Efficacy of a lactylate on production performance and intestinal health

of broilers during a subclinical Clostridium perfringens infection

M. Lensing,* J. D. van der Klis,*1 T. Fabri,† A. Cazemier,‡ and A. J. Else‡

*Schothorst Feed Research, Meerkoetenweg 26, 8218 NA, Lelystad, the Netherlands;
†Animal Health Service, Arnsbergstraat 7, 7400 AA, Deventer, the Netherlands;
and ‡Purac Biochem BV, Arkelsedijk 46, 4206 AC, Gorinchem, the Netherlands

ABSTRACT Clostridium perfringens, an α-toxin pro-
ducing gram-positive bacterium, is an enteric pathogen
for poultry. Because subclinical C. perfringens infec-
tions often result in damage of the intestinal mucosa,
decreased nutrient digestion, and poor performance, ef-
that a LauL dose higher than 0.15% should be used to
expect positive effects on lesion severity and mortality.
None of the LauL doses led to a significant better re-
sponse on growth performance. In a third trial, efficacy
of LauL was compared with commercial products that

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forts should be taken to find an effective strategy that
controls overgrowth of C. perfringens. For this purpose,
the efficacy of a sodium lauroyl lactylate (LauL) as a
feed additive to prevent C. perfringens colonization in
broilers was determined. First, the effect of LauL was
compared with capric and lauric mono- and diglycer-
ides (MDG) and capric and lauric free fatty acids in
Clostridium-infected chickens. Clostridial lesion scoring
at d 16 showed that MDG and LauL were both effec-
tive in reducing the severity of lesions. When taking
into account results on BW gain and mortality, LauL
was more effective than MDG. For this reason, a dose
response study was made to determine the optimal di-
etary dosage of LauL. In this experiment, it was shown
Key words: Clostridium perfringens, lactylate, intestinal health, broiler
limit bacterial activity in the intestinal tract (Aromabi-
otic Poul 60) or coccidiosis (chemical coccidiostat, Cli-
nacox). None of the products were able to reduce the
number or severity of lesions, and no effect on produc-
tion performance was observed. Thus, despite the clear
positive effect seen in experiment 1, and in experiment
2 with LauL doses higher than 0.15%, supplementing
this lactylate to the diet does not consistently reduce
C. perfringens colonization in broiler chickens because
no such effects were observed in experiment 3. These
results, however, provide a scientific basis for future
studies to further investigate lactylates as potential ad-
ditives to reduce the severity of necrotic enteritis in
broilers in a C. perfringens challenge model.
2010 Poultry Science 89:2401–2409
doi:10.3382/ps.2010-00942

INTRODUCTION tors for Clostridium infections because C. perfringens is

Clostridium perfringens, an α-toxin producing gram-
positive bacterium, is an enteric pathogen for poultry.
Clostridium infections may appear as an acute clinical
or subclinical disease (Løvland and Kaldhusdal, 2001),
with symptoms varying from loss in performance to
mortality. In case of a subclinical infection, the duode-
nal-jejunal mucosa is damaged, which decreases nutri-
ent digestion and absorption, reduces weight gain, and
increases feed conversion ratio (FCR; Elwinger et al.,
1992; Hofshagen and Kaldhusdal, 1992; Kaldhusdal et
al., 2001; Hofacre et al., 2003). Factors that stimulate
intestinal mucus production (e.g., gastrointestinal infec-
tions like coccidiosis) are considered predisposing fac-
©2010 Poultry Science Association Inc.
Received June 13, 2010.
Accepted August 15, 2010.
1 Corresponding author: JDvdklis@schothorst.nl
a mucolytic bacterium (Collier et al., 2003).
Coccidiosis infections can be controlled by coccid-
iostats, which indirectly prevent Clostridium infections.
Some coccidiostats, like ionophores that belong to a
class of antibiotics, can also be used to control Clostrid-
ium infections directly, because they control Clostrid-
ium growth in the gastrointestinal tract. However, a
real alternative to antibiotics or coccidiostats that ef-
fectively fights off Clostridium in a direct manner has
not yet been found. In search for such a product, fatty
acids and fatty acid derivatives provide obvious candi-
dates because they have long been known and used as
antimicrobials (Kabara and Marshall, 2005). Fatty acid
derivatives have detergent or surfactant properties, and
it is these properties that most probably endow them
with antimicrobial activity by way of interacting with
the cell membranes of the microorganisms. Its efficacy,
as an antimicrobial, is determined by the type of de-
rivative and the fatty acid chain length and degree of

