HOME Contact Us

Journal Search Engine

Volume/Issue :
::: Volume :::
::: Issue :::

Year(s) : 2015
to
present
About US +
Search :

For Contributors +

Journal Search +

Most read Article

Current Issue

2015 - Present
2022 Journal of Food Hygiene and Safety Vol.31 No.4 pp.299-303 ISSN : 1229-1153(Print)
2021 DOI : https://doi.org/10.13103/JFHS.2016.31.4.299 ISSN : 2465-9223(Online)
2020
2019
2018 Bactericidal Efficacy of a Disinfectant Spray Containing a
2017
2016
Grapefruit-seed Extract, Citric acid, Malic acid and
2015 Benzalkonium Chloride against Salmonella Typhimurium
Archive and Brucella ovis

Chun-Nam Cha1, Eun-Kee Park2, Ji-Youn Jung3, Chang-Yeul Yoo4, Suk Kim, Hu-Jang Lee*
Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Chinju 52828, Korea
1Engineering Research Institute, Department of Industrial Systems Engineering, Gyeongsang National University, Chinju 52828, Korea

2Department of Medical Humanities and Social Medicine, College of Medicine, Kosin University, Busan 49267, Korea

3Department of Companion and Laboratory Animal Science, Kongju National University, Kongju 32588, Korea

4Department of Smart Information Convergence, Gyeongnam Provincial Namhae College, Namhae 52422, Korea

Correspondence to: Hu-Jang Lee, College of Veterinary Medicine, Gyeongsang National University, 900 Gajwa-dong, Chinju 52828, Korea, +82-55-772-2352,
+82-55-772-2308, hujang@gnu.ac.kr

Received April 30, 2016 Review May 18, 2016 Accepted July 26, 2016

Abstract Other Sections

Salmonella spp. and Brucella spp. can cause considerable diseases on both humans and animals. In addition, these microorganisms cause
the economic loss in animal farming and food industry. In this study, the disinfection efficacy of a disinfectant spray, composed to grapefruit
seed extract, citric acid, malic acid and benzalkonium chloride, was evaluated against S. Typhimurium and B. ovis. A bactericidal efficacy test
by broth dilution method was used to determine the lowest effective dilution of the disinfectant following exposure to test bacteria for 30 min
at 4°C. The disinfectant and test bacteria were diluted with hard water (HW) or organic matter suspension (OM) according to treatment
condition. On HW condition, the bactericidal activity of the disinfectant spray against S. Typhimurium and B. ovis was 5 and 4 fold dilutions,
respectively. On OM condition, the bactericidal activity of the disinfectant spray was 2 and 1 fold dilutions against S. Typhimurium and B. ovis,
respectively. As the disinfectant spray possesses bactericidal efficacy against foodborne pathogens such as S. Typhimurium and B. ovis, the
disinfectant spray can be used to control the spread of bacterial diseases.

Key Words : Disinfectant spray , Salmonella Typhimurium , Brucella ovis , Disinfectant efficacy

This article has been cited by 0 article in

Funding:

