Chapter 4

Organic Acids, Detergents, and Their

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Combination for Inactivation of Foodborne

Pathogens and Spoilage Microorganisms
Dong Chen1 and Tong Zhao*,2
1College of Food Science, Southwest University, 2 Tiansheng Road,
Natural and Bio-Based Antimicrobials for Food Applications

Beibei, Chongqing 400715, China

2Center for Food Safety, College of Agricultural and Environmental

Sciences, University of Georgia,

1109 Experiment Street, Griffin, Georgia 30223, United States
*E-mail: tongzhao@uga.edu.

Organic acids have been broadly used by meat and poultry

industries as a spray treatment of meat carcasses for reduction
of foodborne pathogens. Their benefits, efficacy, and problems
were evaluated by different studies. The combination of
levulinic acid and sodium dodecyl sulfate (SDS) has recently
received attention for its antimicrobial efficacy against
several bacteria and viruses in food matrices or on multiple
food and non-food surfaces. Both ingredients are an FDA
approved flavoring substance and multipurpose food additive,
respectively. Synergism between levulinic acid and SDS was
observed, and the assumed mechanism of action was presented.
The antimicrobial efficacy of levulinic acid and SDS remained
high even when organic materials are present. The other
features, including foamability and readily solubility, extend
its potential applications to decontamination of hard-to-reach
surfaces and control of foodborne pathogens on food contact
surfaces.

© 2018 American Chemical Society

 Inactivation of Foodborne Pathogens with Organic Acids
Organic acids, because of their low pH, have been documented inhibitory
or lethal on various foodborne pathogens. Among them, acetic acid, citric acid,
octanoic acid, lactic acid, levulinic acid, propionic acid, and phenyllactic acid are
revealed possessing bactericidal effects. Lactic acid is often the acid of first choice
because it is odorless compared with other acids (1). The general explanation of
bactericidal mechanism is the reduction of medium pH resulting in a decrease of
the cytoplasmic pH of microbes by ionization of undissociated acid molecules.
In order to maintain intracellular pH close to neutrality, bacterial cells have to
pump out protons by hydrolyzing ATP, which depletes energy causing their death
eventually (2–4).
Results from a recall of ground beef containing Escherichia coli O157:H7
indicate that the contamination level was low and the selective enrichment was
necessary for its isolation (5). Our investigation on the largest outbreak of E. coli
O157:H7 linked to consumption of ground beef revealed that the median most
probable number of the pathogen was 1.5 cells/g (range, <0.3-15 cells/g) or 67.5
cells per patty (range, <13.5-675 cells/patty) (5). Since then the meat industry has
been making an enormous effort trying to eliminate E. coli O157:H7 from meat.
However, very low numbers can still remain in meat, especially in summer months
when shedding from cattle is high.
Meat and poultry processing facilities use a variety of intervention
strategies at critical control points to control E. coli O157:H7 and Salmonella.
These protocols include the application of Food and Drug Administration
(FDA)-approved organic acids surface sprays and rinses to inhibit the growth
and/or kill foodborne pathogens, biosecurity measures, zero tolerance for visible
fecal materials, making sure carcasses are properly chilled, and strict sanitation
procedures. However, because of the nature of meat and poultry processing
facilities and the high speed at which meat and poultry carcasses are produced
and processed, it is difficult to completely prevent the spread of E. coli O157:H7
and/or Salmonella that may be introduced by incoming cattle or birds.
Various organic acids have been applied by meat and poultry industries
for reduction/elimination of E. coli population, especially E. coli O157:H7,
on fresh meats. Studies have revealed that a 2% lactic acid antibacterial rinse
applied for 3 minutes in a poultry slaughter plant reduced aerobic plate count
(APC) and coliform populations by >2 log CFU/g, whereas a solution containing
50-100 ppm of chlorine reduced APC by 0.4 log CFU/g (6). However, studies on
decontamination of E. coli O157:H7 on beef trim with hot organic acid surface
sprays, including acetic, citric, and lactic acid at concentrations up to 1.5%,
revealed that E. coli O157:H7 populations on beef trim did not significantly (P >
0.05) change before and after the hot surface spray treatment (7).
Recent studies on the efficacy of acid-based intervention for reduction of
Shiga toxin-producing E. coli (STEC), especially for E. coli O157:H7, in meat
processing facilities have revealed that these bacteria have unique acid-tolerance
characteristics, and have been associated with various acidic foods, including
apple cider (8), sausages (9), and tomato ketchup (10). Currently, the U.S.
Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS)

64
considers E. coli O157:H7 and six non-O157 serotypes of STEC, including O26,
O45, O103, O111, O121, and O145 strains, as adulterants in both raw ground and
whole muscle cuts (11).