2401
2402 Lensing et al.
saturation. Bayliss (1936) investigated the relation be-
tween the chemical composition of a soap and its germi-
cidal properties. For the saturated soaps, he found the
maximum antimicrobial activity with sodium myristate
(C14:0) fatty acids.
Among the esters of fatty acids, the naturally occur-
ring glycerol esters are the most well known and widely
used. In particular, monoesters with medium-chain
fatty acids (MCFA) have been used as antimicrobials
(Kabara and Marshall, 2005). Other less well-known
esters of fatty acids are the lactylates; these are esters
of lactic acid in which the C2-hydroxy group of lactic
acid is esterified to a fatty acid. The main commercial
use of lactylates is in the bakery industry, with C18
fatty acids (Boutte and Skogerson, 2004). Lactylates
made with MCFA have been used in cosmetics (patent
application EP0595528).
Both components of lactylates, lactic acid and fatty
acids, by themselves are known and used as antimi-
crobials in animal production. Lactic acid can be used
to reduce Campylobacter and Salmonella contamination
of broiler carcasses at processing (Byrd et al., 2001)
and free fatty acids (FFA) such as MCFA have been
shown to reduce Clostridium infections in broiler chick-
ens (Jansman et al., 2006). In vitro, lactylates with
different types of fatty acids have shown already to be
potent inhibitors of C. perfringens (patent application
EP02082739A1), with the lowest minimum inhibitory
concentration of 0.001% (wt/wt) for a lactylate with
C12 and C14 fatty acids. That means that in vitro
they are 100- to 1,000-fold more effective inhibitors
than FFA or lactic acid alone. The series of trials de-
scribed here were made to test a sodium lauroyl lacty-
late (LauL; Puramix 30, Purac Biochem, Gorinchem,
the Netherlands) containing C12 and C14 fatty acids
under in vivo conditions.
The first trial was used to compare the effects of
a LauL to C10–C12 monoglycerides (mono- and dig-
lycerides; MDG) and free C10–C12 MCFA (FFA) in
Clostridium-infected chickens. Because LauL was the
most promising molecule, a dose response study was
made to determine the optimal dietary dosage. In a
third trial, the efficacy of LauL was compared with
commercial products that limit bacterial activity in the
intestinal tract (Aromabiotic Poul 60; Vitamex, Dron-
gen, Belgium; ABP) or coccidiosis (chemical coccid-
iostat; C) and consequently its effect on intestinal mu-
cus production. This series of experiments was made to
determine whether MCFA lactylates can be of help in
reducing problems caused by C. perfringens in broiler
chickens.
MATERIALS AND METHODS
Experimental Design
Experiment 1. The objective of this experiment
was to test the efficacy of a C12–C14 lactylate (LauL)
against a C10–C12 MDG (50:50 mix of C10–C12 fatty
acids bound to MDG) and C10–C12 FFA (50:50 mix
of C10–C12 FFA) on Clostridium-infected chickens. A
total of 600 one-day-old male Ross 308 broiler chickens
were randomly assigned to 5 treatments with 6 repli-
cated cages each. The experiment was carried out from
0 to 20 d of age. From day of arrival to d 9, chicks were
fed a starter diet without any supplements and from d 9
onward, a grower diet was given. After standardization
to 17 birds per cage at d 9, chicks were challenged with
Eimeria maxima and C. perfringens. Control treatments
were infected-nontreated (INT) or noninfected-non-
treated (NINT). Three other infected treatments (IT)
received the grower diet supplemented with 0.3% FFA
(IT + FFA), 0.3% of MDG (IT + MDG), or 0.3% LauL
(IT + LauL). Products were produced and supplied by
Purac Biochem (Gorinchem, the Netherlands). Dosages
were chosen based on results from in vitro studies in
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combination with the expected kinetics of the products
in the intestinal tract. All treatments were randomly
allocated to litter cages except NINT cages that were
grouped together to prevent cross-contamination.
Postmortem clostridial lesion scoring occurred at d
15 and 16 and procedures are described later. Remain-
ing birds were used to collect data on growth perfor-
mance. Birds were weighed per cage at d 0, 9, and 20 to
determine BW gain (BWG) from d 0 to 9 and d 9 to
20. Feed intake (FI) per cage was recorded during the
same periods but daily in the infection period from d 9
to 20. Based on FI and BWG, FCR was calculated on a
cage basis. Mortality with its most probable cause was
recorded from d 0 to the end of the experiment.
Experiment 2. The objective of experiment 2 was
to find the optimal dosage of LauL in a dose response
study, being the most promising molecule from experi-
ment 1, to fight C. perfringens. This experiment was
similar to experiment 1 with regard to breed and sex,
infection protocol, and response parameters. A total
of 1,320 one-day-old Ross 308 male broiler chicks were
randomly assigned to 11 treatments with 6 replicate
cages each. Treatments were designed as noninfected
and treated (NIT) with 0.3% LauL (NIT + 0.3 LauL)
or without (NINT). The infected treatments were set
up as a dose response starting with a 2 times higher
dose as was used in experiment 1 (IT + 0.6 LauL)
and stepwise lowering the supplementation with 50%,
from 0.6% to 0.3% to 0.15%, toward 0% supplementa-
tion of LauL (INT). All diets with or without LauL
supplementation were supplied from d 0 to 37 to see if
a prolonged use of LauL before inoculation was more
effective. Postmortem lesion scoring was performed on
d 15, 16, and 17.
Experiment 3. Experiment 3 was similar to experi-
ment 2 with regard to breed, sex, the infection proto-
col, response parameters, and start and length of the
dietary treatments. A total of 960 one-day-old broiler
chickens were randomly allocated to 8 treatments with
6 replicate cages each. Treatments were designed with