All over the world, Salmonella Typhimurium (S. Typhimurium) is responsible for approximate a third of all cases of foodborne
diseases1). Salmonellosis is an increasingly important health concern and is usually associated with the consumption of
Salmonella-contaminated foods, mainly of animal origin, including beef, pork, poultry and turkeys2). The disease in human is
characterized by self-limiting gastroenteritis that occasionally can cause fever, systemic infection, and severe inflammation of
the intestinal mucosal epithelium3). In food animals, Salmonella infections play a significant role in public health and particularly
in food safety, as food products originated from animals are believed to be the major source of salmonellosis in human4).
Brucellosis is a zoonosis that is transmitted to humans from infected animals’ meat, milk, urine, body fluids, and pregnancy
material, as well as from milk products prepared by infected milk5). The disease is generally transmitted to humans by eating
cheese, made from raw milk, and milk products such as cream and butter6). In addition, the disease may be transmitted by
eating animals’ reticulo-endothelial organs, such as the spleen and liver, without proper cooking7). Brucella organisms are non-
motile gram-negative coccobacilli or short small rods measuring about 0.6- 1.5 × 0.5-0.7 μm without bipolar staining, not
encapsulated bacteria8). There are six recognized species of the genus which include Brucella abortus (B. abortus), Brucella
melitensis (B. melitensis), Brucella ovis (B. ovis), Brucella canis (B. canis), Brucella neotome (B. neotome) and Brucella suis
(B. suis)9). In six recognized Brucella species, B. abortus, B. melitensis, B. ovis and B. suis are responsible for bovine, ovine
and caprine, and swine brucellosis, respectively 10,11).
Due to abuse and overuse of antibiotics in animal agriculture and medicine, antibiotic-resistant strains of Salmonella and
Brucella are increasing12,13). Therefore, the effective cleaning and disinfection are essential measures for the prevention of
infections and outbreaks. The choice of proper biocides is very important for the cleaning and disinfectant regimes. In food
production, hospital settings, and the home, the increase of biocide use is contributing to the emergence of biocide resistant
strains14). Thereby, biocides are often composed of a mixture of ingredients that act upon a wide range of cellular mechanisms
and targets, which makes it difficult for bacteria to become resistant to biocides12).
In the world, salmonellosis and brucellosis cause significant economic loss in agriculture and the food industry15,16). The use of
disinfectant is very effective for successful control of diseases from bacteria, fungi and parasites17,18). In the food industry,
regular cleaning and disinfection are the most efficient way to control pathogenic bacteria that are prevalent in food and the
environment19,20). There are many kinds of disinfectants for food utensils and premises, such as alcoholic solutions and
hypochloric solutions, including organic acid, sodium hypochlorite, alcohol-based products and quaternary ammonium
compounds20,21). However, there is not the efficacy test for the disinfectant spray containing the grapefruit-seed extract, citric
acid, malic acid and benzalkonium chloride (BAC) against foodborne pathogenic bacteria. Therefore, this study was carried out
to examine bactericidal efficacy of a disinfectant spray against S. Typhimurium and B. ovis.

Materials and Methods Other Sections

Bacteria and culture

The test bacteria, S. Typhimurium (G-B-14-21-62) and B. ovis (ATCC 25840) were obtained from the Korean Veterinary Culture
Collection (KVCC, Seoul, Korea). The strains were maintained as frozen glycerol stock. S. Typhimurium were cultured in Luria-
Bertani (LB) broth containing 1.5% agar. B. ovis were spread in Brucella broth containing 5% fetal bovine serum and incubated
at 37°C under 5% CO2 condition.

Disinfectant spray
A disinfectant spray (grapefruit-seed extract 750 mg, citric acid 600 mg, malic acid 187.5 mg and BAC 75 mg per 300 mL) was
provided by Korea Thumb Vet Co. Ltd. (Iksan, Korea). The disinfectant was stored in the dark in room temperature and
prepared for dilution on the day of evaluation. Determination of the bactericidal efficacy of the disinfectant was based on Animal
and Plant Quarantine Agency (Anyang, Korea), Regulation No. 2013-3422).

Diluents and treatment condition

Testing was based on bactericidal effects of the disinfectant diluents in two treatment conditions (hard water (HW) and organic
matter (OM) condition) and pathogen control (sanitizer negative control) in Table 1. HW, an ingredient of HW treatment
condition, was made by adding anhydrous CaCl2 0.305 g and MgCl2·6H2O 0.139 g into one liter distilled water. Organic
suspension, an ingredient of OM treatment condition, is a solution of 5% (w/v) yeast extract in HW. The test organisms were
prepared by titration of each cultural broth into at least 108 colony-forming units (CFU)/ mL viable organisms with the same
kind of diluents of treatment condition.