Reduction of Detergents on Foodborne Pathogens

Based on their chemical structure, detergents can be classified into three
species, including 1) anionic detergents with a structure of alkylbenzenesulfonate,
2) cationic detergents with a structure of quaternary ammonium, and 3) non-ionic
and zwitterionic detergents with a structure of the uncharged hydrophilic group.
The main purpose of their application in the food industry is for cleaning of food
facilities with low inhibitory effect on foodborne pathogens.
In 1961, the United States Public Health Service recommended that personnel
need to wash their hands with soap and water for 1-2 minutes before and after
client contact. The result indicated that the antibacterial soaps can remove 65
to 85% bacteria from human skin (12). Food handlers, including those who
deliver and serve foods, should follow this recommendation to reduce potential
cross-contamination. A recent study indicates that some bacterial strains, such as
Pseudomonas aeruginosa, may become detergent-resistant, which leads to their
survival even in the presence of high concentration of soaps (13).

Combination of Levulinic Acid plus Sodium Dodecyl Sulfate

Background Information
One chemical treatment that in recent years has shown considerable
promise as an antimicrobial intervention is a combination of levulinic acid
and sodium dodecyl sulfate (SDS). Levulinic acid (4-oxopentanoic acid,
CH3C(O)CH2CH2CO2H, molecular mass: 116.12 Da) is a 5-carbon organic acid
which has been designated as Generally Recognized as Safe (GRAS, 21 CFR,
172.515) by FDA for direct addition to food products as a flavoring agent (14).
It can be produced at a low cost yet in a high yield from renewable feedstocks
(15). Addition of sodium levulinate in ready-to-eat (RTE) meats was effective
at inhibiting growth of spoilage bacteria and Listeria monocytogenes without
a noticeable different flavor (16, 17). S. Enteritidis populations in pure culture
held at 21°C were reduced by 3.4 log CFU/ml when exposed to 0.3% levulinic
acid for 30 minutes (18). SDS, an anionic surfactant, is designated by FDA
as a multipurpose food additive (21 CFR 172.822). It is approved for use in a
variety of foods, including egg whites, fruit juices, vegetable oils, and gelatin as
a whipping or wetting agent, and also widely used in household products such as
toothpastes, shampoos, shaving creams, and bubble baths (18).
Various microbial isolates have been individually evaluated for their
sensitivity to levulinic acid with SDS. Our results reveal that all bacterial isolates
tested are killed instantly within 10-15 seconds of contact with levulinic acid
with SDS while retaining the quality of treated products. The microorganisms
65
tested include but not limited to 1) yeasts and molds, such as Candida magnoliae,
Debaryomyces hansenii, Geotrichum candidum, Saccharomyces cerevisiae,
and Zygosaccharomyces bailii; 2) viruses, such as norovirus; 3) bacterial
spores, such as Bacillus anthracis spores; 4) bacteria, such as Aerococcus
viridans, B. anthracis, Campylobacter jejuni, Citrobacter freundii, E. coli
O26:H11, O111:NM and O157:H7, L. monocytogenes, Klebsiella oxytoca and K.
pneumoniae, S. Enteritidis, S. Medegridis, S. Montevideo, and S. Typhimurium,
Staphylococcus aureus, Streptococcus gordonii, S. mitis, S. mutans and S. oralis,
Vibrio cholerae, Yersinia pestis and Y. pseudotuberculosis; 5) plant pathogens,
such as Acidovorax avenae subsp. citrulli, Erwinia carotovora, Xanthomonas
campestris, Xanthomonas axonopodis, and Pantoea ananatis. Unfortunately,
this novel disinfectant cannot be used to inactivate foodborne parasites (19, 20).
Cryptosporidium and microsporidia are not inactivated when treated for various
periods of time with 2% levulinic acid and 1% SDS or 3% levulinic acid and 2%
SDS at 20°C (20).

Inactivation on Yeasts and Molds

The pure culture of five isolates of yeasts, including Saccharomyces
cerevisiae, Debaryomyces hansenii, Candida magnoliae, Zygosaccharomyces
bailii, and Geotrichum candidum, was individually determined at 21°C for
its sensitivity to the solution containing levulinic acid plus SDS at different
concentrations. Results revealed that the inactivation of levulinic acid plus SDS
on yeasts was concentration- and species-dependent. As shown in Table 1, the
killing effect of 0.5% levulinic acid plus 0.05% SDS is weak, and a longer contact
time (>10 minutes) was required for a significant (P < 0.05) reduction. Some
of them, especially Z. bailii, require high concentrations of 2% levulinic acid
plus 1% SDS and long contact time (20 minutes) to reach a reduction of >5 log
CFU/ml.
The pure culture of four mold isolates, including Mucor hiemalis, Penicillium
pubeses, P. expansum, and Paecylomyces variotri, was individually determined at
21°C for its sensitivity to levulinic acid plus SDS at different concentrations. As
shown in Table 2, the killing effect on molds was various and species-dependent.
Generally, the combination with higher concentrations would achieve a greater
reduction. Some of them (e.g. M. hiemalis) was more resistant, suggesting that
other factors, like heating, could be considered if mold contamination is the sole
concern.

