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

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

INTRODUCTION tors for Clostridium infections because C. perfringens is

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

2401
2402 Lensing et al.

saturation. Bayliss (1936) investigated the relation be- acids bound to MDG) and C10–C12 FFA (50:50 mix
tween the chemical composition of a soap and its germi- of C10–C12 FFA) on Clostridium-infected chickens. A
cidal properties. For the saturated soaps, he found the total of 600 one-day-old male Ross 308 broiler chickens
maximum antimicrobial activity with sodium myristate were randomly assigned to 5 treatments with 6 repli-
(C14:0) fatty acids. cated cages each. The experiment was carried out from
Among the esters of fatty acids, the naturally occur- 0 to 20 d of age. From day of arrival to d 9, chicks were
ring glycerol esters are the most well known and widely fed a starter diet without any supplements and from d 9
used. In particular, monoesters with medium-chain onward, a grower diet was given. After standardization
fatty acids (MCFA) have been used as antimicrobials to 17 birds per cage at d 9, chicks were challenged with
(Kabara and Marshall, 2005). Other less well-known Eimeria maxima and C. perfringens. Control treatments
esters of fatty acids are the lactylates; these are esters were infected-nontreated (INT) or noninfected-non-
of lactic acid in which the C2-hydroxy group of lactic treated (NINT). Three other infected treatments (IT)
acid is esterified to a fatty acid. The main commercial received the grower diet supplemented with 0.3% FFA
use of lactylates is in the bakery industry, with C18 (IT + FFA), 0.3% of MDG (IT + MDG), or 0.3% LauL
fatty acids (Boutte and Skogerson, 2004). Lactylates (IT + LauL). Products were produced and supplied by
made with MCFA have been used in cosmetics (patent Purac Biochem (Gorinchem, the Netherlands). Dosages
application EP0595528). were chosen based on results from in vitro studies in

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Both components of lactylates, lactic acid and fatty combination with the expected kinetics of the products
acids, by themselves are known and used as antimi- in the intestinal tract. All treatments were randomly
crobials in animal production. Lactic acid can be used allocated to litter cages except NINT cages that were
to reduce Campylobacter and Salmonella contamination grouped together to prevent cross-contamination.
of broiler carcasses at processing (Byrd et al., 2001) Postmortem clostridial lesion scoring occurred at d
and free fatty acids (FFA) such as MCFA have been 15 and 16 and procedures are described later. Remain-
shown to reduce Clostridium infections in broiler chick- ing birds were used to collect data on growth perfor-
ens (Jansman et al., 2006). In vitro, lactylates with mance. Birds were weighed per cage at d 0, 9, and 20 to
different types of fatty acids have shown already to be determine BW gain (BWG) from d 0 to 9 and d 9 to
potent inhibitors of C. perfringens (patent application 20. Feed intake (FI) per cage was recorded during the
EP02082739A1), with the lowest minimum inhibitory same periods but daily in the infection period from d 9
concentration of 0.001% (wt/wt) for a lactylate with to 20. Based on FI and BWG, FCR was calculated on a
C12 and C14 fatty acids. That means that in vitro cage basis. Mortality with its most probable cause was
they are 100- to 1,000-fold more effective inhibitors recorded from d 0 to the end of the experiment.