2 controls, INT and NINT without supplementation
of LauL or with 0.3% LauL (IT + 0.3 LauL or NIT
 ANTI-CLOSTRIDIAL EFFECTS OF SODIUM LAUROYL LACTYLATE
+ 0.3 LauL), to test if LauL would benefit in healthy
and infected chickens. The efficacy of 0.3% LauL in in-
fected chickens was tested against 4 IT treatments with
0.2% LauL dosage (IT + 0.2 LauL), the combination
of LauL with a chemical coccidiostat (Clinacox: 100 g/
ton, Janssen Animal Health, Beerse, Belgium; IT + 0.3
LauLC), a chemical coccidiostat alone (IT + C), and a
commercial MCFA product (Aromabiotic Poultry: 1.6
kg/ton in starter and 1.2 kg/ton in grower phase, Vita-
mex, Drongen, Belgium; IT + ABP).
Birds and Housing
One-day-old male Ross 308 broiler chickens were sup-
plied by Probroed & Sloot B.V. (Meppel, the Nether-
lands). At d 0, broilers arrived at the poultry facilities
of Schothorst Feed Research B.V. (experiments 1 and
3; Lelystad, the Netherlands) or the Animal Health
Service (experiment 2; Deventer, the Netherlands) and
were housed in 2-tier litter floor digestibility cages af-
ter individual weighing (surface area 0.90 × 0.65 m).
Twenty birds were allocated to cages based on a weight
class system such that mean weight per cage was simi-
lar at the start of the experiment. Broilers were housed
in these cages throughout the entire experiment. At d
9, before first inoculation, the number of chickens was
standardized to 17, and bird weight was measured be-
fore and after standardization. Lighting and tempera-
ture schedule throughout the experimental period was
22L:2D in the time period from d 0 to 9 and 18L:6D
from d 9 to the end of the experiment. The ambient
temperature gradually decreased from 32°C at the start
to 25°C at the end of the experiment. Feed was sup-
plied for ad libitum intake from d 0 onward with the
exception of the 5-h feed withdrawal period before in-
oculations (on d 9 and 14). Water was available for
ad libitum intake throughout the experiment. Broilers
were not vaccinated during the experiment.
Experimental Diets
Broilers were supplied a wheat-soybean meal-based
starter diet for ad libitum intake from day of arrival
until d 9. At d 9, a wheat-barley-based basal grower
diet was fed until the end of the experiment (Table 1).
Grower diets were fed as mash because of the necessity
of homogeneously mixing in the test products after feed
production. Diets did not contain any nonstarch poly-
saccharide enzymes, coccidiostats, or antimicrobial feed
additives other than the test products. The nutrient
composition of the experimental diets was according
to Dutch standards to meet nutrient requirements of
broilers (CVB, 2008).
Challenge
To induce the onset of a C. perfringens infection, a
mild E. maxima infection was used as pretrigger. Birds
were orally inoculated at d 9 with 1 mL of E. maxi-
2403
ma (Central Veterinary Laboratory, Weybridge, UK;
~10,000 sporulated oocysts per mL; 1 mL per bird)
or with sterile saline after a 5-h feed withdrawal pe-
riod. This inoculation with E. maxima has consistently
shown to induce C. perfringens with an average of 50%
of chickens scored positive for clostridial lesions. More-
over, reddish E. maxima lesions are distinctly different
from white C. perfringens lesions. After the 5-d incuba-
tion time for coccidiosis to develop, birds were orally
inoculated on d 14 with C. perfringens type A [~108
cfu/mL; 1 mL of liver broth (Difco, Detroit, MI) per
bird] or with 1 mL of sterile liver broth after a 5-h feed
withdrawal period to induce necrotic enteritis. After
inoculations, birds had immediate access to feed. The
pathogenic C. perfringens strain (code GD 5.11.53) was
obtained from the Animal Health Service (Deventer,
the Netherlands). The strain was grown on an agar
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of sheep blood and the culture was typed by the Cen-
tral Institute of Animal Disease Control (Lelystad, the
Netherlands) as C. perfringens producing type α and
β2 toxins.
Pathological Parameters
During the peak of Clostridium infection at d 15
and 16 (1 and 2 d postinfection), 4 random selected
birds per cage were used for postmortem coccidial and
clostridial lesion scoring in the duodenal and jejunal
segment of the small intestine (48 birds per treatment).
In experiments 2 and 3, a third scoring was done at
d 17 (3 d postinfection). Birds were killed by an in-
tracardial injection with a euthasate (T61; Intervet,
Mechelen, Belgium). The abdomen was opened and the
gastrointestinal tract was exposed. The gastrointestinal
tract was segmented into the duodenum and jejunum
(the segment from the gizzard to the 10 cm preced-
ing Meckel’s diverticulum). All birds were scored blind
(i.e., the person scoring for lesions had no knowledge of
the bird’s treatment). The following method was used
to score the C. perfringens lesions: 0 = no lesions; 1 =
1 to 5 small white lesions (spots of less than 1 mm in
diameter); 2 = more than 5 small white lesions (spots
of less than 1 mm in diameter) or 1 to 5 larger lesions
(spots of 1 to 2 mm in diameter); 3 = more than 5 larg-
er lesions (1 to 2 mm in diameter) or erosive zones; 4
= dead birds with positive necrotic enteritis diagnoses
postmortem. Based on the clostridial lesion scores, the
percentage of Clostridium-infected birds was calculated
by the following equation:
(sum of birds scored >0/total number
of birds scored) × 100%.
The percentage of birds affected indicated the inci-
dence of C. perfringens. By categorizing the severity
of lesions from 0 to 4, an average lesion score could be
calculated for each treatment, giving an indication of
the severity of the C. perfringens infection besides its
incidence.
2404 Lensing et al.
Table 1. Composition of the basal diets in experiments 1, 2, and 3
Starter (d 0 to 9) Grower (d 9 to end of experiment)