Experimental procedures
For the efficacy test against S. Typhimurium, the disinfectant was diluted 3, 4, 5, 6, 7 and 8 times with HW, and diluted 1, 2, 3,
4, 5 and 6 times with OM, respectively. For the efficacy test against B. ovis, the disinfectant was also diluted 2, 3, 4, 5, 6 and 7
times with HW, and diluted 1, 2, 3, 4, 5 and 6 times with OM, correspondingly.
To verify the lowest effective dilution of the disinfectant, five serial dilutions of the disinfectant were prepared and placed at 4°C
prior to test reaction. 2.5 mL of each the disinfectant diluent was mixed with the same amount of test organism followed by
contact time of 30 min at 4°C. During this period, the mixture was shaken at 10 min interval. At the end of 30 min contact time,
one mL of the mixture was neutralized with 9 mL of nutrient broth containing 5% inactivated horse serum (BD Korea Co., Ltd.,
Incheon, Korea) at 37°C. 0.1 mL of the neutralized reaction mixture was subcultured into 10 mL of each recovery cultural broth
at 37°C for 48 h in incubator. For the test of each bacteria control, 2.5 mL of HW was mixed with the same amount of each test
organism followed by contact time of 30 min at 4°C, and then all procedure were undertaken in parallel for the disinfection test.
The valid dilution of the disinfectant was determined that the greatest dilution showing no growth in four or more in the five
replicates was confirmed. The final dilution time was statistically determined by a median value among three valid dilution of
the triplicate test, but each value of which should be within 20% experimental error. In each bacteria control, the number of
bacterial growth in the five replicates was counted.

Results Other Sections

Table 2 shows the final valid dilution of the disinfectant spray. When the bactericidal effect on HW condition was evaluated, the
antibacterial activity of the disinfectant showed on 5 and 2 fold dilutions against S. Typhimurium and B. ovis, respectively. With
the investigation of the bactericidal effect of the disinfectant on OM condition, S. Typhimurium and B. ovis were inactivated on 4
and 1 fold dilutions, individually. Because the organic material interferes with the efficacy by either inactivating the disinfectant
or blocking it from surface contact, the bactericidal activity of the disinfectant on the OM condition was lowered against
pathogenic bacteria compared with HW condition.
Comparing the results of the disinfectant against two pathogenic bacteria in the present study, the bactericidal effect against S.
Typhimurium was higher than that against B. ovis on the HW and OM condition. In each bacterial control, the growth of S.
Typhimurium and B. ovis was verified in all replicates.

Discussion Other Sections

In food-processing environments, the ability to persist and multiply makes contamination by foodborne pathogens difficult to
control and eradicate. Therefore, food-processing facilities must be designed carefully, with an emphasis on effective cleaning
and disinfection operations in the production line23). The use of efficient cleaning agents and disinfectants on surface-attached
microbes minimizes contamination of the product, enhances shelf life and reduces the risks of foodborne illness24).
Various methods are used during cleaning and disinfection processes, to sanitize food-processing environments through the
use of alkaline and acid detergents, and to control the organism residing in the food and the processing devices through the
use of a wide range of chemical disinfectants25).
In a previous study26), a grapefruit-seed extract had antimicrobial properties against a wide range of gram-negative and gram-
positive organisms at dilutions found to be safe. In addition, organic acids, including citric acid and malic acid have been used
to control foodborne pathogenic bacteria such as Listeria monocytogenes, Escherichia coli and S. Typhimurium27,28).
Furthermore, BAC is a quaternary ammonium compound that is widely used as disinfectant and cationic surface active agent
for sanitation in foodprocessing lines and surfaces in the food industry29), as clinical disinfectant and antiseptic in health care
facilities and domestic households30).
A previous study reported that the log-reduction was 6.72 after 99 mL of an antiseptic containing 85% ethanol was interacted
with 1 mL of S. Typhimurium suspension for 15 sec31). In another previous study using a disinfectant containing 10% (w/v)
povidone-iodine11), the bactericidal activity against S. Typhimurium and B. ovis on HW condition was 400 and 150 fold
dilutions, respectively and 5 and 20 fold dilutions on OM condition, respectively. In addition, a disinfectant (chlorine dioxide
0.01% (v/v), betaine HCl 0.5% (v/v) and propylene glycol 0.30% (v/v)) had a bactericidal activity against S. Typhimurium and B.
ovis with 2.5 and 1.1 fold dilutions on HW and OM condition, respectively32).
Considering the ingredient content and components of above disinfectants, bactericidal activity of the disinfectant spray used
for this study was greater than that of the antiseptic composed of ethanol and the disinfectant composed of chlorine dioxide,
betaine HCl and propylene glycol. On the other hand, compared with a disinfectant containing 10% povidone-iodine, the
disinfectant spray had a low bactericidal activity on both HW and OM condition.
With the results from this study, the disinfectant spray as an effective and safe disinfectant can be applied to control foodborne
pathogens in food-processing environments. In the present study, the disinfectant efficacy of the disinfectant spray has a
limitation that the results are based on in vitro test. Organic material in suspension (OM condition) could not represent all
possible parameters of Salmonella and Brucella contaminated food-industry environment.

