Dong-Hyun Kang, School of Food Science, Washington State University, Pullman, WA 99164-6376, USA. E-mail: email@example.com
Aim: To investigate the efficacy of steam pasteurization for reducing Salmonella serotype Enteritidis on raw almond surfaces.
Methods and Results: Nonpareil almonds were inoculated to 107–8 CFU g−1 with a Salm.Enteritidis cocktail (Salm. Enteritidis 43353, ME-13, ME-14) or Salm.Enteritidis phage type 30, dried overnight and subjected to steam treatments through a pilot-sized vertical pasteurization machine for 5, 15, 25, 35, 45, 55 and 65 s to investigate the effect of steam on a single layer of almond. Survival of Salm.Enteritidis was evaluated with tryptic soy agar and xylose lysine desoxycholate overlay for total and healthy cells, respectively. No significant differences (P > 0·05) in reduction were observed between the Salm. Enteritidis cocktail and Salm. Enteritidis PT 30 inoculum. Reduction of Salm. Enteritidis increased as a function of treatment time, with 25 s being sufficient to achieve a 5-log reduction. Discolouration and visible formation of wrinkles were observed following steam pasteurization of more than 35 s.
Conclusions: Steam pasteurization of 25 s is sufficient to achieve a 5-log reduction of Salm. Enteritidis inoculated on raw almonds without visual quality degradation.
Significance and Impact of the Study: Steam pasteurization is an effective alternative to reduce or prevent Salm. Enteritidis contamination on raw almonds.
Salmonellosis is one of the leading causes of diarrhoea, fever and abdominal cramps according to the Center of Disease and Control (CDC 2008). Severe cases of salmonellosis often lead to human morbidity and mortality (Poppe 1994). Salmonella serotype Enteritidis, in conjunction with Salmonella Typhimurium, are the most commonly reported micro-organisms linked to salmonellosis (Poppe 1994). In 2002, Salm. Enteritidis was identified as the causative agent of 15·8% salmonellosis incidents (5116 of 32 308 total cases) in the United States (Ahmed et al. 2000; CDC 2003; Marcus et al. 2004).
Salmonella Enteritidis phage type 30 (PT-30) are usually isolated from raw liquid egg, chicken, poultry sewage, chicken feed and human diarrhoea (Chart et al. 1991). Recent outbreaks of salmonellosis associated with raw almonds in Canada and United States have also been attributed to Salm. Enteritidis PT-30 (CDC 2004; Isaacs et al. 2005). Cross-contamination of raw almonds can easily occur at harvesting, drying and hulling-shelling (CDC 2004).
Steam pasteurization is effective for reducing naturally occurring and pathogenic bacteria in foods (Nutsch et al. 1998). The effectiveness of steam pasteurization is because of the large amount of heat transferred to targeted foods when steam condenses, increasing the surface temperature promptly (James et al. 2000). Steam has a greater heat capability than the same amount of water at a given temperature (James and James 1997) and can effectively penetrate cavities, crevices and feather follicles that may provide protection for surface-attached micro-organisms (Morgan et al. 1996).
Current outbreaks of salmonellosis because of the consumption of contaminated raw almonds have initiated development of effective processing methods to reduce Salm. Enteritidis on raw almonds while preserving the texture and sensory qualities of the raw almonds. The objective of this study was to investigate the efficacy of steam pasteurization for reducing Salm. Enteritidis on raw almond surfaces as an alternative control procedure.
Materials and methods
Salmonella Enteritidis strains 43353, ME-13 and ME-14 were obtained from the Food and Drug Administration (Rockville, MD, USA), and PT-30 was kindly provided by Dr Linda Harris (University of California Davis, Davis, CA, USA). All bacteria were stored in Tryptic Soy Broth (TSB; Difco, Detroit, MI, USA) with 15% glycerol (w/v) at −20°C prior to use.
Preparation of Salmonella Enteritidis inoculum
Prior to each experiment, frozen cultures of Salm.Enteritidis were streaked onto Tryptic Soy Agar (TSA; Difco) and incubated at 37°C for 24 h. A single isolated colony was transferred into 9-ml TSB and incubated at 37°C for an additional 24 h. A subsequent loop transfer and overnight (18 h) incubation at 37°C was performed to ensure healthy cell growth. The overnight cultures (0·3 ml) were then dispensed onto TSA to form bacterial lawns. Four plates per 500 g of almonds were prepared for each bacterial strain. Following incubation at 37°C for 24 h, 8–9 ml of 0·2% peptone water (Difco) was added to each petri dish and bacterial cells were harvested by loosening the cells with sterile swabs. Cell suspensions of Salm.Enteritidis 43353, ME-13 and ME-14 were combined to make a cocktail inoculum while PT-30 was prepared as a single strain inoculum. For every 500 g of almonds to be inoculated, collected cell suspensions were centrifuged at 4000 g for 30 min, the supernatant discarded and the cell pellet resuspended to 25 ml with fresh 0·2% peptone water. The appropriate number of 25-ml inoculum preparations were pooled and vigourously vortexed prior to inoculating the almonds. Inoculum levels were determined by tenfold serial dilutions in 9-ml 0·2% peptone water and spread plating the inoculum onto TSA and Xylose Lysine Desoxycholate (XLD; Difco).
