Effect of Different Sanitizers on Vegetables in Bangladesh

Effect of Different Sanitizer
on Freshly cut Vegetables in
Bangladesh
Md. Ashfaq Aziz
Research Associate
Food Analysis and Research Laboratory,
Center for Advanced Research in Sciences,
University of Dhaka

INTRODUCTION
 Bangladesh is an agriculture based country, agriculture is the single largest producing
sector of the economy of our country.
 Various types of crops, fruits and vegetables are grown in Bangladesh. Rice and jute are
the primary crops, maize and vegetables are assuming greater importance.
 The most common and demandable fresh vegetables in Bangladesh are: Potato, tomato,
cucumber, carrot, teasel gourd(Kakrol), green papaya, green chilli, cauliflower, cabbage,
bitter gourd(Korola), bottle gourd(Laow), staring bean(Borboti), lettuce, spinach.
 These vegetables contain different types of nutrient, such as- vitamins, calcium, iron,
zinc, mineral etc.

FOODBORNE PATHOGEN IN VEGETABLES
 Fresh vegetables are very important and necessary for health. Due to the
unhygienic cutting, handling, transporting, washing and improper storage many
foodborne pathogenic microorganisms can grow on raw vegetables. And those
microorganisms can cause serious illness. Sometimes pathogenic
microorganisms can transmit from soil.
 Some foodborne pathogenic microorganisms are found in raw vegetables are
given below-
1. Escherichia coli O157:H7
2. Salmonella spp.
3. Shigella spp.
4. Enterobacter cloacae

DIFFERENT SANITIZERS
 Sanitizer is a type of antimicrobial that kills or irreversibly inactivates at least 99.9
percent of all bacteria, fungi, and viruses (called microbials, microbiological,
microorganisms) present on a surface.
 Different types of sanitizers can be used for cleaning and washing vegetables and
keeping safe from foodborne pathogen. Some names of sanitizers are give below-
1. Hypochlorite
2. Chlorine Dioxide
3. Iodophors
4. Peroxyacetic Acid or Peracetic Acid (PAA)
5. Quaternary Ammonium Compounds (QACs or Quats)
6. Scallop Powder or SP (Calcinated calcium).

AIMS AND OBJECTIVES
 To keep vegetables safe from pathogenic microorganisms, different sanitizers can be
used.
 By washing vegetables with sanitizer properly pathogenic microorganisms are washed
out and removed.
 Important goal of this study is to detect the pathogen and confirm identification by
API 20E method from lettuce, cucumber and carrot.
 Observing the measurement of the antibiotic resistance and sensitivity of different
isolate is another important goal of this study.

MATERIALS AND METHOD
➢ Materials:
 Sterile knife
 Sterile perforated pot
 Sterile plastic pot
 Chopping board
 Different sanitizer (ex: Peracetic acid,
Scallop powder solution)
 Sterile distilled water
 Sterile normal saline
 Stomacher bag
 Measuring cylinder
 Measuring machine
 Sterile falcon tube with normal saline
 Test tube rack
 Micropipette(1000µ and 200µ)
 Micropipette tips(1000µ and 200µ)
 Different selective and non-selective
agar medium (Plate Count Agar,
Chromocult, Bismuthsulfite Agar,
Sabouraud dextrose Agar, Sensitivity
Test Agar)
 Inoculating loop
 Glass spreader
 API 20E kit
 Antibiotic discs

 The working strategy for lettuce, cucumber and carrot pathogens was as follows-
1. Lettuce,
cucumber and
carrot sample
collection
2. Preparation of
sanitizers
7. Put the 10 gm
lettuce, cucumber
and carrot in
separate
stomacher bag
with 90 ml NS and
mash 30-60 sec
3. Washing all
equipment
with sanitizer
properly
4. Chopping of
Vegetables
5. Washing
vegetables with
sanitizers and
autoclaved distilled
water
6. Dry sanitizer
and distilled water
washed vegetables
for 4 hours under
laminar air flow
8. Serial dilution
and spread plate
method on PCA,
CHROMAGAR, BSA
and SDA
9. Incubation for
24 hour at 37°C
10. Adding TSB
broth into remaining
sample in stomacher
bag for enrichment
before incubation
11. Counting
colonies from the
spread plate
12. If growth absent
in plate again
streaking for single
colony into new CHR
& BSA
15. Stocking the
bacteria with
glycerol at -20°C
14. Performing
Biochemical test
API 20E
13. Again single
colony streaking
on PCA plate
16. Rest of the
vegetables store
at -4 ̊C for further
work
 The working strategy for lettuce, cucumber and carrot pathogens was as follows-