Conclusions Other Sections

In the food industry, salmonellosis and brucellosis were very important diseases because of high mortality for farmed animals,
zoonoses and economic loss. In the study of the bactericide efficacy test of the disinfectant spray, the results suggest that the
disinfectant has a safe and potential bactericidal activity against S. Typhimurium and B. ovis. So, the disinfectant spray can be
used to control the spread of foodborne pathogens.

국문요약 Other Sections

살모넬라증과 부루셀라증은 가축 및 사람에 심각한 피 해를 유발하는 질병으로, 축산업과 식품산업에 많은 경제 적 손실을 초래

하고 있다. 본 연구에서는, 자몽종자추출물, 구연산, 사과산 그리고 염화벤잘코늄을 주성분으로 하는 스프레이형 소독제의
Salmonella Typhimurium과 Brucella ovis에 대한 효력시험을 수행하였다.
살균효력시험은 배지희석법에 따라 수행하였으며, 스프 레이형 소독제와 시험 세균들을 처리조건에 따라 경수와 유기물로 희석
하여 반응을 시켰다. 유기물 조건에서, Salmonella Typhimurium과 Brucella ovis에 대한 스프레이 형 소독제의 살균력은 경수조
건에서의 살균력과 비교하여 낮게 나타났는데, 이는 유기물들에 의한 소독제의 살균 유 효성분에 대한 저해작용에 따른 것으로
사료된다.
스프레이형 소독제는 Salmonella Typhimurium과 Brucella ovis와 같은 인수공통전염병 유발 병원균들에 대해 살균효 과를 나타
내어, 살모넬라증과 브루셀라증과 같은 세균성 질병의 확산을 제어하는데 효과적으로 이용될 수 있을 것 으로 사료된다.

Acknowledgements Other Sections

This work was financially supported by Korea Thumb Vet Co. Ltd. (Iksan, Korea).

Table Other Sections

Table 1..Experimental design for the determination of the bactericidal efficacy of the disinfectant spray
1)HW, standard hard water; OM, organic matter.
2)+, presence; -, absence

Table 2..Final valid dilution of the disinfectant spray against S. Typhimurium and B. ovis
1)HW, standard hard water; OM, organic matter; BC, bacterial control; DT, dilution time; VD, valid dilution.
2)The number of examinations.
3)○, growth inhibition, no growth of more than four in the five replicates.
 4)×, no growth inhibition, growth of more than two in the five replicates
5)+, all growth in each replicate.