‘Nonpareil’ California raw shelled whole almonds used in this study were obtained from Stewart and Jasper Marketing Inc. (Newman, CA, USA). Almond samples (500 ± 1 g) were weighed into 30·5 × 30·5-cm plastic polyethylene bags (Ziploc®, Racine, WI, USA), and 25 ml of the pooled inoculum was added. The bag was sealed and inverted for 60 s to submerge the almonds in the Salm. Enteritidis inoculum. Inoculated almonds were evenly spread onto two double-layered sheets of 46 × 57-cm filter paper (Fisherbrand Qualitative P8; Fisher Scientific, Pittsburgh, PA, USA), covered with an additional double layer of filter paper, placed inside a cardboard box and dried for 24 h at room temperature (22 ± 2°C).
Effect of steam pasteurization on Salmonella Enteritidis on raw almond surfaces
A custom made vertical steam pasteurization machine (Fig. 1) designed and built by Dr José Reyes-De-Corcuera was used in this study. The steam pasteurization machine consisted of a treatment chamber (10·2-cm diameter by 20·3-cm long 304 stainless steel tube) and holds a retractable stainless steel wire basket that serves as sample holder. A diffuser plate at the top of the chamber ensured the production of a uniform steam flow.
Dried inoculated almonds (25 ± 1 g; 3–4 kernels) were spread into a single layer in the treatment basket and placed into the steam chamber. A rubber ring was placed on top of the treatment basket to ensure steam flow through the basket, and the chamber was securely sealed with two clamps. Steam was passed through the machine by opening the steam valve while maintaining an absolute pressure of 143 kPa. The almonds were steam pasteurized for 5, 15, 25, 35, 45, 55 and 65 s with treatment time starting as the lower temperature indicator reached 95°C. After each treatment, steam was closed, and 22 ± 1°C compressed air (WSU Central Stores, Pullman, WA, USA) was injected for 5 s to cool almond surfaces. The almonds were then removed, placed onto a drying basket with a fan attached underneath and fan dried at 22 ± 1°C for 10 min. Dried almonds were transferred to a stomacher bag containing 50-ml 0·2% peptone water and vigorously rubbed for 1 min. The suspension was tenfold serially diluted with 0·2% peptone water to the appropriate dilution and plated onto TSA and XLD and incubated for 24 h at 37°C for the enumeration of total and healthy cells, respectively. In addition to plating 100 μl of the lowest dilution, 1 ml was distributed over four plates (250 μl each) to lower the detection limit. Colonies from randomly selected plates were subjected to serological confirmation as Salmonella using Latex agglutination test kits (RIM Salmonella Latex Agglutination Test; Remel, Lenexa, KA, USA). All treatments were triplicated.
Triplicate data were analysed using analysis of variance with the GLM procedure of sas (Version 8.1; SAS Institute Inc., Cary, NC, USA) for a completely randomized block design. When the main effect was significant (P < 0·05), means separation was separated by Duncan’s multiple-range test.
Inoculated almonds subjected to different steam durations were used to assess the effectiveness of steam against Salm. Enteritidis. Reduction levels increased with steam treatment time regardless of inocula used. The recovery of Salm. Enteritidis after each steam treatment interval is summarized in Table 1. Among treatments where cell survival was observed, significant differences (P < 0·05) existed between treatments with treatment durations >15 s. Steam treatment of 15 s also reduced Salm. Enteritidis compared to the 5-s treatment though statistical differences could not be determined. A 5-log reduction of Salm. Enteritidis was achieved after 25 s of steam pasteurization and was further reduced to undetectable levels after 65 s. In addition to the effect of steam pasteurization, the two Salm. Enteritidis inocula were compared for differences in heat resistance. No significant difference in heat resistance was observed between Salm. Enteritidis cocktail and PT-30.
Table 1. Survival (log10 CFU g−1)* of inoculated Salmonella Enteritidis on almond surfaces treated with steam pasteurization
*Mean ± standard deviation; n = 3 for all treatments.
†Refers to almonds subjected to steam until chamber temperature stabilized at 95°C.
‡Means with the same lower case letter within a column are not significantly different (P > 0·05).
§Detection limit of assay was 0·3 log10 CFU g−1 almond.