 Antibiotic discs with their resistance and sensitivity zone.
Name of Antibiotics Code Disc
Concentration
Zone Diameter Nearest Whole mm
Resistance (≤) Intermediate Sensitive (≥)
Chloramphenicol C 30 µg 12 13-17 18
Gentamicin CN 10 µg 12 13-14 15
Azithromycin AZM 15 µg 13 14-17 18
Erythromycin E 15 µg 13 14-22 23
Kanamycin K 30 µg 13 14-17 18
Ciprofloxacin CIP 5 µg 15 16-20 21
Penicillin G P 10 µg 28 - 29
Tetracycline TE 30 µg 11 12-14 15
Ampicillin AMP 10 µg 13 14-16 17
Cephalexin CL 30 µg 14 15-17 18
Piperacillin PRL 75 µg 17 18-20 21
Amoxicillin AML 10 µg 13 14-17 18
Nalidixic Acid NA 30 µg 13 14-18 19
Nitrofurantoin F 300 µg 14 15-16 17
Ceftazidime CAZ 30 µg 14 15-17 18
Doxycycline DO 30 µg 10 11-13 14
Amikacin AK 30 µg 14 15-16 17
Clindamycin DA 2 µg 14 15-20 21
Vancomycin VA 30 µg 14 - 15
Norfloxacin NOR 10 µg 12 13-15 16
Aztreonam ATM 30 µg 15 16-21 22
Trimethoprim W 5 µg 10 11-15 16
Rifampicin Rif 30 µg 16 17-19 20

RESULT AND MICROBIAL ANALYSIS
 Microbial analysis of lettuce before sanitizer wash and after sanitizer wash-
Table-1: Microbial count of lettuce at 0 day.
Table-2: Microbial count of lettuce after 3 day at 4 ̊C.
Result of Day 0
Microorganisms
Control Distilled Water Washed SP Washed PAA Washed
BE AE BE AE BE AE BE AE
TABC 6.3×104 N/A 4.1×104 N/A 9.7×103 N/A <1.0 N/A
TCC <1.0 P <1.0 P <1.0 P <1.0 P
E.coli <1.0 A <1.0 P <1.0 A <1.0 A
E.cloacae 2×103 N/A <1.0 A <1.0 P <1.0 A
Yeast and mold 5.6×104 N/A 4×104 N/A 5.6×103 N/A <1.0 N/A
Result of Day 3 (-4 ̊C)
Microorganisms
TABC 8.2×104 N/A 7×103 N/A 7.2×103 N/A 2.5×103 N/A
TCC 2×102 N/A <1.0 ND 3×102 N/A 3×102 N/A
E.coli <1.0 ND <1.0 ND <1.0 ND <1.0 ND
E.cloacae <1.0 ND <1.0 ND 2×102 N/A 1×102 N/A
Yeast and mold 3×103 N/A 2×103 N/A 1.2×103 N/A 6×102 N/A

Table-3: Microbial count of lettuce after 6 day at 4 ̊C.
Microorganisms
TABC 3×105 N/A 7.4×104 N/A 4.5×103 N/A 9×102 N/A
TCC <1.0 P <1.0 P <1.0 P <1.0 P
E.coli <1.0 P <1.0 P <1.0 A <1.0 A
E.cloacae 7×102 N/A <1.0 P <1.0 P <1.0 A
Yeast and mold 2×105 N/A 5.4×104 N/A 5.2×103 N/A 7×102 N/A