Item Experiment 1 Experiments 2 and 3   Experiment 1 Experiments 2 and 3

Ingredient (%)          
  Maize 19.35 18.54   — —
  Soybean meal, Hipro1 25.73 25.73   19.50 19.53
  Sunflower seed meal — —   5.26 5.26
  Wheat 47.76 48.57   35.88 35.77
  Wheat middlings — —   1.09 1.09
  Barley — —   24.24 24.24
  Maize starch — —   3.83 3.82
  Soybean oil 1.19 1.19   4.81 4.59
  Animal fat 1.77 1.77   1.86 1.86
  Limestone 1.34 1.83   1.12 1.13
  Monocal 0.61 0.62   0.78 0.78
  Salt 0.11 0.28   0.12 0.13
  Premix vitamins and minerals2 1.50 1.50   0.50 0.70
  Premix Lys (HCl 79%) 0.28 0.28   0.31 0.33
  Premix Met (dl 99%) 0.22 0.22   0.27 0.28
  Premix Thr (l 98%) 0.08 0.08   0.09 0.15

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  Natrium carbonate 0.06 0.06   0.34 0.34
Calculated nutrient (g/kg)          
  Moisture 109 122   117 117
  Ash 55 55   49 50
  CP 199 199   187 185
  Crude fat 51 51   80 83
  Crude fiber 26 26   36 40
  Starch 384 392   360 361
  AME broiler (kcal) 2,759 2,798   2,864 2,865
  Calcium 8.9 9.0   6.7 6.7
  Phosphorus 5.1 5.1   5.7 5.7
  Sodium 1.4 1.4   1.6 1.6
  Chloride 2.7 2.7   1.9 2.0
  Potassium 8.5 8.5   7.8 8.0
  Apparent digestible Lys 10.4 10.4   9.6 9.6
  Apparent digestible Met + Cys 7.6 7.6   7.7 7.7
  Apparent digestible threonine 6.5 6.6   6.9 6.9
1Hipro (Brazilian soybeans; Cargill, Amsterdam, the Netherlands).
2The mineral-vitamin premix contained the following per kilogram: Ca, 185 g; Na, 80 g; Cl, 100 g; Cu, 1,200 mg; Fe, 4,500 mg; Mn, 7,000 mg;
Zn, 3,700 mg; I, 100 mg; Se, 15 mg; vitamin A, 1,000,000 IE; vitamin D3, 200,000 IE; vitamin E, 2,500 IE; vitamin B1, 50 mg; vitamin B2, 500 mg;
pantothenic acid, 800 mg; niacin, 4,000 mg; vitamin B6, 300 mg; folic acid, 100 mg; vitamin B12, 1,500 µg; biotin, 10,000 µg; vitamin K3, 125 mg;
choline, 20,000 mg.

Three in vivo experiments were performed in suc-
cession. The experimental protocols and all procedures
complied with the guidelines of the Ethical Committee
for Animal Experiments (Lelystad, the Netherlands).
Statistical Analysis
The incidence of C. perfringens (% of birds scored
>0) and mortality rate were analyzed by Fisher’s ex-
act test, whereas the severity of lesions, measurements
on production performance, and other parameters were
analyzed by ANOVA using GenStat statistical software
(GenStat version 12, Hemel Hempstead, UK). Raw
data of BW and feed intake were analyzed for outliers.
Outliers were marked significant when exceeding 2.5 ×
SD of the treatment mean and were excluded from the
data set. Treatment means were compared by the least
significant difference. The statistical model(s) used per
experiment were as follows, based on the entire data
sets or subsets for each experiment.
The model used for experiments 1, 2, and 3 was as
follows:
Yij = μ + Blocki + Treatmentj + eij,
with Yij = response parameter; μ = mean; Blocki =
effect of block (i = 1 to 6); Treatmentj = effect of
treatment (j = 5, 11, and 8 for experiments 1, 2, and 3,
respectively); and eij = residual error.
The model used for experiments 2 and 3 was as fol-
lows:
Yij = μ + Blocki + Dosej + eij,
with Yij = response parameter; μ = mean; Blocki =
effect of block (i = 1 to 6); Dosej = effect of dose (j =
9 and 3 for experiments 2 and 3, respectively); and eij
= residual error.
The model used for experiment 3 was as follows:
Yijk = μ + Blocki + Coccidiostatj + Testk
+ Interactionjk + eijk,
with Yijk = response parameter; μ = mean; Blocki =
effect of block (i = 1 to 6); Coccidiostatj = effect of coc-
 ANTI-CLOSTRIDIAL EFFECTS OF SODIUM LAUROYL LACTYLATE 2405
Table 2. The percentage of birds scored positive for Clostridium perfringens lesions and the lesion
severity in the infected-nontreated (INT) group and noninfected-nontreated group (NINT) presented
as mean values of lesion scoring at d 15 and 16 (1 and 2 d postinfection, n = 48)
Lesion severity
Item Positive birds (%) (scale 0 to 4) P-value