than FFA or lactic acid alone. The series of trials de- Experiment 2. The objective of experiment 2 was
scribed here were made to test a sodium lauroyl lacty- to find the optimal dosage of LauL in a dose response
late (LauL; Puramix 30, Purac Biochem, Gorinchem, study, being the most promising molecule from experi-
the Netherlands) containing C12 and C14 fatty acids ment 1, to fight C. perfringens. This experiment was
under in vivo conditions. similar to experiment 1 with regard to breed and sex,
The first trial was used to compare the effects of infection protocol, and response parameters. A total
a LauL to C10–C12 monoglycerides (mono- and dig- of 1,320 one-day-old Ross 308 male broiler chicks were
lycerides; MDG) and free C10–C12 MCFA (FFA) in randomly assigned to 11 treatments with 6 replicate
Clostridium-infected chickens. Because LauL was the cages each. Treatments were designed as noninfected
most promising molecule, a dose response study was and treated (NIT) with 0.3% LauL (NIT + 0.3 LauL)
made to determine the optimal dietary dosage. In a or without (NINT). The infected treatments were set
third trial, the efficacy of LauL was compared with up as a dose response starting with a 2 times higher
commercial products that limit bacterial activity in the dose as was used in experiment 1 (IT + 0.6 LauL)
intestinal tract (Aromabiotic Poul 60; Vitamex, Dron- and stepwise lowering the supplementation with 50%,
gen, Belgium; ABP) or coccidiosis (chemical coccid- from 0.6% to 0.3% to 0.15%, toward 0% supplementa-
iostat; C) and consequently its effect on intestinal mu- tion of LauL (INT). All diets with or without LauL
cus production. This series of experiments was made to supplementation were supplied from d 0 to 37 to see if
determine whether MCFA lactylates can be of help in a prolonged use of LauL before inoculation was more
reducing problems caused by C. perfringens in broiler effective. Postmortem lesion scoring was performed on
chickens. d 15, 16, and 17.
Experiment 3. Experiment 3 was similar to experi-
ment 2 with regard to breed, sex, the infection proto-
MATERIALS AND METHODS
col, response parameters, and start and length of the
Experimental Design dietary treatments. A total of 960 one-day-old broiler
chickens were randomly allocated to 8 treatments with
Experiment 1. The objective of this experiment 6 replicate cages each. Treatments were designed with
was to test the efficacy of a C12–C14 lactylate (LauL) 2 controls, INT and NINT without supplementation
against a C10–C12 MDG (50:50 mix of C10–C12 fatty of LauL or with 0.3% LauL (IT + 0.3 LauL or NIT
 ANTI-CLOSTRIDIAL EFFECTS OF SODIUM LAUROYL LACTYLATE 2403
+ 0.3 LauL), to test if LauL would benefit in healthy ma (Central Veterinary Laboratory, Weybridge, UK;
and infected chickens. The efficacy of 0.3% LauL in in- ~10,000 sporulated oocysts per mL; 1 mL per bird)
fected chickens was tested against 4 IT treatments with or with sterile saline after a 5-h feed withdrawal pe-
0.2% LauL dosage (IT + 0.2 LauL), the combination riod. This inoculation with E. maxima has consistently
of LauL with a chemical coccidiostat (Clinacox: 100 g/ shown to induce C. perfringens with an average of 50%
ton, Janssen Animal Health, Beerse, Belgium; IT + 0.3 of chickens scored positive for clostridial lesions. More-
LauLC), a chemical coccidiostat alone (IT + C), and a over, reddish E. maxima lesions are distinctly different
commercial MCFA product (Aromabiotic Poultry: 1.6 from white C. perfringens lesions. After the 5-d incuba-
kg/ton in starter and 1.2 kg/ton in grower phase, Vita- tion time for coccidiosis to develop, birds were orally
mex, Drongen, Belgium; IT + ABP). inoculated on d 14 with C. perfringens type A [~108
cfu/mL; 1 mL of liver broth (Difco, Detroit, MI) per
Birds and Housing bird] or with 1 mL of sterile liver broth after a 5-h feed
withdrawal period to induce necrotic enteritis. After
One-day-old male Ross 308 broiler chickens were sup- inoculations, birds had immediate access to feed. The
plied by Probroed & Sloot B.V. (Meppel, the Nether- pathogenic C. perfringens strain (code GD 5.11.53) was
lands). At d 0, broilers arrived at the poultry facilities obtained from the Animal Health Service (Deventer,
of Schothorst Feed Research B.V. (experiments 1 and the Netherlands). The strain was grown on an agar

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3; Lelystad, the Netherlands) or the Animal Health of sheep blood and the culture was typed by the Cen-
Service (experiment 2; Deventer, the Netherlands) and tral Institute of Animal Disease Control (Lelystad, the
were housed in 2-tier litter floor digestibility cages af- Netherlands) as C. perfringens producing type α and
ter individual weighing (surface area 0.90 × 0.65 m). β2 toxins.