Reference Other Sections

1. Gupta A , Fontana J , Crowe C , Bolstorff B , Stout A , Van Duyne S , Hoekstra MP , Whichard JM , Barrett TJ , Angulo FJ
(2003) Emergence of multidrug-resistant Salmonella enterica serotype Newport infections resistant to expandedspectrum
cephalosporins in the United States , J. Infect. Dis, Vol.188 ; pp.1707-1716
2. Bagheryan Z , Raoof JB , Golabi M , Turner AP , Beni V (2016) Diazonium-based impedimetric aptasensor for the rapid
label-free detection of Salmonella typhimurium in food sample Biosens , Bioelectron, Vol.80 ; pp.566-573
3. Fournier B , Williams IR , Gewirtz AT , Neish AS (2009) Toll-like receptor 5-dependent regulation of inflammation in
systemic Salmonella enterica Serovar typhimurium infection , Infect. Immun, Vol.77 ; pp.4121-4129
4. Ibrahim MA , Emeash HH , Ghoneim NH , Abdel- Halim MA (2013) Seroepidemiological studies on poultry
salmonellosisand its public health importance , J. World's Poult. Res, Vol.3 ; pp.18-23
5. Akpinar O (2016) Historical perspective of brucellosis: a microbiological and epidemiological overview , Infez. Med, Vol.24 ;
pp.77-86
6. Yumuk Z , O'Callaghan D (2012) Brucellosis in Turkey – an overview , Int. J. Infect. Dis, Vol.16 ; pp.e228-e235
7. Memish ZA , Balkhy HH (2004) Brucellosis and international travel , J. Travel Med, Vol.11 ; pp.49-55
8. Hubálek Z , Rudolf I (2011) Microbial zoonoses and sapronoses, Springer, ; pp.214-215
9. Kaltungo BY , Saidu SNA , Musa IW , Baba AY (2014) Brucellosis: A neglected zoonosis , Br. Microbiol. Res. J, Vol.4 ;
pp.1551-1574
10. Cloeckaert A , Grayon M , Gr pinet O , Bounedine KS (2003) Classification of Brucella strains isolated from marine
mammals by infrequent restriction site-PCR and development of specific PCR identification tests , Microb. Infect, Vol.5 ;
pp.593-602
11. Park EK , Cho Y , Lee HJ (2014) Bactericidal efficacy of a disinfectant solution composed of povidine-iodine against
Salmonella typhimurium and Brucella ovis , J. Fd Hyg. Safety, Vol.29 ; pp.165-169
12. Whitehead RN , Overton TW , Kemp CL , Webber MA (2011) Exposure of Salmonella enteric serovar Typhimurium to high
level biocide challenge can select multidrug resistant mutants in a single step , PLoS On, Vol.6 ; pp.e22833
13. Turkmani A , Psaroulaki A , Christidou A , Samoilis G , Mourad TA , Tabaa D , Tselentis Y (2007) Uptake of ciprofloxacin
and ofloxacin by 2 Brucella strains and their fluoroquinolone- resitant variants under different conditions. An in vitro study ,
Dign. Microbiol. Infect. Dis, Vol.59 ; pp.447-451
14. Russell AD (2003) Biocide use and antibiotic resistance: the relevance of laboratory findings to clinical and environmental
situations , Lancet Infect. Dis, Vol.3 ; pp.794-803
15. Giammanco G , Pignato S , Giammanco GM (1999) Recent trends in salmonellosis epidemiology , J. Prev. Med. Hyg,
Vol.40 ; pp.19-24
16. Munozdel RM , Montano MF , Renteria TB , Sanchez E , Moreno JF , Perez A , Saucedo S (2007) Assessment of the
economic impact of a brucellosis control program in a dairy herd using the partial budget method , J. Anim. Vet. Adv, Vol.6 ;
pp.146-151
17. Kahrs RF (1995) General disinfection guidelines , Rev. Sci. Tech, Vol.14 ; pp.105-163
18. Ahmad K (2005) Control of animal diseases caused by bacteria: Principles and approaches , Pak. Vet. J, Vol.25 ; pp.200-