Experiment 1
  NINT 0 0.0 <0.001
  INT 42 1.3  
Experiment 2
  NINT 0 0.0 <0.001
  INT 62 1.8  
Experiment 3
  NINT 0 0.0 <0.001
  INT 83 2.2  

cidiostat (j = yes, no); Testk = effect of test product (k the NINT control (Table 3), as expected, whereas the
= with or without LauL); Interactionjk = coccidiostat INT treatment had the lowest BWG. All dietary treat-
× test effect; and eijk = residual error. ments, FFA, MDG, and LauL resulted in a higher FI

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Effects were considered significant at P ≤ 0.05,
whereas 0.05 < P ≤ 0.10 was considered to be a near
significant trend.
RESULTS
General
The INT groups in all experiments were scored posi-
tive for C. perfringens. As an average of lesion scoring
performed at d 15 and 16, the INT groups resulted in
42, 62, and 83% of birds scored positive for clostridial
lesions in experiments 1, 2, and 3, respectively. In NINT
groups, all birds were scored negative. Also, clostridial
lesions were more severe (P < 0.001; Table 2). Calcu-
lating the scores from 0 to 4 in all necropsied birds, a
lesion severity of 1.3, 1.8, and 2.2 for the 3 successive
experiments was determined.
Experiment 1
On d 15 and 16, the number of birds scored positive
for C. perfringens was not affected by dietary treat-
ments FFA, MDG, and LauL compared with the INT
control (Table 3). The severity of lesions was not sig-
nificantly affected by any of the treatments on d 15,
whereas on peak of infection at d 16, dietary supple-
mentation of MDG and LauL resulted in lesions that
were less severe compared with the INT and IT + FFA
groups (P < 0.001; Table 3). Three days later (6 d
postinfection), no significant differences were observed
between treatments (data not shown) as all treatments
recovered from necrotic enteritis, based on macroscopi-
cal evaluation. A similar pattern was observed for mor-
tality, which was higher in the INT control, FFA, and
MDG treatment compared with the NINT treatment
(P ≤ 0.05). The LauL treatment was intermediate, not
being significantly different from any treatment (Table
3).
Production parameters from d 9 to 20 were affected
by dietary treatments. Body weight gain was highest in
and BWG compared with INT (P < 0.001).
Experiment 2
The percentage of birds scored positive for C. per-
fringens was significantly influenced by challenge and
dietary treatments. No lesions were observed in NINT
and NIT treatments, whereas 56% was infected in the
INT control treatment (mean of d 15, 16, and 17; Table
4). Supplementation of 0.6% LauL (IT + 0.6 LauL) re-
sulted in 35% of birds scored positive. The other LauL-
supplemented treatments resulted in similar percent-
ages of broiler scored positive for necrotic enteritis as
INT.
With regard to severity of lesions, birds supplement-
ed with 0.6% LauL had less severe lesions than birds in
the INT control (P < 0.001). A dose of 0.3% LauL did
reduce lesion severity as well toward a near-significant
reduction in comparison to the INT control group (P ≤
0.10). All other LauL-supplemented treatments did not
improve results on lesion severity (Table 4). A sigmoid
dose response curve was fitted to the data, explaining
81% of the variation (Figure 1). It was shown that a
dose higher than 0.15% should be used to expect any
positive effect of LauL on lesion severity.
A similar pattern was observed for mortality (data
not shown), being highest in the INT control (18%)
but reduced in 0.6% LauL to 5% (P ≤ 0.05) and 11%
in 0.3% LauL (P ≤ 0.10).
Production parameters were only affected by dietary
treatments from d 9 to 20. Body weight gain was high-
est in the NINT control (574 g) and NIT + 0.3 LauL
(591 g), as expected, whereas all infected treatments
had a 150 to 200 g lower BWG from d 9 to 20 (data not
shown). None of the LauL doses led to a significantly
better response on final weight at d 37 (Table 4).
Experiment 3
Like in experiment 2, the percentage of birds scored
positive for C. perfringens was significantly influenced
2406 Lensing et al.
Table 3. The percentage of birds observed with necrotic enteritis and the severity of lesions scored in all necropsied birds at d 15
and 16 (1 and 2 d postinfection, n = 48) and the results of the remaining birds on BW gain (BWG) and feed intake (FI) from d 9 to
20 (experiment 1)
Positive birds (%) Severity (scale 0 to 4)