Twenty birds were allocated to cages based on a weight
class system such that mean weight per cage was simi- Pathological Parameters
lar at the start of the experiment. Broilers were housed
in these cages throughout the entire experiment. At d During the peak of Clostridium infection at d 15
9, before first inoculation, the number of chickens was and 16 (1 and 2 d postinfection), 4 random selected
standardized to 17, and bird weight was measured be- birds per cage were used for postmortem coccidial and
fore and after standardization. Lighting and tempera- clostridial lesion scoring in the duodenal and jejunal
ture schedule throughout the experimental period was segment of the small intestine (48 birds per treatment).
22L:2D in the time period from d 0 to 9 and 18L:6D In experiments 2 and 3, a third scoring was done at
from d 9 to the end of the experiment. The ambient d 17 (3 d postinfection). Birds were killed by an in-
temperature gradually decreased from 32°C at the start tracardial injection with a euthasate (T61; Intervet,
to 25°C at the end of the experiment. Feed was sup- Mechelen, Belgium). The abdomen was opened and the
plied for ad libitum intake from d 0 onward with the gastrointestinal tract was exposed. The gastrointestinal
exception of the 5-h feed withdrawal period before in- tract was segmented into the duodenum and jejunum
oculations (on d 9 and 14). Water was available for (the segment from the gizzard to the 10 cm preced-
ad libitum intake throughout the experiment. Broilers ing Meckel’s diverticulum). All birds were scored blind
were not vaccinated during the experiment. (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 =
Experimental Diets 1 to 5 small white lesions (spots of less than 1 mm in
Broilers were supplied a wheat-soybean meal-based diameter); 2 = more than 5 small white lesions (spots
starter diet for ad libitum intake from day of arrival of less than 1 mm in diameter) or 1 to 5 larger lesions
until d 9. At d 9, a wheat-barley-based basal grower (spots of 1 to 2 mm in diameter); 3 = more than 5 larg-
diet was fed until the end of the experiment (Table 1). er lesions (1 to 2 mm in diameter) or erosive zones; 4
Grower diets were fed as mash because of the necessity = dead birds with positive necrotic enteritis diagnoses
of homogeneously mixing in the test products after feed postmortem. Based on the clostridial lesion scores, the
production. Diets did not contain any nonstarch poly- percentage of Clostridium-infected birds was calculated
saccharide enzymes, coccidiostats, or antimicrobial feed by the following equation:
additives other than the test products. The nutrient
composition of the experimental diets was according (sum of birds scored >0/total number
to Dutch standards to meet nutrient requirements of of birds scored) × 100%.
broilers (CVB, 2008).
The percentage of birds affected indicated the inci-
Challenge dence of C. perfringens. By categorizing the severity
of lesions from 0 to 4, an average lesion score could be
To induce the onset of a C. perfringens infection, a calculated for each treatment, giving an indication of
mild E. maxima infection was used as pretrigger. Birds the severity of the C. perfringens infection besides its
were orally inoculated at d 9 with 1 mL of E. maxi- 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- Yij = μ + Blocki + Treatmentj + eij,
cession. The experimental protocols and all procedures
complied with the guidelines of the Ethical Committee with Yij = response parameter; μ = mean; Blocki =
for Animal Experiments (Lelystad, the Netherlands). 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.
