Minimally processed lettuce is sensitive to microbial attack caused by the loss of natural resistance and their high water content and nutrients. The use of natural anti-microbial agents such as the lactoperoxidase system (LPS) represents an interesting alternative to the use of chemical treatments. This study focused on the importance of LPS and its efficiency as a bioprotective agent. The effect of washing with LPS, at two different concentrations (4 and 5 IU) and four different treatment times (1, 2, 3 and 4 h) on microbial growth in fresh-cut lettuce, was determined at 30°C and compared with a 80 ppm chlorine at 4°C for 20 min. The results deduced that both doses of LPS showed significant reductions in microbial growth when applied during 4 h, but the highest dose proved to be more effective. No significant differences were found between the LPS treatment and chlorine considering native microflora reduction and quality markers during storage.
Minimally processed lettuce form an important component of a healthy diet and is a convenient way to increase the consumption of this fresh vegetable [
Disinfection using hypochlorite is currently used by a majority (76%) of fresh produce manufacturers to enhance safety and shelf-life profiles. Water containing 50 - 200 ppm of chlorine is used extensively in food processing plants to disinfect whole fruits and vegetables as well as fresh-cut produce. However, chlorine does not ensure elimination or even an efficient reduction in the number of bacteria [
LPS name points out the dairy origin [
The main objective of this work was to optimize the application of lactope- roxidase system (LPS) for minimally processed lettuce decontamination. Moreover, the effect of the LPS system on the control of spoilage microflora and improving shelf-life characteristics during lettuce storage was determined.
Lactoperoxidase enzyme (200 IU/mg) was donated by Sigma-Aldrich (France), glucose oxidase (Sigma-Aldrich, France) and glucose were used as a peroxide generator system and sodium thiocyanate (NaSCN) were purchased from Sigma-Aldrich (France).
Biological iceberg lettuce were harvested at optimal maturity. Samples were then transferred to the laboratory within 1 h under refrigeration conditions and stored up to 24 h at 4˚C before treatment. Three external leaves were removed by hand (using sterile gloves) and the other parts of the lettuce were shredded in pieces of approximately 2 cm2, using a sterile blade. Three separate treatment solutions were prepared using distilled water. The concentrations were 4 and 5 IU for LPS, 80 ppm for chlorine and temperatures were 30 and 4˚C respectively [
Microbiological analysis was performed on hour, 1, 2, 3 and 4 to determine the effect of LPS concentration and contact time applied.
The enumeration of the microbial population was made according to Guireaud et al. (1998) [
To investigate the LPS effect on the preservation of fresh lettuce, samples were stored in refrigerator (4˚C) 7 days and microbiological analyses were conducted on treatment day and on 1st, 2nd, 3rd, 5th and 7th days of the refrigerated storage.
The objective was to study the effect of LPS on the quality of minimally pro- cessed lettuce. Weight loss, colour and pH were evaluated as indicators of shelf-life. Shelf-life is defined as the length of time at which the vegetable can maintain the appearance and safety [
Samples were weighed immediately after treatment (IW, initial weight) and then, after removal from refrigerated storage (SW, storage weight). Weight loss of each individual sample was calculated as:
W L ( % ) = ( 1 − S W / I W ) × 100
WL was expressed as percentage of weight loss with respect to initial mass.
Color measurements of lettuce were performed using a colorimeter (Minolta CR-300, Japan) to measure the CIE color space co-ordinates, L*, a* and b* (lightness, red value and yellow value, respectively, on the Hunter scale). The colorimeter was calibrated with a white tile standard.
Ten grams of lettuce was blended for 2 min in 20 mL of deionised water. The pH of the slurry was determined at room temperature using a pH-meter (Hach Company Electrochemical Meters).
The mean values obtained from the microbiological evaluation of lettuce were analyzed by one one-way ANOVA procedure of SPSS® 17.0. Duncan’s multiple range tests were used to determine any significant difference between mean values and evaluations as based on a significance level of p < 0.05.