Treatment1 Dosage (%) d 15 d 16 d 15 d 16

Lesion scoring            
  NINT — 0b 0b   0.0 0.0c
  INT — 16ab 68a   0.5 2.1a
  IT + FFA 0.3 23a 60a   0.5 2.0a
  IT + MDG 0.3 33a 58a   0.5 1.7b
  IT + LauL 0.3 17ab 41a   0.4 1.1b
  SEM   NA2 NA   0.18 0.25
  P-value   ≤0.05 ≤0.05   0.30 <0.001
BWG FI Mortality
Performance   d 9 to 20 (g) d 9 to 20 (g)   d 9 to 20 (%)  

  NINT — 539a 741a   0.0b  

  INT — 318c 510c   14.6a  
  IT + FFA 0.3 377b 567b   14.8a  

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  IT + MDG 0.3 368b 558b   11.8a  
  IT + LauL 0.3 375b 554b   5.1ab  
  SEM   10.6 10.9   NA  
  P-value   <0.001 <0.001   ≤0.05  
a–cValues with no common superscripts in a column differ significantly (P ≤ 0.05).
1NINT = not infected and not treated; INT = infected and not treated; IT + FFA = infected and treated with 0.3% free fatty acids; IT + MDG =
infected and treated with 0.3% mono- and diglycerides; IT + LauL = infected and treated with 0.3% lauroyl lactylate.
2NA = not available in Fisher’s exact test as a nonparametric statistical method.

by challenge (P < 0.001). No lesions were observed in
NINT and NIT treatments, whereas 80% was infected
in the INT control treatment (mean of d 15, 16, and
17). Supplementation of LauL (0.3 or 0.2%), ABP, or C
or the combination of 0.3% LauL and C did not reduce
the number of infected birds when compared with the
INT control. Lesion severity of infected birds treated
with ABP had more severe lesions (2.5) than the INT
control and 0.3 or 0.2% LauL treatments (varied from
2.0 to 2.2). Supplementation of C with or without 0.3%
LauL gave intermediate results (2.2 and 2.3, respective-
ly), being lower than the ABP group but slightly higher
than the LauL treatments and the INT control (data
not shown). The highest mortality rate of 32.4% was
observed in the ABP treatment, which was higher than
in the 0.2% LauL or 0.3% LauLC group (P ≤ 0.05).
Supplementing 0.3% LauL or C resulted in a similar
mortality rate as the INT control (Table 5).
Results of production parameters of the infected
treatments are presented in Table 5. No differences
in growth performance were observed between treat-
ments.

Table 4. Birds observed with necrotic enteritis (%) and the mean severity of lesions scored in all
necropsied birds at d 15, 16, and 17 (1 to 3 d postinfection, n = 72) and the final BW of the remain-
ing birds at d 37 (experiment 2)
Dosage Positive Severity BW d 37
Treatment1 (%) birds (%) (scale 0 to 4) (g)

NINT — 0c 0.0f 2,249

NIT + 0.3 LauL 0.3 0c 0.0f 2,302
INT — 56ab 1.6abcd 2,246
IT + 0.005 LauL 0.005 69a 2.0a 2,151
IT + 0.010 LauL 0.010 51ab 1.5bcd 2,229
IT + 0.019 LauL 0.019 58ab 1.7abc 2,305
IT + 0.038 LauL 0.038 54ab 1.6abcd 2,147
IT + 0.075 LauL 0.075 60ab 1.8ab 2,169
IT + 0.15 LauL 0.15 57ab 1.6abcd 2,259
IT + 0.3 LauL 0.3 43ab 1.2de 2,275
IT + 0.6 LauL 0.6 35b 0.9e 2,305
SEM   NA2 0.17 16.6
P-value   ≤0.05 <0.001 0.193
a–fValues with no common superscripts in a column differ significantly (P ≤ 0.05).
1NINT = not infected and not treated; NIT = not infected and treated with 0.3% lauroyl lactylate; INT =
infected and not treated; IT + x LauL = infected and treated with x% lauroyl lactylate.
2NA = not available in Fisher’s exact test as a nonparametric statistical method.
 ANTI-CLOSTRIDIAL EFFECTS OF SODIUM LAUROYL LACTYLATE 2407
DISCUSSION
Colonization of C. perfringens in the intestinal tract of
birds produces a strong inflammatory response, which
leads to a severe damage of the intestinal epithelium
(Van Immerseel et al., 2004). Consequently, this results
in poor nutrient digestibility and growth performance.
The negative effect of colonization of C. perfringens on
health and growth performance was confirmed in all 3
broiler experiments of the current study. Experimental
infection of 9-d-old broilers with E. maxima followed
by an infection with C. perfringens at 14 d of age in-
duced mild to severe clostridial lesions and pronounced
reduction of performance from the day after infection
until approximately 6 d after. Body weight gain in the
healthy control (NINT) was in all experiments around
550 to 620 g from d 9 to 20 or 21, whereas BW gain was

