 and Melo et al., 2019). Eggshell surface count of yeast and mould was significantly (p<0.05) lower in ‘HP’ group than ‘C’ group. Sheldon and Brake (1991) reported 97.1 percent reduction in yeast and mould count of eggshell surfaces treated with 5 percent Hydrogen peroxide. Maktabi et al. (2018) reported effect of Hydrogen peroxide on yeast and mould counts of eggshell surfaces.
The comparison of mean Yeast and Mould count of combination treatment groups (‘C’, ‘HP+UV-1’, ‘HP+UV-2’ and ‘HP+4’) reported significantly (p<0.05) lower count in ‘HP+UV-4’ group. Rotem et al. (1985) reported that UV light was effective in killing fungal spores. Comparison of effect of various treatments on yeast and mould counts indicates non-significant difference except for HP+UV-4 this shows superiority of ‘HP+UV-4’ treatment over other treatment groups
In current study none of the eggshell surface sample from Control (‘C’) as well as from all Treatment groups of experiment were found positive for Salmonella spp. Earlier Berrang et al. (1995); Gao et al. (1997); Coufal et al. (2003) and Keklik et al. (2010) reported that ultraviolet radiation was safe decontamination agent for reduction of Salmonella spp. from egg surface. Various researchers reported that Hydrogen peroxide is effective in reduction of Salmonella spp. from eggshell surfaces (Padron, 1995; Cox et al., 2002 and Buhr et al., 2013). Researchers observed that effectively used Hydrogen peroxide and UV radiation to reduce Salmonella spp. contamination from eggshell surface (Gottselig, 2011 and Morouj et al., 2016). Chemical and physical methods are used since long for egg sanitation (Shane and Faust, 1996, Chavez et al., 2002; Coufal et al., 2003, Reu et al., 2006; Wells et al., 2010; Patil, 2013 and Maktabi et al., 2018).
The findings of this study imply that combination of hydrogen peroxide (1.5% v/v) with UV radiation (4 minutes) is an effective method to reduce shell surface microflora of table eggs. Moreover, combination of various physical and chemical methods needs to be evaluated for their effectiveness in reducing shell surface microflora.
The authors are grateful to Associate Dean, College of Veterinary and Animal Sciences, MAFSU, Parbhani for providing necessary facilities and guidance at various stages of the experiment.