Virucidal Efficacy
Virucidal efficacy of levulinic acid plus SDS was tested, with such that 5 %
levulinic acid plus 2 % SDS inactivated murine norovirus (MNV-1) surrogate
by 2.5 1og PFU/ml, and stainless steel-inoculated MNV-1 by >1.50 log PFU/ml,
after 1 min of exposure (21). Lower concentrations of 0.5% levulinic acid plus
0.5% SDS induced 2.7-, 1.4-, and 2.4-log PFU/ml reductions for hepatitis A virus
66
(HAV), MNV-1, and MS2 bacteriophage, respectively, after 2 min of exposure
(22). The virucidal efficacy of this combination was not significantly affected by
the presence of organic matters (up to 10%), indicating this combination could be
effective for reducing infectious virus in a clinical matrix, where stool or vomit,
which can protect viruses from inactivation by a sanitizer, often exists (21). High
concentrations of 5% levulinic acid plus 2% SDS was also determined to be
virucidal for influenza A H3N2 virus (23). Thus, this novel disinfectant may
be an alternative sanitizer that can be used as a part of an egg decontamination
strategy and as a tool to mitigate human and avian influenza transmission by eggs.

Inactivation on Bacterial Spores

Isolates of B. cereus, B. subtilis, B. circulans and Alicyclobacilli

acidoterrestris were individually induced to form spores by standard procedures.
These spores were treated by heat at 65°C for 30 minutes for inactivation of
germinated bacteria. Results revealed that the killing effect of this formulation
(levulinic acid plus SDS) on Bacillus species was variable, even among the
same species. The killing effect is very high on spores induced from isolates of
B. subtilis (ATCC #82), even at low concentrations of 0.5% levulinic acid plus
0.05% SDS. All tested spores (>7 log CFU/ml) were instantly inactivated (12-15
seconds of processing time) at 21°C. However, the killing effect at 21°C on
spores induced from isolates of B. subtilis (ATCC #31028) was very weak, even
at high concentrations of 20% levulinic acid plus 3% SDS for an extended contact
time (120 minutes). Similar results were demonstrated for spores induced from
isolates of B. circulans (#47-10), B. cereus (ATCC #10987), and A. acidoterrestris
(#SAC, #OS-CAJ, and #N-110). Inactivation of spores by 3% levulinic acid
plus 2% SDS with the treatment of raised temperatures at 62, 70, and 80°C for
different contact times was also determined. Results revealed that the treatment
of 3% levulinic acid plus 2% SDS at 62°C instantly killed all the tested spores
induced from different Bacillus species, but the spores induced from isolates
of A. acidoterrestris species were more resistant to the same treatment. The
killing effect was directly related to the temperature. At 80°C, all tested spores
even induced from isolates of A. acidoterrestris were inactivated within 1 min.
These results indicate that the treatment composed of levulinic acid plus SDS is a
powerful disinfection agent for inactivation of spores when it is used with heating.

67
 Table 1. Effect of Levulinic Acid Plus Sodium Dodecyl Sulfate (SDS) at Different Concentrations at 21°C on Various Yeast Species
Yeasta Treatments Yeast counts (log CFU/ml) at minute:
0b 1 2 5 10 20 30 60
Saccharomyces cerevisiae 0.1 M PBS (control) 5.2 5.3 5.5 5.3 5.3 5.2 5.3 5.3
2.0% levulinic acid 5.4 5.3 5.5 5.4 5.3 5.3 5.5 5.0
1.0% SDS 2.7 2.4 2.6 2.3 2.8 2.7 2.3 2.4
0.5% levulinic acid 4.9 4.5 3.9 3.2 2.7 1.7 1.3 <0.7
plus 0.05% SDS
2.0% levulinic acid 0.7 -c - - - - - -
plus 1.0% SDS
Debaryomyces hansenii 0.1 M PBS (control) 4.8 4.9 4.9 4.8 4.8 4.8 4.8 4.8
68

2.0% levulinic acid 4.9 4.8 4.9 4.6 4.4 4.7 3.0 1.3
1.0% SDS 4.5 4.5 4.1 4.5 4.4 4.5 4.5 4.4
0.5% levulinic acid 4.9 4.9 4.7 4.5 4.3 3.7 3.1 1.7
plus 0.05% SDS
2.0% levulinic acid 1.0 - - - - - - -
plus 1.0% SDS
Candida magnoliae 0.1 M PBS (control) 5.9 5.8 6.1 5.9 5.9 5.9 5.9 5.7
2.0% levulinic acid 6.0 5.9 5.9 5.9 6.0 6.0 5.9 5.8
1.0% SDS 3.5 3.5 3.3 3.2 3.2 2.7 3.0 3.1
0.5% levulinic acid 4.0 3.6 3.2 2.1 1.3 <0.7 <0.7 <0.7
plus 0.05% SDS
 Yeasta Treatments Yeast counts (log CFU/ml) at minute:
0b 1 2 5 10 20 30 60
2.0% levulinic acid 2.1 0.7 - - - - - -
plus 1.0% SDS
Zygosaccharomyces bailii 0.1 M PBS (control) 5.4 5.5 5.6 5.5 5.3 5.4 5.6 5.5
2.0% levulinic acid 5.4 5.4 5.5 5.4 5.4 5.4 5.4 5.3
1.0% SDS 4.6 4.7 4.6 4.6 4.6 4.5 4.5 4.4
0.5% levulinic acid 5.0 5.0 4.8 3.6 3.8 2.3 2.6 <0.7
plus 0.05% SDS
2.0% levulinic acid 4.6 4.2 3.9 2.9 2.0 <0.7 <0.7 <0.7
plus 1.0% SDS
69