202
19. Todd EC , Michaels BS , Holah J , Smith D , Greig JD , Bartleson CA (2010) Outbreaks where food workers have been
implicated in the spread of foodborne disease Part 10. Alcohol-based antiseptics for hand disinfection and a comparison of
their effectiveness with soaps , J. Food Prot, Vol.73 ; pp.2128-2140
20. Walton JT , Hill DJ , Protheroe RG , Nevill A , Gibson H (2008) Investigation into the effect of detergents on disinfectant
susceptibility of attached Escherichia coli and Listeria moncytogenes , J. Appl. Microbiol, Vol.105 ; pp.309-315
21. Carson RT , Larson E , Levy SB , Marshall BM , Aiello AE (2008) Use of antibacterial consumer products containing
quaternary ammonium compounds and drug resistance in the community , J. Antimicrob. Chemother, Vol.62 ; pp.1160-1162
22. (2013) Animal and Plant Quarantine Agency (QIA) The guideline for disinfectant efficacy tests against bacteria , QIA
Regulation No. 2013-34. QIA Anyang Korea,
23. Zoz F , Iaconelli C , Lang E , Iddir H , Guyot S , Grandvalet C , Gervais P , Beney L (2016) Control of relative air humidity as
a potential means to improve hygiene on surfaces: Preliminary approach with Listeria monocytogenes , PLoS One, Vol.11 ;
pp.e0148418
24. Wirtanen G , Salo S (2003) Disinfection in food processing - ecacy testing of disinfectants , Rev. Environ. Sci. Biochenol,
Vol.2 ; pp.293-306
25. Van Houdt R , Michiels CW (2010) Biofilm formation and the food industry, a focus on the bacterial outer surface , J. Appl.
Microbiol, Vol.109 ; pp.1117-1131
26. Heggers JP , Cottingham J , Gusman J , Reagor L , McCoy L , Carino E , Cox R , Zhao JG (2002) The effectiveness of
processed grapefruit-seed extract as an antibacterial agent: II. Mechanism of action and in vitro toxicity , J. Altern.
Complement Med, Vol.8 ; pp.333-340
27. Over KF , Hettiarachchy N , Johnson MG , Davis B (2009) Effect of organic acids and plant extracts on Escherichia coli
O157:H7, Listeria monocytogenes, and Salmonella Typhimurium in broth culture model and chicken meat systems , J. Food
Sci, Vol.74 ; pp.M515-M521
28. Kang S , Jang A , Lee SO , Min JS , Kim IS , Lee M (2003) Effect of organic acids on microbial populations and Salmonella
typhimurium in pork loins , Asian-Aust. J. Anim. Sci, Vol.16 ; pp.96-99
29. Kuda T , Yano T , Kuda MT (2008) Resistances to benzalkonium chloride of bacteria dried with food elements on stainless
steel surface , LWT, Vol.41 ; pp.988-993
30. Mangalappalli-Illathu AK , Korber DR (2006) Adaptive resistance and differential protein expression of Salmonella enterica
serovar Enteritidis biofilms exposed to benzalkonium chloride , Antimicrob. Agents Chemother, Vol.50 ; pp.3588-3596
31. Kampf G , Hollingsworth A (2008) Comprehensive bactericidal activity of an ethanol-based hand gel in1 5 seconds , Ann.
Clin. Microbiol. Antimicrob, Vol.7 ; pp.2
32. Cha CN , Lee YE , Kang IJ , Yoo CY , An S , Kim S , Lee HJ (2012) Bactericidal efficacy of Vital-Oxide®, disinfectant
solution against Salmonella typhimurium and Brucella ovis , J. Fd Hyg. Safety, Vol.27 ; pp.50-54

Copyright © The Korean Society of Food Hygiene and Safety. All rights reserved.
#1110, Huanghua Building, 320, Gangnam-daero, Gangnam-gu, Seoul 06252, Korea

Tel: 82-2-566-0417 Fax: 82-2-566-4417 E-mail: foodhygiene@paran.com