 Microbial analysis of cucumber before sanitizer wash and after sanitizer wash-
Table-1: Microbial count of cucumber at 0 day.
Table-2: Microbial count of cucumber after 3 day at 4 ̊C.
Result of Day 0
Microorganisms
TABC 7.5×104 N/A 1×104 N/A 1.1×103 N/A <1.0 N/A
TCC 1×103 N/A <1.0 P <1.0 P <1.0 P
E.coli <1.0 A <1.0 A <1.0 A <1.0 A
E.cloacae 1×105 P 1×102 P 3×102 P <1.0 A
Yeast and mold 3×104 N/A 1×104 N/A 6×102 N/A <1.0 N/A
Microorganisms
TABC 7×103 N/A 2×103 N/A 2.7×103 N/A 2×103 N/A
TCC <1.0 ND <1.0 ND 2.7×103 N/A 9×102 N/A
E.coli <1.0 ND <1.0 ND <1.0 ND <1.0 ND
E.cloacae 3×102 N/A <1.0 ND 5×102 N/A <1.0 ND
Yeast and mold 3×103 N/A 2×102 N/A 6×102 N/A <1.0 N/A

Microorganisms
TABC 7×104 N/A 1×102 N/A 6×103 N/A 1.1×104 N/A
TCC 1×102 N/A <1.0 P <1.0 P <1.0 P
E.coli <1.0 P <1.0 A <1.0 A <1.0 P
E.cloacae 1×102 N/A <1.0 A <1.0 P <1.0 P
Yeast and mold 2.5×103 N/A 1×102 N/A 4×102 N/A 1×104 N/A
Microorganisms
TABC 5×103 N/A <1.0 N/A <1.0 N/A <1.0 N/A
TCC <1.0 P <1.0 P <1.0 P <1.0 P
E.coli <1.0 A <1.0 A <1.0 A <1.0 A
E.cloacae <1.0 P <1.0 P <1.0 P <1.0 P
Yeast and mold 1.1×103 N/A 2×102 N/A 3×102 N/A 6×102 N/A

 Microbial analysis of carrot before sanitizer wash and after sanitizer wash-
Table-1: Microbial count of carrot at 0 day.
Table-2: Microbial count of carrot after 5 day at 4 ̊C.
Result of Day 0
Microorganisms
TABC 8×103 N/A 5×103 N/A <1.0 N/A 2×103 N/A
TCC 1.2×104 P <1.0 P <1.0 P <1.0 P
E.coli <1.0 A <1.0 P <1.0 A <1.0 A
E.cloacae 1×103 P <1.0 P <1.0 P <1.0 P
Yeast and mold 1.9×104 N/A 2×103 N/A <1.0 N/A <1.0 N/A
Microorganisms
TABC 4×103 N/A 4×103 N/A 2×102 N/A 1×102 N/A
TCC 3×102 P <1.0 P <1.0 P <1.0 P
E.coli <1.0 A <1.0 A <1.0 A <1.0 A
E.cloacae <1.0 P <1.0 P <1.0 P <1.0 P
Yeast and mold 6×103 N/A 1.5×103 N/A 9×102 N/A 9×102 N/A

Microorganisms
TABC 8×104 N/A 7.4×104 N/A 5×102 N/A <1.0 N/A
TCC 6×103 N/A 1×103 N/A <1.0 P <1.0 P
E.coli <1.0 A <1.0 A <1.0 A <1.0 A
E.cloacae 2×103 N/A 2×102 N/A <1.0 P <1.0 P
Yeast and mold 3.6×105 N/A 7.7×104 N/A 6×102 N/A 1×103 N/A
Microorganisms
TABC 4.4×104 N/A 1.5×104 N/A 7×102 N/A 9×103 N/A
TCC <1.0 P <1.0 P <1.0 P <1.0 P
E.coli <1.0 A <1.0 A <1.0 A <1.0 A
E.cloacae <1.0 P <1.0 P <1.0 P <1.0 P
Yeast and mold 4.8×104 N/A 1.3×104 N/A 2×103 N/A 8×103 N/A

 Microbial count on different agar plate(Representative):
Plate Count Agar Plate Chromocult Agar Plate
Bismuthsulfite Agar Plate Sabouraud Dextrose Agar Plate

 Streaking after enrichment and isolation of single colony(Representative):
Single Colony on Chromocult Plate Single Colony on Chromocult Plate
Single Colony on BSA Plate Single Colony on PCA Plate