Geotrichum candidum 0.1 M PBS (control) 4.6 4.7 4.8 4.7 4.7 4.5 4.6 4.6
2.0% levulinic acid 4.6 4.4 4.4 4.3 4.1 3.8 3.4 2.0
1.0% SDS 3.6 3.8 3.3 3.5 3.7 3.5 3.4 3.3
0.5% levulinic acid 3.0 2.6 2.6 2.4 <0.7 <0.7 <0.7 <0.7
plus 0.05% SDS
2.0% levulinic acid 3.3 <0.7 - - - - - -
plus 1.0% SDS
a Initial inoculation level: Saccharomyces cerevisiae: ca. 7.9 log CFU/ml; Debaryomyces hansenii: ca. 7.9 log CFU/ml; Candida magnoliae: ca. 8.5 log
CFU/ml; Zygosaccharomyces bailii: ca. 7.5 log CFU/ml; Geotrichum candidum: ca. 7.1 log CFU/ml. b The time “0” was delayed by 5 to 10 seconds due
to sample processing. c Negative by both direct plating and enrichment cultures.
 Table 2. Effect of Levulinic Acid plus Sodium Dodecyl Sulfate (SDS) at Different Concentrations at 21°C on Various Mold Species
Molda Treatments Mold counts (log CFU/ml) at minute:
0b 1 2 5 10 20 30 60
Mucor hiemalis 0.1 M PBS (control) 6.1 6.1 6.1 6.2 6.4 6.1 6.1 6.2
3.0% levulinic acid 6.1 6.2 6.2 6.0 5.8 5.1 4.8 4.1
2.0% SDS 6.0 5.8 5.8 6.0 5.9 5.8 5.8 5.4
0.5% levulinic acid plus 5.8 5.9 6.0 6.1 5.5 5.8 5.9 5.4
0.05% SDS
2.0% levulinic acid plus 5.1 5.0 4.9 4.9 4.5 3.7 3.4 2.5
1.0% SDS
3.0% levulinic acid plus 5.6 5.6 5.5 5.0 4.7 4.6 3.2 2.4
70

2.0% SDS
Penicillium pubeseus 0.1 M PBS (control) 4.9 4.8 4.8 4.8 4.9 4.8 4.9 4.7
3.0% levulinic acid 5.2 4.9 4.8 5.2 4.0 3.2 2.7 1.7
2.0% SDS 4.4 4.3 4.4 4.4 4.5 4.5 4.4 4.4
0.5% levulinic acid plus 5.1 5.1 5.1 5.0 5.0 4.9 4.9 4.5
0.05% SDS
2.0% levulinic acid plus 5.2 5.2 5.1 5.0 4.8 4.6 4.4 4.2
1.0% SDS
3.0% levulinic acid plus 3.5 3.2 <0.7 <0.7 <0.7 <0.7 <0.7 <0.7
2.0% SDS
Penicillium expansum 0.1 M PBS (control) 4.4 4.4 4.5 4.8 4.6 4.0 4.5 4.5
 Molda Treatments Mold counts (log CFU/ml) at minute:
0b 1 2 5 10 20 30 60
3.0% levulinic acid 4.3 4.4 4.2 4.0 3.7 3.3 3.3 2.4
2.0% SDS 4.2 4.1 3.8 3.4 3.5 3.5 3.5 3.6
0.5% levulinic acid plus 4.5 3.9 3.6 3.2 2.8 2.7 2.0 2.0
0.05% SDS
2.0% levulinic acids plus 4.1 3.7 3.6 3.4 3.3 3.0 2.5 1.7
1.0% SDS
3.0% levulinic acid plus 3.9 <0.7 <0.7 <0.7 <0.7 <0.7 <0.7 <0.7
2.0% SDS
Paecylomyces variotri 0.1 M PBS (control) 5.5 5.5 5.7 5.4 5.5 5.5 5.5 5.5
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3.0% levulinic acid 5.6 5.6 5.4 5.6 5.6 5.3 5.4 4.6
2.0% SDS 5.6 5.5 5.5 5.4 5.4 5.6 5.6 5.6
0.5% levulinic acid plus 5.2 5.2 4.8 4.5 4.1 4.4 3.8 3.4
0.05% SDS
2.0% levulinic acid plus 4.3 3.8 3.4 3.0 2.7 2.3 1.5 0.7
1.0% SDS
3.0% levulinic acid plus 4.6 4.4 4.4 3.6 2.9 2.2 <0.7 <0.7
2.0% SDS
a Initial inoculation level: Mucor hiemalis: ca. 7.5 log CFU/ml; Penicillium pubeseus: ca. 7.8 log CFU/ml; Penicillium expansum: ca. 7.5 log CFU/ml;

Paecylomyces variotri: ca. 7.4 log CFU/ml. b The time “0” was delayed by 5 to 10 seconds due to sample processing.
Bactericidal Efficacy on Food Products