Statistical Analysis The model used for experiments 2 and 3 was as fol-
lows:
The incidence of C. perfringens (% of birds scored
>0) and mortality rate were analyzed by Fisher’s ex- Yij = μ + Blocki + Dosej + eij,
act test, whereas the severity of lesions, measurements
on production performance, and other parameters were with Yij = response parameter; μ = mean; Blocki =
analyzed by ANOVA using GenStat statistical software effect of block (i = 1 to 6); Dosej = effect of dose (j =
(GenStat version 12, Hemel Hempstead, UK). Raw 9 and 3 for experiments 2 and 3, respectively); and eij
data of BW and feed intake were analyzed for outliers. = residual error.
Outliers were marked significant when exceeding 2.5 × The model used for experiment 3 was as follows:
SD of the treatment mean and were excluded from the
data set. Treatment means were compared by the least Yijk = μ + Blocki + Coccidiostatj + Testk
significant difference. The statistical model(s) used per
experiment were as follows, based on the entire data + Interactionjk + eijk,
sets or subsets for each experiment.
The model used for experiments 1, 2, and 3 was as with Yijk = response parameter; μ = mean; Blocki =
follows: 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, and BWG compared with INT (P < 0.001).
whereas 0.05 < P ≤ 0.10 was considered to be a near
significant trend. Experiment 2
The percentage of birds scored positive for C. per-
RESULTS
fringens was significantly influenced by challenge and
General dietary treatments. No lesions were observed in NINT
and NIT treatments, whereas 56% was infected in the
The INT groups in all experiments were scored posi- INT control treatment (mean of d 15, 16, and 17; Table
tive for C. perfringens. As an average of lesion scoring 4). Supplementation of 0.6% LauL (IT + 0.6 LauL) re-
performed at d 15 and 16, the INT groups resulted in sulted in 35% of birds scored positive. The other LauL-
42, 62, and 83% of birds scored positive for clostridial supplemented treatments resulted in similar percent-
lesions in experiments 1, 2, and 3, respectively. In NINT ages of broiler scored positive for necrotic enteritis as
groups, all birds were scored negative. Also, clostridial INT.
lesions were more severe (P < 0.001; Table 2). Calcu- With regard to severity of lesions, birds supplement-
lating the scores from 0 to 4 in all necropsied birds, a ed with 0.6% LauL had less severe lesions than birds in
lesion severity of 1.3, 1.8, and 2.2 for the 3 successive the INT control (P < 0.001). A dose of 0.3% LauL did
experiments was determined. reduce lesion severity as well toward a near-significant
reduction in comparison to the INT control group (P ≤
Experiment 1 0.10). All other LauL-supplemented treatments did not
improve results on lesion severity (Table 4). A sigmoid
On d 15 and 16, the number of birds scored positive dose response curve was fitted to the data, explaining
for C. perfringens was not affected by dietary treat- 81% of the variation (Figure 1). It was shown that a
ments FFA, MDG, and LauL compared with the INT dose higher than 0.15% should be used to expect any
control (Table 3). The severity of lesions was not sig- positive effect of LauL on lesion severity.
nificantly affected by any of the treatments on d 15, A similar pattern was observed for mortality (data
whereas on peak of infection at d 16, dietary supple- not shown), being highest in the INT control (18%)
mentation of MDG and LauL resulted in lesions that but reduced in 0.6% LauL to 5% (P ≤ 0.05) and 11%
were less severe compared with the INT and IT + FFA in 0.3% LauL (P ≤ 0.10).
groups (P < 0.001; Table 3). Three days later (6 d Production parameters were only affected by dietary
postinfection), no significant differences were observed treatments from d 9 to 20. Body weight gain was high-
between treatments (data not shown) as all treatments est in the NINT control (574 g) and NIT + 0.3 LauL
recovered from necrotic enteritis, based on macroscopi- (591 g), as expected, whereas all infected treatments
cal evaluation. A similar pattern was observed for mor- had a 150 to 200 g lower BWG from d 9 to 20 (data not
tality, which was higher in the INT control, FFA, and shown). None of the LauL doses led to a significantly
MDG treatment compared with the NINT treatment better response on final weight at d 37 (Table 4).