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reduced by 150 to 200 g in the INT groups.
A necrotic enteritis infection reduces growth perfor-
mance that is usually caused by a decrease in feed in-
take during infection. A reduction in feed intake was
also observed in these experiments. The effect of a
clostridial infection on daily feed intake in experiment
2 is presented in Figure 2, which is a typical response
for all 3 experiments. Although a mild coccidiosis infec-
tion was introduced at d 9, no difference in feed intake
was observed until after the inoculation of C. perfrin-
gens at d 14. From d 14 onward, daily feed intake de-
creases until the peak of infection at d 16. After d 16,
daily feed intake recovers but stays consistently lower
than the healthy control group until d 19 (Figure 2).
Between d 9 and 20, a 150 g lower FI was measured
for the infected birds in comparison to the noninfected
Figure 1. Estimation of the dose response relationship between the
dietary lauroyl lactylate (LauL) concentration and the lesion severity
(experiment 2). The closed dots indicate the infected-treated and open
dot indicates the infected-nontreated group. Clostridial lesions were
scored on a scale of 0 to 4 (0 = no lesions and 4 = most severe lesions
and death due to necrotic enteritidis). Dots sharing no common letter
differ significantly from each other (P ≤ 0.05). Color version available
in the online PDF.
birds. This lower FI does not fully explain the 150 to
200 g loss in BWG after infection (assuming a FCR of
1.4). The stronger effect of the infection on BW may
be related to reduced nutrient digestion and absorption
after C. perfringens colonization as well as to inflamma-
tion. Bacterial pathogens, such as C. perfringens, are
known to cause mucosal damage of the intestinal gut,

Table 5. Production performance of infected birds not treated (INT) or treated with test products (experiment 3)1
Treatment INT IT + 0.3 LauL IT + 0.2 LauL IT + ABP IT + C IT + 0.3 LauLC SEM P-value