The levulinic acid plus SDS solution is substantially effective against bacterial
foodborne pathogens on the surface of a variety of foods (18). As indicated in
Table 3, the mean count of S. Typhimurium on apples treated with water only for
1, 2, and 5 min was 2.7, 2.7, and 2.7 log CFU/apple, respectively. The mean count
of S. Typhimurium on apples treated with 0.5% levulinic acid plus 0.05% SDS
was <0.7, 1.4, and <0.7 log CFU/apple, with reductions of >2.0, 1.3, and >2.0
log CFU/apple, respectively. Similar trends were obtained with the observation of
aerobic bacterial counts. However, it took 5 minutes to achieve a significant (P <
0.05) reduction of yeast and mold on apples. Following the treatment, the counts
in the treated solution containing 0.5% levulinic acid plus 0.05% SDS were <0.7
log CFU/ml of S. Typhimurium and 1.7 log CFU/ml of yeast and mold (Y&M);
the treated solution containing 50 ppm of chlorine were <0.7 log CFU/ml of S.
Typhimurium and 1.6 log CFU/ml of Y&M; and the treated solution with water
only were 2.7 log CFU/ml of S. Typhimurium and 1.6 log CFU/ml of Y&M. The
counts of S. Typhimurium on apples treated for 1, 2, and 5 min with 50 ppm
of chlorine was 3.3, 3.1, and 2.8 log CFU/apple, respectively, without showing
significant (P > 0.05) reductions.
The mean count of S. Typhimurium on celery treated with water only for
1, 2, and 5 min was 3.7, 3.6, and 3.5 log CFU/celery, respectively; on celery
treated with 0.5% levulinic acid plus 0.05% SDS was 1.1, 1.0, and 1.3 log CFU/
celery, respectively. Thus, reductions of S. Typhimurium for 2.6, 2.6, and 2.2
log CFU/celery, respectively, were achieved on celery after 1, 2, and 5 min of
the treatment of low concentrations of levulinic acid and SDS. Similar results
were obtained for aerobic bacterial count and yeast and mold reductions (Table
4). The S. Typhimurium count in groups treated with 50 ppm of chlorine on
celery for 1, 2, and 5 min was 3.4, 3.1, and 3.0 log CFU/celery, respectively.
Therefore, the reduction of S. Typhimurium for <0.5 log CFU/celery was achieved.
Following treatments, the microbial counts in the treated solution containing 0.5%
levulinic acid plus 0.05% SDS were <0.7 log CFU/ml of S. Typhimurium and 1.3
log CFU/ml of Y&M; the treated solution containing 50 ppm of chlorine was <0.7
log CFU/ml of S. Typhimurium and 2.3 log CFU/ml of Y&M; and the treated
solution containing water only was 3.2 log CFU/ml of S. Typhimurium and 3.5
log CFU/ml of Y&M.
A reduction of 4.2 log CFU/g of Salmonella on lettuce was achieved after
a 1-min treatment of 0.3% levulinic acid and 0.05% SDS (18). Treatment of
alfalfa seeds with 0.05% levulinic acid and 0.05% SDS for 5 min at 21°C reduced
populations of S. Typhimurium and E. coli O157:H7 by 6.4 and 5.6 log CFU/g,
respectively (24). Lethality of levulinic acid plus SDS on pecans is also tested.
The population of Salmonella on in-shell nuts was reduced by 3.4 log CFU/g
after treating with 2% levulinic acid plus 0.05% SDS, compared to 2.8 log CFU/g
caused by chlorinated water (1,000 µg/ml) (25).
Reductions of Salmonella on poultry carcasses with feathers (26) and poultry
skin (18) by 5-min immersion at 21°C with higher concentrations of 3% levulinic
acid and 2% SDS were greater than 4 log CFU/9 cm2 and 5 log CFU/g, respectively,
indicating the antibacterial efficacy of this combination could maintain even in the

72
presence of organic matters. S. Enteritidis on chicken wings was reduced by >5
log CFU/g by the treatment with 3% levulinic acid plus 1% SDS at 8°C (18).
When chicks were challenged with S. Heidelberg, drinking water containing 0.5%
levulinic acid and 0.0125% SDS significantly (P < 0.05) reduced the percentage
of crops infected with S. Heidelberg, but did not significantly (P > 0.05) affect the
percentage of S. Heidelberg-positive ceca (27). The same combination of levulinic
acid and SDS reduced planktonic E. coli O157:H7 by >7 log CFU/ml within 30
minutes (20).
Lower concentrations of 1% levulinic acid plus 0.1% SDS had a great effect
on reducing Salmonella (0.70 log CFU/g) on beef patties, but levulinic acid and
SDS-treated beef patties had increased growth of psychrotrophic organisms and
reduced color scores after 3 days of retail display (28). On the contrary, when it
was used as a spray treatment on beef trim, the combination did not change any
meat quality (29).
Using a single hurdle approach, treatment of cantaloupes with high
concentrations of levulinic acid and SDS provided greater reductions of
Salmonella and L. monocytogenes than 200 ppm of chlorine for netted rind and
stem scar tissues (30). Incorporation of double hurdles of chlorine and levulinic
acid/SDS treatments resulted in the elimination of S. Poona from 50% of samples
(31). Another double hurdle approach employing 7.5% levulinic acid plus 0.5%
SDS, followed by either chlorine or levulinic acid/SDS provided additional
reductions of L. monocytogenes at stem scars and rinds compared to the treatment
of the cantaloupe with chlorine only (32). Inoculated stem scars of tomatoes and
blueberries were also tested, with 2.6-, <1.5-log reductions of Salmonella and E.
coli O157:H7, respectively, achieved by low concentrations of levulinic acid and
SDS (33, 34).