Treatment | MES | PSC | TC | LAB | YM |
---|---|---|---|---|---|
NTC | 6.5 ± 6.2A | 4.4 ± 4.2A | 4.9 ± 3.9A | 5.3 ± 4.7A | 5.9 ± 4.2A |
1 LPS 1 | 6.0 ± 4.8B | 4.3 ± 4.2B | 4.7 ± 4.1B | 5.3 ± 4.7A | 5.6 ± 4.2B |
1 LPS 2 | 6.0 ± 4.8BC | 4.0 ± 1.8B | 4.0 ± 3.0C | 5.2 ± 4.8A | 5.1 ± 1.8C |
2 LPS 1 | 5.9 ± 4.6CD | 4.1 ± 3.8C | 4.4 ± 4.2D | 5.2 ± 4.6A | 4.9 ± 3.8CD |
2 LPS 2 | 5.7 ± 4.8E | 3.1 ± 2.2D | 3.9 ± 3.2E | 4.2 ± 3.7BC | 4.2 ± 2.2D |
3 LPS 1 | 5.9 ± 4.6D | 4.0 ± 3.4C | 3.7 ± 3.0F | 4.9 ± 4.2D | 4.0 ± 3.4D |
3 LPS 2 | 4.1 ± 3.3F | 2.9 ± 2.0EF | 2.9 ± 2.0G | 4.0 ± 2.8BC | 3.0 ± 2.0E |
4 LPS 1 | 5.6 ± 5.4E | 3.0 ± 1.8DE | 2.3 ± 2.0H | 4.4 ± 4.2B | 3.7 ± 1.8F |
4 LPS 2 | 3.6 ± 2.9G | NDG | NDI | 3.8 ± 3.0C | 2.2 ± 0.0E |
Chlorine | 3.9 ± 3.1H | 2.8 ± 2.0F | NDI | 2.4 ± 1.9C | 1.8 ± 2.0E |
MES: total mesophyllic bacteria count, PSC: total psychrophilic bacteria count, TC: total coliforms, LAB: lactic acid bacteria and YM: total yeast and molds count. Counts are expressed in Log10 CFU g−1 (±standard deviation). Different letters in the same column indicate significant differences (p < 0.05). The concentrations used for each treatment were the following: LPS 1 (4 IU), LPS 2 (5 IU) and chlorine (80 ppm). The number used before LPS is the number of hours of treatment. NTC: non-treated control.
fact, this difference in sensitivity can probably explained by the differences in cell wall structure and their different barrier propreties [
Changes in the mean values of CFU g−1 in Log10, the main groups of lettuce contaminant bacteria, measured at 4 h post activation of LPS, indicate the best decrease of the total amount, independently of the applied dose. For example, reduction of YM with 0.3, 1, 1.9 and 2.2 log units were obtained after 1, 2, 3 and 4 h of treatment with the lowest dose of LPS (4 IU), respectively. These results confirm a previous report in which LPS was effective on reducing microorgan- isms growth in vegetable juices, the reduction becomes important after 4 h of contact [
The use of two doses of LPS has been determinant for the effective selection of appropriate concentration, since for all microbial group differed between 4 and 5 IU of lactoperoxidase used for 4 h. Indeed, the best reductions were observed for the higher dose (5 IU).
When LPS was used for 4 h, the effect on TC, LAB and YM was the same as that observed with the Chlorine (p > 0.05). For MES and PSC, a significant inhibitory effect was found with LPS in comparison to the chlorinated water. Thus, from a microbiological point of view, LPS is a viable alternative to chlorine as decontamination treatments.
Sagoua et al. (2011) [
Finally, we selected 5 IU for lactoperoxidase dose and 4 h of contact for the rest of our study.