(P ≤ 0.05). The LauL treatment was intermediate, not
being significantly different from any treatment (Table Experiment 3
3).
Production parameters from d 9 to 20 were affected Like in experiment 2, the percentage of birds scored
by dietary treatments. Body weight gain was highest in 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 ly), being lower than the ABP group but slightly higher
NINT and NIT treatments, whereas 80% was infected than the LauL treatments and the INT control (data
in the INT control treatment (mean of d 15, 16, and not shown). The highest mortality rate of 32.4% was
17). Supplementation of LauL (0.3 or 0.2%), ABP, or C observed in the ABP treatment, which was higher than
or the combination of 0.3% LauL and C did not reduce in the 0.2% LauL or 0.3% LauLC group (P ≤ 0.05).
the number of infected birds when compared with the Supplementing 0.3% LauL or C resulted in a similar
INT control. Lesion severity of infected birds treated mortality rate as the INT control (Table 5).
with ABP had more severe lesions (2.5) than the INT Results of production parameters of the infected
control and 0.3 or 0.2% LauL treatments (varied from treatments are presented in Table 5. No differences
2.0 to 2.2). Supplementation of C with or without 0.3% in growth performance were observed between treat-
LauL gave intermediate results (2.2 and 2.3, respective- 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- Figure 1. Estimation of the dose response relationship between the
mance that is usually caused by a decrease in feed in- dietary lauroyl lactylate (LauL) concentration and the lesion severity
(experiment 2). The closed dots indicate the infected-treated and open
take during infection. A reduction in feed intake was dot indicates the infected-nontreated group. Clostridial lesions were
also observed in these experiments. The effect of a scored on a scale of 0 to 4 (0 = no lesions and 4 = most severe lesions
clostridial infection on daily feed intake in experiment and death due to necrotic enteritidis). Dots sharing no common letter
2 is presented in Figure 2, which is a typical response differ significantly from each other (P ≤ 0.05). Color version available
in the online PDF.
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- birds. This lower FI does not fully explain the 150 to
gens at d 14. From d 14 onward, daily feed intake de- 200 g loss in BWG after infection (assuming a FCR of
creases until the peak of infection at d 16. After d 16, 1.4). The stronger effect of the infection on BW may
daily feed intake recovers but stays consistently lower be related to reduced nutrient digestion and absorption
than the healthy control group until d 19 (Figure 2). after C. perfringens colonization as well as to inflamma-
Between d 9 and 20, a 150 g lower FI was measured tion. Bacterial pathogens, such as C. perfringens, are
for the infected birds in comparison to the noninfected 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 A chemical coccidiostat was preferred over an iono-
Immerseel et al., 2004). Besides that, the inflammatory phore because the latter has strong antibiotic proper-
response and activation of the immune system result in ties. Adding an ionophore to the diet could have killed
high energy costs and reduced efficiency of protein uti- off the inoculated C. perfringens even before causing
lization that may lead to a reduced supply of nutrients intestinal damage, creating too big of a risk of failure
for maintenance and growth. of the infection model. In this third experiment, neither
To be able to determine whether the lactylate used in an effect of LauL nor a coccidiostat was observed. The
the current study has potential to be effective against absence of an effect of LauL and the coccidiostat is
C. perfringens, the results on incidence and lesion sever- hard to explain but could have been caused by a stron-
ity were compared with the INT controls. In the first ger infection. The infection was worse in this study, re-
experiment, LauL containing C12–C14 fatty acids es- sulting in 83% of birds showing an average lesion score
terified to lactic acid was tested against C10–C12 MDG of 2.2, whereas experiments 1 and 2 resulted in 42 and
or C10–C12 FFA. Based on lesion severity and mortal- 56% of birds with lesions of 1.3 and 1.6, respectively
ity rate, it seemed that this lactylate had the greatest (Table 2). The infection in the third study might have
potential to reduce negative effects of a C. perfringens been too strong to find a preventive effect of LauL or a
infection. This led to a dose response study with LauL, coccidiostat against an infection of C. perfringens.