Inactivation on Bacterial Biofilm

Levulinic acid and SDS have also shown a potential to be used as an

anti-biofilm agent. The combination was effective in a concentration-dependent
manner against biofilms grown on stainless steel formed by three major
foodborne pathogens (Salmonella, E. coli O157:H7 and L. monocytogenes)
compared to other commonly used sanitizers, including a commercial quaternary
ammonium-based sanitizer (150 ppm), lactic acid (3%), sodium hypochlorite
(100 ppm), and hydrogen peroxide (2%), with mature biofilms possessing greater
resistance (35, 36). In another study, the killing effect of levulinic acid plus SDS
on well-formed Salmonella biofilms (48 h at 37°C) on coupons (4 by 2.5 cm)
composed of various materials was extensively evaluated and compared with
acidified sodium chlorite (500 ppm, pH 2.8). As indicated in Table 5, levulinic
acid plus SDS treatment achieved greater reductions than acidified sodium
chlorite (500 ppm, pH 2.8) treatment on Salmonella biofilms formed on a variety
of abiotic surfaces commonly used in the food industry.

73
 Table 3. Microbial Counts on Apples Treated by Different Chemicals at 21°C for Different Times in A 4-L Tank
Treated by 0.5% levulinic acid plus Treated with 50 ppm acidified sodium
Treated with water only
0.05% SDS, pH 3.1 chlorite, pH 4.6
Apple Timing
group a (Min) Microbial counts (log CFU/whole apple) b

Salmonella ABCc Y&M Salmonella ABC Y&M Salmonella ABC Y&M

1 1 2.7 3.6 3.4 0.7 <1.7 3.1 3.3 3.7 3.2
2 2.7 3.6 2.8 1.3 2.7 3.9 3.4 3.7 3.5
5 3.0 5.0 4.0 <0.7 <1.7 2.3 3.2 4.9 3.9
2 1 2.5 3.1 2.8 <0.7 2.7 3.2 3.2 3.4 3.2
2 2.8 3.0 2.8 1.3 1.4 1.0 2.6 2.7 3.6
74

5 2.8 3.0 3.0 1.2 <1.7 1.3 2.9 3.2 3.7

3 1 2.7 2.3 3.0 <0.7 <1.7 3.5 3.3 3.6 2.9
2 2.7 3.1 2.9 1.5 2.5 3.9 3.3 3.4 3.9
5 2.5 2.9 2.9 <0.7 <1.7 2.4 2.4 3.9 3.2
4 1 2.7 2.8 3.0 <0.7 <1.7 3.1 3.2 3.6 3.0
2 2.6 2.9 3.0 1.3 <1.7 3.4 3.1 4.5 3.5
5 2.3 3.4 3.2 <0.7 2.3 2.1 2.7 2.9 3.3
aBackground bacterial counts before inoculation for apple 1: 4.0 log CFU/apple; apple 2: 3.6 log CFU/apple. Following inoculation, S. Typhimurium counts
for apple 1: 4.0 log CFU/apple; apple 2: 4.8 log CFU/apple. b Inoculation level for S. Typhimurium is 6.0 log CFU/ml, and for yeast and mold (Y&M) is
4.6 log CFU/ml. c ABC: aerobic bacterial counts.
 Table 4. Microbial Counts on Celery Treated by Different Chemicals at 21°C for Different Times in A 4-L Tank
Treated by 0.5% levulinic acid plus Treated with 50 ppm acidified
Treated with water only
0.05% SDS, pH 3.1 sodium chlorite, pH 4.6
Celery Timing
groupa (Min) Microbial counts (log CFU/celery) b

Salmonella ABCc Y&M Salmonella ABC Y&M Salmonella ABC Y&M

1 1 3.8 6.0 5.5 2.0 4.5 3.4 3.6 6.4 5.5
2 3.8 6.3 5.5 0.7 4.9 2.3 3.1 5.9 5.1
5 3.7 6.0 4.9 1.5 3.3 3.4 2.8 4.5 4.9
2 1 4.0 5.8 4.4 0.7 3.1 4.1 3.3 5.4 5.2
2 3.6 6.2 5.5 1.4 2.8 4.1 3.1 4.6 5.0
75