Reduction of the indigenous flora of lettuce because of decontamination with watery suspensions of LPS and chlorine are shown in
If decontamination of lettuce sample was performed with LPS solution a reductions of 3.3, 4.9 4.6, 3.1 and 3.6 Log10 CFU g−1, respectively, were observed for MES, PSC, TC, LAB and YM. These reductions were important throughout the cold storage. The sensibility of MES and PSC to LPS has been previously cited for Björck (1978) [
Time (days) | Bacterial population | Treatment | ||
---|---|---|---|---|
LPS | Chlorine | NTC | ||
0 | MES PSC TC LAB YM | 3.5 ± 3.5A NDA NDA 1.6 ± 1.2A 2.1 ± 1.8A | 3.8 ± 3.8A 1.1 ± 0.8A NDA 1.5 ± 1.0A 1.1 ± 0.8A | 6.8 ± 6.0B 4.9 ± 4.2B 4.6 ± 4.2B 4.7 ± 3.8B 5.7 ± 5.7B |
1 | MES PSC TC LAB YM | 3.5 ± 3.5A 1.4 ± 0.8A NDA 1.7 ± 1.3A 2.2 ± 2.1A | 3.8 ± 3.8A 1.5 ± 1.0A NDA 1.7 ± 1.5A 1.7 ± 1.4A | 8.6 ± 8.6B 4.8 ± 4.5B 4.6 ± 4.1B 5.7 ± 5.5B 5.8 ± 5.7B |
2 | MES PSC TC LAB YM | 3.8 ± 3.6A 2.5 ± 2.2A NDA 2.9 ± 2.3A 2.5 ± 1.8A | 4.1 ± 3.9A 2.0 ± 1.0A NDA 3.6 ± 3.5A 1.7 ± 1.4A | 8.7 ± 8.6B 4.5 ± 4.0B 4.7 ± 4.1B 6.8 ± 6.4B 6.6 ± 6.7B |
3 | MES PSC TC LAB YM | 4.2 ± 3.9A 3.7 ± 3.3A NDA 2.8 ± 2.4A 2.8 ± 2.4A | 4.2 ± 4.1A 3.1 ± 2.2A NDA 2.9 ± 2.5A 3.7 ± 3.6A | 10.0 ± 9.5B 6.4 ± 5.8B 5.0 ± 4.0B 7.7 ± 7.6B 6.4 ± 6.4B |
5 | MES PSC TC LAB YM | 5.5 ± 5.6A 5.8 ± 5.2A 1.0 ± 1.0A 3.9 ± 3.4A 4.9 ± 3.8A | 5.6 ± 5.6A 5.4 ± 5.2A 1.0 ± 1.0A 3.9 ± 3.4A 4.6 ± 4.2A | 10.8 ± 10.9B 7.7 ± 7.3B 5.9 ± 5.4B 8.6 ± 7.8B 8.5 ± 8.6B |
7 | MES PSC TC LAB YM | 7.8 ± 7.3A 6.6 ± 6.6A 2.3 ± 2.3A 4.9 ± 4.0A 5.8 ± 5.5A | 7.6 ± 7.4A 6.6 ± 6.5A 2.5 ± 2.3A 4.8 ± 4.4A 5.6 ± 5.3A | 12.2 ± 11.6B 8.7 ± 8.2B 7.7 ± 7.5B 10.8 ± 10.1B 8.8 ± 8.3B |
MES: total mesophyllic bacteria count, PSC: total psychrophilic bacteria count, TC: total coliforms, LAB: lactic acid bacteria and YM: total yeast and molds count; LPS, Chlorine: lettuce samples treated by the LPS and chlorine respectively; NTC: non-treated control; ND: No detected; Values are means of three replicates and counts are expressed in Log10 CFU g−1 (±standard deviation); Means followed by different letters are significantly different (p < 0.05) for each bacterial population.