 Biochemical Test:
Purple Colony on Chromocult Plate
Black Colony on BSA Plate
Blue Colony on Chromocult Plate
Pink Colony on Chromocult Plate
API 20E for Biochemical Test
-Purple Colony on Chromocult Plate : 99.3% E.coli.
-Black Colony on BSA Plate : 98.6% Enterobacter cloacae
-Blue Colony on Chromocult Plate : 99.7% E.coli
-Pink Colony on Chromocult Plate : 96.9% Enterobacter cloacae

 Some STA plate of antibiogram:
E.Coli 99.7% E.coli 99.3%
Enterobacter cloacae 98.6% Enterobacter cloacae 96.9%

 Antibiotic Sensitivity Test
Name of Antibiotics E.coli (99.7%) E.coli (99.3%) Enterobacter cloacae
(98.6%)
Enterobacter cloacae
(96.9%)
Diameter
(mm)
R/S/I Diameter
(mm)
R/S/I Diameter
(mm)
R/S/I Diameter
(mm)
R/S/I
Chloramphenicol 20 S 22 S 20 S 20 S
Gentamicin 10 R 10 R 7 R 9 R
Azithromycin 8 R 8 R 8 R 8 R
Erythromycin 7 R 7 R 7 R 7 R
Kanamycin 15 I 16 I 9 R 11 R
Ciprofloxacin 20 S 25 S 30 S 23 S
Penicillin G 7 R 7 R 7 R 7 R
Tetracycline 14 I 16 S 12 I 10 R
Ampicillin 7 R 7 R 7 R 7 R
Bacitracin 7 R 7 R 7 R 7 R
Cephalexin 7 R 7 R 7 R 7 R
Piperacillin 12 R 22 S 20 I 20 I
Amoxicillin 7 R 7 R 7 R 7 R
Nalidixic Acid 15 I 12 R 17 I 12 R
Nitrofurantoin 13 R 14 R 12 R 10 R
Ceftazidime 18 S 20 S 20 S 18 S
Novobiocin 7 R 7 R 7 R 7 R
Doxycycline 14 S 14 S 10 R 7 R
Amikacin 15 I 17 S 15 I 15 I
Clindamycin 7 R 7 R 7 R 7 R
Vancomycin 7 R 7 R 7 R 7 R
Oxacillin 7 R 7 R 7 R 7 R
Norfloxacin 16 S 17 S 22 S 18 S
Aztreonam 22 S 23 S 25 S 26 S
Trimethoprim 7 R 7 R 7 R 7 R
Oxytetracycline 7 R 7 R 7 R 7 R
Rifampicin 7 R 7 R 7 R 7 R

DISCUSSION
 These results imply that fresh lettuce, cucumber and carrot from vegetable market were heavily
contaminated with bacteria, which is a serious concern after harvest and during consumption.
 Aerobic bacteria and Enterobacter cloacae are mostly present in unwashed raw vegetable.
Pathogenic strain of E.coli also found in vegetable. But after sanitizer wash most of them are
removed.
 Chloramphenicol, ciprofloxacin, piperacillin and aztreonam are highly sensitive to E.coli.
Ciprofloxacin, norfloxacin and aztreonam are highly sensitive to Enterobacter cloacae.
Gentamicin, azithromycin, erythromycin, ampicillin, cephalexin, amoxicillin, clindamycin,
vancomycin, trimethoprim and rifampicin are highly resistance to E.coli and Enterobacter
cloacae.
 The effects of sanitizers on fresh vegetables are follows:
-PAA(Peracetic Acid), SP and distilled water all can remove pathogenic microorganisms from fresh
vegetables.
-SP and PAA is more effective than distilled water.

CONCLUSION
 To minimize human pathogens in fresh production, good agricultural
practices and good hygiene measures should be implemented from farm
to fork during production, harvesting, processing, packaging,
transporting, and storing.
 Moreover sanitizers can also be harmful and the effects of sanitizers on
lettuce, cucumber and carrots can make the pathogens resistant to be
washed off that are reported by this study.
 In this research work we expect to reduce the microbial load from fresh
lettuce, cucumber and carrot.
 This study will give a clear idea of pathogen detection in lettuce,
cucumber and carrot.

Effect of Different Sanitizers on Vegetables in Bangladesh

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