5 3.3 5.1 5.2 1.4 2.8 4.1 2.8 5.4 5.0

3 1 3.1 5.3 5.4 1.0 2.7 3.9 3.3 6.1 5.5
2 3.2 4.8 5.1 0.7 3.0 3.9 3.1 5.9 5.4
5 3.4 5.9 5.3 1.4 5.3 4.6 2.9 3.7 5.1
4 1 3.7 5.6 4.9 0.7 3.5 4.5 3.4 6.0 5.5
2 3.7 5.1 4.8 1.2 4.7 3.4 3.2 5.2 5.2
5 3.6 5.1 5.2 1.0 3.6 3.4 3.6 6.2 5.4
aBackground bacterial counts before inoculation for celery 1: 7.0 log CFU/celery; celery 2: 7.0 log CFU/celery. Following inoculation, S. Typhimurium DT
104 counts for celery 1: 5.2 log CFU/celery; celery 2: 4.8 log CFU/celery. b Inoculation level for S. Typhimurium is 6.1 log CFU/ml, and for yeast and mold
(Y&M) is 5.0 log CFU/ml. c ABC: aerobic bacterial counts.
 Table 5. Inactivation of Salmonella Enteritidis in Biofilms by 3% Levulinic Acid plus 2% Sodium Dodecyl Sulfate (SDS)
Coupon type Chemical Counts of Salmonella Enteritidis (log CFU/cm2) at minutes
solution
0a 1 2 5 10 20
Stainless steel PBS, pH 7.2 8.0 8.4 8.5 8.6 8.2 8.1
Acidified sodium chlorite (500 7.5 5.9 5.7 5.4 6.2 6.0
ppm), pH 2.8
3% levulinic acid plus 2% <0.7b <0.7 <0.7 <0.7 <0.7 <0.7
SDS, pH 3.0
Polyvinyl chloride PBS, pH 7.2 8.8 9.0 8.1 8.8 8.0 8.3
Acidified sodium chlorite (500 6.9 5.5 5.8 5.3 4.2 2.9
ppm), pH 2.8
76

3% levulinic acid plus 2% 2.3 1.7 2.0 2.2 <0.7 <0.7

SDS, pH 3.0
Nitrile rubber PBS, pH 7.2 7.8 8.0 8.5 7.7 7.9 7.7
Acidified sodium chlorite (500 7.2 5.2 2.7 2.6 1.3 <0.7
ppm), pH 2.8
3% levulinic acid plus 2% 4.1 1.7 1.7 <0.7 <0.7 <0.7
SDS, pH 3.0
Glass PBS, pH 7.2 8.2 8.7 8.4 8.4 8.4 8.4
Acidified sodium chlorite (500 6.8 3.3 0.7 0.7 <0.7 <0.7
ppm), pH 2.8
3% levulinic acid plus 2% <0.7 <0.7 <0.7 <0.7 <0.7 <0.7
SDS, pH 3.0
 Coupon type Chemical Counts of Salmonella Enteritidis (log CFU/cm2) at minutes
solution
0a 1 2 5 10 20
Ultra-high molecular weight PBS, pH 7.2 8.4 8.4 8.6 8.4 8.4 8.4
polyethylene
Acidified sodium chlorite (500 6.8 6.1 2.1 0.7 <0.7 <0.7
ppm), pH 2.8
3% levulinic acid plus 2% <0.7 <0.7 <0.7 <0.7 <0.7 <0.7
SDS, pH 3.0
a The time “0” was delayed by 35 to 45 seconds due to sample processing. b Not detected by the direct plating method (<0.7 log CFU/cm2).
77
Antimicrobial Mechanism

The application of levulinic acid and SDS alone has limited antimicrobial
efficacy. However, the combination greatly increases the bactericidal (20, 22,
25, 36–39) and virucidal (21, 22) activities of these two chemicals. When
0.5% levulinic acid and 0.05 to 1% SDS used individually, cell numbers of
Salmonella and E. coli O157:H7 were reduced by ≤2 log CFU/ml within 20 min
at 21°C, whereas the combination achieved >7-log CFU/ml inactivation of both
pathogens within 10 seconds (18). Synergism between levulinic acid and SDS
was also found in the case of their antimicrobial efficacy targeting biofilms (36).
Levulinic acid, besides its antimicrobial mechanism of action as described above,
is capable to affect electrostatic charges of SDS molecules and cell surfaces,
chelate metal ions, disrupt substrate transport, reduce proton motive force, and
release lipopolysaccharide from the outer membrane of Gram-negative bacteria
(21, 40–45). All these factors contribute to an increase in cell permeability,
leading to an increase of SDS absorption into cells and damage of the cytoplasmic
membrane (46). SDS can denature proteins, chelate divalent cations, such as
Ca2+ and Mg2+, and damage cell membranes, and its bactericidal and virucidal
effects can be increased when pH is reduced to between 1.5 and 3.0 (40, 47–61).
Therefore, levulinic acid and SDS complement each other enabling penetration
of cells, leading to increased susceptibility (40, 62). When the combination was
applied to inactivate biofilms, SDS, due to its amphiphilic property (63, 64)
and wettability (46, 65), can act as antiadhesive agents enhancing the release of
bacterial cells and viruses tightly bound on the surface into surroundings in the
planktonic state. Levulinic acid may also assist in removal of the attachment
polymer by chelating divalent cations required to link the polymer at the surface
(66, 67). Microscopic images indicate that the cells are detached from biofilm
matrices and the integrity of cell envelopes are decreased after the treatment of
levulinic acid plus SDS (36). Hence, the combination of an organic acid and
surfactant may promote each other to detach bacterial cells from a biofilm matrix.
Once the cells are directly exposed to the chemicals without the protection of
EPS, they can be easily inactivated.