8·77 ± 0·38a‡
8·16 ± 0·33a
8·15 ± 0·16a
7·52 ± 0·10a
5·19 ± 0·22b
4·35 ± 0·40b
4·65 ± 0·66b
4·12 ± 0·81b
4·50 ± 0·43bc
3·85 ± 0·40b
3·96 ± 0·52bc
2·85 ± 1·23b
4·10 ± 0·25c
2·83 ± 0·57c
3·11 ± 0·06c
0·73 ± 0·63c
1·92 ± 0·71d
0·65 ± 0·56d
2·23 ± 0·45d
0·48 ± 0·42d
0·60 ± 0·15e
0·88 ± 0·39e
0·50 ± 0·26e
Although steam pasteurization was effective in reducing Salm. Enteritidis, almond quality was also reduced as a result of steam pasteurization (Fig. 2). Detachment of almond skin was observed after 5 s of steam pasteurization. The skin reattached after 10 min of drying, but was visibly more wrinkled. As steam pasteurization time increased, bleaching of the almond skin was observed in addition to difficulties in skin reattachment. Small grey spots were first detected on almonds pasteurized for 25 s and became more prominent with increased steam treatment time.
The effectiveness of steam pasteurization has been demonstrated against various pathogenic and nonpathogenic bacteria (Nutsch et al. 1998; Minihan et al. 2003; Retzlaff et al. 2004, 2005; Corantin et al. 2005; Murphy et al. 2005, 2006). This study confirms that steam pasteurization is an effective alternative for improving microbial safety of raw almonds. Reduction of inoculated Salm. Enteritidis was a function of treatment time, and a >5-log reduction was achieved with a 25-s steam pasteurization treatment without severely compromising the visual quality of raw almonds.
The effectiveness of steam treatment on Salm. Enteritidis is dependent on the condition of steam applied. When steam was applied by boiling water below inoculated almonds (conventional steaming), Salm. Enteritidis reductions of 2·8 log were observed after a 25-s steaming treatment (Lee et al. 2006). In contrast, in the experiments reported in this article, a >5-log reduction of Salm. Enteritidis was achieved at 25 s when steam was applied as a directed steam flow (steam pasteurization) (Table 1). Salmonella Enteritidis concentrations on raw almonds were further reduced to undetectable levels when steam pasteurized for 65 s. The enhanced reduction of Salm. Enteritidis by steam pasteurization may be because of rapid increase of temperature within the enclosed chamber compared to heat dissipation in open air with conventional steaming.
Other methods have been investigated for reducing of Salm. Enteritidis on raw almonds. Propylene oxide (PPO; C3H6O) is commonly used to reduce micro-organisms on raw whole almonds and raw/blanched sliced, diced or coarsely ground almonds in the United States tree nut industry (Danyluk et al. 2005). When PPO (0·5 kg m−3 for 4 h) was applied to raw whole almonds, reductions were consistently >5-log after 5 days of storage (Danyluk et al. 2005). The effect of high hydrostatic pressure against Salm. Enteritidis on almonds has been reported by Goodridge et al. (2005). A 0·41 MPa per 25°C per 5 min treatment was sufficient to reduce planktonic Salm. Enteritidis to undetectable levels. On the other hand, pressurizing almonds at under identical conditions achieved a limited reduction of 0·83 log CFU g−1 (Goodridge et al. 2005). The decreased ability of HHP (hydrostatic high pressure) to inactivate Salm. Enteritidis cells inoculated on raw almonds compared to those suspended in 0·1% peptone water was attributed to the low water activity (aw) of the almonds. HHP is much more effective at reducing foodborne pathogens in foods with higher aw (Palou et al. 1998; Goodridge et al. 2005). Compared to PPO and HHP treatments, treatment of raw almonds through steam pasteurization offers an effective, economical and chemical-free alternative for ensuring the safety of raw almonds.
The major disadvantage of steam pasteurizing is the loss of visual quality with increased treatment time. As shown in Fig. 2, detachment of almond skin, wrinkled almond surfaces and the presence of small grey spots on almond skin were visible after a 35-s steam pasteurization treatment. Discolouration by steam pasteurization was also observed in black pepper and paprika (Almela et al. 2002; Kispeter et al. 2003; Waje et al. 2008). Salmonella spp. contamination levels naturally found in raw almonds are c. 1·2–2·9 MPN (100 g)−1 (Danyluk et al. 2007). Thus, it is unlikely that raw almonds will be treated with prolonged steam that results in such visual degradations as observed within this study. Nonetheless, an alternative to minimizing potential discolouration on raw almonds by steam pasteurization is the use of combined treatments. Enhanced effectiveness of steam pasteurization against Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Heidelberg has been reported when used in combination with other methods such as acid, high pressure or vacuum treatment (Kozempel et al. 2000; Huang 2004; Murphy et al. 2006). Using combined treatments may be a potential solution towards reducing steam pasteurization-induced discolouration, but further studies are required to establish optimum combinations and treatment times to achieve desired safety limits without compromising the quality of the product.
Steam pasteurization is an applicable and effective alternative for inactivating Salm. Enteritidis inoculated onto raw almonds. A 5-log reduction of Salm. Enteritidis can be achieved through steam pasteurization for 25 s without deteriorating the visual quality of almonds.
This work was supported by grant No. R32-2008-000-10183-0 from the World Class University (WCU) project of the Ministry of Education, Science & Technology (MEST) and the KOSEF through Seoul National University.