French Food Safety Agency [
The mean aerobic mesophilic counts were 5.5 and 5.6 Log10 CFU g−1 for LPS and chlorines samples respectively after 5 days of storage, indicating that all treated samples were acceptable for consumption because the aerobic bacterial count for lettuce fresh is less than 7.7 Log10 CFU g−1 [
Fresh-cut lettuce is very susceptible to weight loss which is due to moisture loss. Yet, the relative humidity is generally very high inside the bags, so dehydration is not a common problem when lettuce are packaged [
Colour was monitored in all the samples during the entire storage (
Another colour parameter related to browning [
The initial pH of the lettuce is 6.52 (
A general decrease of pH was observed over storage, which could be due to an increase in the bacterial growth [
The results showed that the variable time of exposure to different LPS doses
Time (days) | Parameter | Treatment | ||
---|---|---|---|---|
LPS | Chlorine | NTC | ||
0 | pH WL L a b | 6.38 ± 0.01A 0.00 ± 0.00A 61.71 ± 1.15A −16.39 ± 0.47A 30.60 ± 0.01A | 6.25 ± 0.09B 0.00 ± 0.00A 64.59 ± 2.17A −15.54 ± 0.03A 28.91 ± 0.30B | 6.52 ± 0.09C 0.00 ± 0.00A 64.43 ± 1.11A −16.19 ± 1.86A 30.44 ± 0.28A |
1 | pH WL L a b | 6.36 ± 0.17A 0.66 ± 0.01A 61.89 ± 0.10A −16.28 ± 0.27A 30.05 ± 0.54A | 6.21 ± 0.06B 0.82 ± 0.08A 63.67 ± 0.10A −14.07 ± 0.55B 28.30 ± 0.91B | 6.35 ± 0.76A 5.51 ± 0.25B 59.79 ± 0.10B −15.03 ± 1.55AB 30.12 ± 0.44A |
2 | pH WL L a b | 6.33 ± 0.10A 2.72 ± 0.01A 60.05 ± 0.10A −16.20 ± 0.13A 29.63 ± 0.33A | 6.13 ± 0.30B 3.41 ± 0.36A 60.05 ± 0.10A −12.97 ± 0.70B 27.49 ± 0.50B | 6.08 ± 0.44B 11.45 ± 0.78B 54.61 ± 0.10B −14.69 ± 0.38C 28.93 ± 0.13A |
3 | pH WL L a b | 6.32 ± 0.08A 4.47 ± 0.12A 59.72 ± 0.41A −13.15 ± 1.01A 28.82 ± 0.42A | 6.10 ± 0.09B 5.84 ± 0.56B 57.90 ± 1.48A −12.55 ± 1.19A 25.43 ± 0.56B | 5.84 ± 0.09C 15.07 ± 0.57C 49.89 ± 1.02B −13.43 ± 1.05A 27.26 ± 0.69C |
5 | pH WL L a b | 6.24 ± 0.08A 8.82 ± 0.18A 56.38 ± 1.80A −11.49 ± 1.41A 26.50 ± 0.40A | 6.02 ± 0.08B 11.88 ± 0.11B 55.86 ± 1.46A −10.52 ± 0.77A 24.20 ± 0.26B | 5.30 ± 0.08C 16.87 ± 1.70C 44.59 ± 0.06B −11.83 ± 0.62A 25.21 ± 0.25C |
7 | pH WL L a b | 6.20 ± 0.08A 10.43 ± 0.67A 51.15 ± 0.17A −10.16 ± 0.89A 25.27 ± 0.25A | 6.00 ± 0.08B 12.64 ± 0.51B 53.39 ± 1.45B −10.10 ± 0.27A 22.50 ± 0.52B | 5.05 ± 0.08C 16.30 ± 1.08C 38.96 ± 1.07C −11.79 ± 0.83B 23.72 ± 0.62C |
pH, WL, L, a, b: pH, weight loss, colour parameter; LPS, Chlorine: lettuce samples treated by the LPS and chlorine respectively; NTC: non-treated control; Values are means of three replicates and counts are expressed in Log10 CFU g−1 (±standard deviation); Means followed by different letters are significantly different (p < 0.05) for each bacterial population.
affected most of the microbial quality. Based on this quality tested 4 h and 5 IU can be considered the best time and the best dose for the application for keeping fresh-cut lettuce microbial quality. The effectiveness of LPS as a decontamination treatment was comparable with that of chlorine. Application of LPS according to the proposed protocol were effective in inhibiting the growth and survival of spoilage microorganisms associated to minimally processed vegetables, without affecting its Quality markers.
However, in order to increase and improve the efficiency of LPS, coupling with other substances with antimicrobial potential can be considered.
Telmoudi, A. and Hassouna, M. (2017) Effect of Lactoperoxidase System on Microbiological and Quality Markers of Minimally Processed Lettuce. Technology and Investment, 8, 121-130. https://doi.org/10.4236/ti.2017.82010