Application of Levulinic Acid Plus SDS

The combination of levulinic acid and SDS exhibits desirable properties

besides its high antimicrobial efficacy. First, both of the two chemicals are
GRAS, so the concern for their safety is limited. Second, levulinic acid can be
cost-effective since it can be produced from cellulose-containing waste materials
and thereby costs can be low (14). The estimated cost of a 3% levulinic acid
preparation is 20 cents per liter based on biosynthesis (15), and because the
antimicrobial activity of the formula is not readily neutralized in the presence of
organic materials, unlike many chemical treatments such as sodium hypochlorite,
the levulinic acid plus SDS treatment can be reused to minimize cost and water
usage (26). Third, the two compounds are readily soluble in water, a property that
allows it to be used in various formats such as solutions, pastes, gels, and foams.
78
Since the format of cleaning and sanitizing chemicals can influence the length
of contact time, it affects the final antimicrobial efficacy. For example, foaming
solutions generally have higher viscosity and hence cling to surfaces longer. They
also access some hard-to-reach surfaces like niches where liquid solutions can
not due to surface tension. Application of levulinic acid and SDS as a foam on
contaminated deli slicers substantially reduced larger populations of Salmonella,
L. monocytogenes, and E. coli O157:H7 than the liquid treatment (38). Fourth,
the antimicrobial activity of the combination of levulinic acid and SDS is not
mitigated by the presence of organic materials, which is a very important feature
for a potential disinfectant applied in hospitals, restaurants, and other public
facilities, where organic matters (e.g. stool, vomit, food soils, debris, and etc.)
and biofilms often exist. Therefore, the combination of levulinic acid and SDS
has a potential to be massively used as an alternative sanitizer in the industry.
Although the combination of levulinic acid and SDS has shown substantial
antimicrobial activity in some studies, currently levulinic acid plus SDS do not
have regulatory approval for application as a sanitizer. Additional studies are
needed to validate the efficacy of levulinic acid and SDS in actual production
operations (18). For reduction of bacterial loads of fresh produce at pre-harvest,
spray treatment of whole tomato plants one hour pre-harvest revealed that total
average aerobic bacterial counts were 5.8 and 4.1 log CFU/tomato before and
after treatment, respectively, with an average reduction of 1.7 log CFU/tomato.
Average coliform counts before and after treatment were 4.2 and 3.0 log CFU/
tomato, respectively, with an average reduction of 1.2 log CFU/tomato. The results
revealed that application of this food-grade bactericide was effective for reduction
of foodborne pathogens and microbial loads on pre-harvested tomatoes (Figure 1).
The combination of levulinic acid and SDS was incorporated into FIT® L
Food and Vegetable Wash products (composed of water, levulinic acid, naturally
derived surfactants, grapefruit oil, and other natural ingredients) by a University
of Georgia Research Foundation’s licensee in 2010. It was granted a utility
patent by the U.S. Patent and Trademark Office (patent NO. 8,722,123) in 2014
(68). Significant reductions of E. coli O157:H7, Salmonella and Shigella on
preinoculated strawberry, cantaloupe, and broccoli with or without presence of
organic matters were observed by a 5-min treatment of FIT® L produce wash
which was diluted 1:88 for usage in the study (69). Inoculated samples were
treated with the diluted levulinic acid wash after storage at 10°C for 24 h. A
200-ppm free available chlorine wash was used as a control. Wash solutions were
prepared using potable water and water with an increased organic content of 2.5
g/liter total dissolved solids and total organic carbon. More than 4.3-log CFU/g
and 3.81- to 3.91-log CFU/g reductions of E. coli O157:H7 on the samples were
achieved in wash solutions containing potable and challenge water, respectively.
The Salmonella counts were reduced by 2.04 - >4.63 and 3.26 to 4.11 log CFU/g
on the fruits and vegetables treated with potable and challenge water, respectively.
As for Shigella, reductions of 1.16 - >4.13 log CFU/g were achieved treated with
diluted FIT® L produce wash in potable water, while the counts of 2.09 to 3.31
log CFU/g were reduced after the treatment with the levulinic acid wash in water
with organic matters (69).

79
Figure 1. Field spray of levulinic acid plus SDS on a tomato farm at pre-harvest.

Future Development

Despite the extensive research already completed on the spectrum of activity

and the levels required for successful inactivation of foodborne bacteria, more
research is needed to better elucidate the mechanisms of antimicrobial activity of
levulinic acid and SDS, the efficacy of the combination applied on food products
at concentrations that do not have adverse sensory effects, as well as an approach
to effectively reduce the cost of the application of levulinic acid and SDS in actual
food processing facilities.

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