BW d 0 44.1 43.9 44.2 44.0 43.6 43.7 0.096 0.205

0 to 9 d                
  Growth (g) 172 190 187 184 181 188 2.2 0.241
  Feed intake (g) 229 235 231 237 234 236 2.3 0.966
  FCR2 1.332 1.239 1.295 1.290 1.293 1.254 0.0077 0.014
9 to 21 d                
  Growth (g) 455 475 474 456 485 497 8.3 0.610
  Feed intake (g) 660 672 677 661 685 686 8.0 0.868
  FCR 1.469 1.426 1.429 1.453 1.412 1.386 0.0153 0.836
21 to 30 d                
  Growth (g) 761 715 740 792 765 760 12.4 0.401
  Feed intake (g) 1,211 1,188 1,190 1,266 1,207 1,215 15.6 0.610
  FCR 1.595 1.675 1.610 1.599 1.584 1.599 0.0150 0.441
30 to 36 d                
  Growth (g) 638 598 636 666 635 633 14.7 0.850
  Feed intake (g) 1,063 992 1,015 1,116 1,059 1,049 16.3 0.314
  FCR 1.667 1.744 1.596 1.677 1.665 1.696 0.0277 0.807
0 to 36 d                
  Final weight (g) 2,075 2,025 2,075 2,144 2,114 2,159 26.2 0.558
  Feed intake (g) 3,262 3,189 3,213 3,383 3,286 3,294 35.7 0.532
  FCR 1.610 1.614 1.583 1.612 1.587 1.600 0.0089 0.866
Mortality (%) 19.4ab 21.3ab 17.6b 32.4a 27.8ab 15.7b NA ≤0.05
a,bValues with no common superscripts in a column differ significantly (P ≤ 0.05).
1INT = infected and not treated; IT + x LauL = infected and treated with x% lauroyl lactylate; IT + ABP = infected and treated with Aromabiotic
Poul 60 (Vitamex, Drongen, Belgium); IT + C = infected and treated with Clinacox (Janssen Animal Health, Beerse, Belgium); IT + x LauLC =
infected and treated with x% lauroyl lactylate and Clinacox.
2FCR = feed conversion ratio.
3NA = not available in Fisher’s exact test as a nonparametric statistical method.
2408 Lensing et al.
which impedes nutrient absorption and digestion (Van
Immerseel et al., 2004). Besides that, the inflammatory
response and activation of the immune system result in
high energy costs and reduced efficiency of protein uti-
lization that may lead to a reduced supply of nutrients
for maintenance and growth.
To be able to determine whether the lactylate used in
the current study has potential to be effective against
C. perfringens, the results on incidence and lesion sever-
ity were compared with the INT controls. In the first
experiment, LauL containing C12–C14 fatty acids es-
terified to lactic acid was tested against C10–C12 MDG
or C10–C12 FFA. Based on lesion severity and mortal-
ity rate, it seemed that this lactylate had the greatest
potential to reduce negative effects of a C. perfringens
infection. This led to a dose response study with LauL,
which showed a clear significant reduction in lesion se-
verity with higher concentrations of LauL in the diets,
but did not reduce the number of birds scored positive
for C. perfringens. From a sigmoid curve, it was con-
cluded that only LauL doses higher than 0.15% were
to be expected to improve lesion severity. In the third
study, it was therefore decided to repeat a 0.3% dose.
A 0.2% dose was also tested because the latter might
have an effect but was not tested as such in experiment
2. Both doses of LauL could be used as dietary supple-
mentation in practice and to determine their potential
in the feed industry, efficacy of LauL was compared
with some commercial products that are used in prac-
tice to control bacterial overgrowth directly or indi-
rectly. Aromabiotic Poul 60 contains MCFA that have
antibacterial properties and should have a direct effect
on bacteria such as C. perfringens. Clinacox was used
to see if LauL would have any advantages in compari-
son to or in combination with a chemical coccidiostat.
Figure 2. The effect of the subsequent infection of Eimeria maxima
at d 9 and Clostridium perfringens at d 14 on daily feed intake between
9 and 20 d of age (experiment 2). NINT = noninfected-nontreated;
INT = infected-nontreated. An asterisk (*) indicates significant effects
of infection on a specific day (P ≤ 0.05). NS = no significant effects of
infection. Color version available in the online PDF.
A chemical coccidiostat was preferred over an iono-
phore because the latter has strong antibiotic proper-
ties. Adding an ionophore to the diet could have killed
off the inoculated C. perfringens even before causing
intestinal damage, creating too big of a risk of failure
of the infection model. In this third experiment, neither
an effect of LauL nor a coccidiostat was observed. The
absence of an effect of LauL and the coccidiostat is
hard to explain but could have been caused by a stron-
ger infection. The infection was worse in this study, re-
sulting in 83% of birds showing an average lesion score
of 2.2, whereas experiments 1 and 2 resulted in 42 and
56% of birds with lesions of 1.3 and 1.6, respectively
(Table 2). The infection in the third study might have
been too strong to find a preventive effect of LauL or a
coccidiostat against an infection of C. perfringens.
Although no effect of lactylate was seen in the third
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study, a slight positive effect was seen in the first 2
experiments. The positive effect of lactylate can be at-
tributed to their chemical composition. Acyl lactylates
are produced by the reaction between the acyl group of
fatty acids from 6 to 22 carbon atoms and lactic acid
in the presence of an alkali metal. This reaction pro-
duces the salts of the corresponding fatty acid ester of
lactylic acid using the appropriate fatty acid and lactic
acid. By controlling the fatty acid used and the degree
of condensation of the lactylic acid component, differ-
ent effects can be obtained. One of the characteristics
of LauL is its strong bacteriostatic property. Sodium
lauroyl lactylate has been shown to have a broad range
of antimicrobial activity against bacteria, molds, and
yeast (Marnett et al., 1966; Osipow et al., 1969). In
these studies, bacteriostatic effects were shown against
Staphylococcus aureus, Aspergillus terreus, and Asper-
gillus niger and several Bacillus and Saccharomyces
species.
In a Clostridium infection study performed in the
Netherlands by the Product Board for Livestock Pro-
duction, it was concluded that a mixture of C10 and
C12 fatty acids had an anticlostridial effect, showing
a reduction in the number of broilers infected and a
milder lesion score (Jansman et al., 2006). Sodium lau-
royl lactylate does contain C12 fatty acids as part of its
structure and, based on these data, the mechanism of
action for LauL could be attributed to the anticlostrid-
ial effect of C12 fatty acids alone. However, to attri-
bute the anticlostridial effect of LauL only on the an-
ticlostridial effect of C12 is too straightforward. Other
chain lengths of fatty acids are just as bacteriostatic,
but they are not as soluble in water as short- or medi-
um-chain fatty acids, making it difficult or impossible
to achieve the most active concentration in the cell.
The intrinsic antimicrobial effect of the molecule may
be different from its effective concentration in aque-
ous solution. The C12 fatty acids present in LauL are,
therefore, a nice compromise between chain length and
solubility but are by itself probably not the strongest
antimicrobial. A lactylate, however, in combination
with fatty acids with different chain lengths, has strong
 ANTI-CLOSTRIDIAL EFFECTS OF SODIUM LAUROYL LACTYLATE
antimicrobial properties. In the data given in patent
EP2082739A1, the minimum inhibitory concentration
values of C12 and C14 lactylates are the lowest. The
minimum inhibitory concentration values for these lac-
tylates tested for C. perfringens are, respectively, 0.002
and 0.001% in comparison to 0.04% for C10 lactylates.
The mechanism of action is most probably due to the
slow metabolization of the lactylate. Lactylates will
break down on ingestion, releasing FFA and lactic acid,
which will be metabolized as normal. There is some
evidence that the metabolic degradation or intestinal
absorption of lactylates may be delayed in comparison
to lactic acid or FFA (Phillips et al., 1981). This de-
lay may be sufficient to allow the lactylate to have a
stronger effect on bacterial infection in the intestinal
tract than either FFA or lactic acid alone. But even
when it does hydrolyze, both of the molecules released
(lactic acid and MCFA) still have some antibacterial
activity. The combination between the strong antimi-
crobial lactylate, its bacteriostatic products in the form
of MCFA and lactic acid, and its slow metabolization
is the most probable explanation for the observed ef-
fects on clostridial lesion scores and mortality in broil-
ers seen in experiments 1 and 2. Still, more attention
should be paid to the molecule and its physiological
effects because none of its expected properties did im-
prove results in experiment 3.
It can be concluded from this study that higher con-
centrations of LauL in broiler diets have a potential to
reduce the severity of necrotic enteritis in broilers in an
E. maxima and C. perfringens challenge model.
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