which showed a clear significant reduction in lesion se- Although no effect of lactylate was seen in the third

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verity with higher concentrations of LauL in the diets, study, a slight positive effect was seen in the first 2
but did not reduce the number of birds scored positive experiments. The positive effect of lactylate can be at-
for C. perfringens. From a sigmoid curve, it was con- tributed to their chemical composition. Acyl lactylates
cluded that only LauL doses higher than 0.15% were are produced by the reaction between the acyl group of
to be expected to improve lesion severity. In the third fatty acids from 6 to 22 carbon atoms and lactic acid
study, it was therefore decided to repeat a 0.3% dose. in the presence of an alkali metal. This reaction pro-
A 0.2% dose was also tested because the latter might duces the salts of the corresponding fatty acid ester of
have an effect but was not tested as such in experiment lactylic acid using the appropriate fatty acid and lactic
2. Both doses of LauL could be used as dietary supple- acid. By controlling the fatty acid used and the degree
mentation in practice and to determine their potential of condensation of the lactylic acid component, differ-
in the feed industry, efficacy of LauL was compared ent effects can be obtained. One of the characteristics
with some commercial products that are used in prac- of LauL is its strong bacteriostatic property. Sodium
tice to control bacterial overgrowth directly or indi- lauroyl lactylate has been shown to have a broad range
rectly. Aromabiotic Poul 60 contains MCFA that have of antimicrobial activity against bacteria, molds, and
antibacterial properties and should have a direct effect yeast (Marnett et al., 1966; Osipow et al., 1969). In
on bacteria such as C. perfringens. Clinacox was used these studies, bacteriostatic effects were shown against
to see if LauL would have any advantages in compari- Staphylococcus aureus, Aspergillus terreus, and Asper-
son to or in combination with a chemical coccidiostat. 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-
Figure 2. The effect of the subsequent infection of Eimeria maxima ous solution. The C12 fatty acids present in LauL are,
at d 9 and Clostridium perfringens at d 14 on daily feed intake between therefore, a nice compromise between chain length and
9 and 20 d of age (experiment 2). NINT = noninfected-nontreated; solubility but are by itself probably not the strongest
INT = infected-nontreated. An asterisk (*) indicates significant effects
of infection on a specific day (P ≤ 0.05). NS = no significant effects of antimicrobial. A lactylate, however, in combination
infection. Color version available in the online PDF. with fatty acids with different chain lengths, has strong
 ANTI-CLOSTRIDIAL EFFECTS OF SODIUM LAUROYL LACTYLATE 2409
antimicrobial properties. In the data given in patent feed withdrawal on Salmonella and Campylobacter contamination
EP2082739A1, the minimum inhibitory concentration of broilers. Poult. Sci. 80:278–283.
Collier, C. T., J. D. van der Klis, B. Deplancke, D. B. Anderson, and
values of C12 and C14 lactylates are the lowest. The H. R. Gaskins. 2003. Effects of tylosin on bacterial mucolysis,
minimum inhibitory concentration values for these lac- Clostridium perfringens colonization, and intestinal barrier func-
tylates tested for C. perfringens are, respectively, 0.002 tion in a chick model of necrotic enteritis. Antimicrob. Agents
Chemother. 47:3311–3317.
and 0.001% in comparison to 0.04% for C10 lactylates. CVB. 2008. Tables values. Animal Nutrition 2008: Feeding values
The mechanism of action is most probably due to the of different feedstuffs and nutrient requirements for production
slow metabolization of the lactylate. Lactylates will animals. Central Bureau for Livestock Production, Lelystad, the
break down on ingestion, releasing FFA and lactic acid, Netherlands.
Elwinger, K., C. Schneitz, E. Berndtson, O. Fossum, B. Teglöf, and
which will be metabolized as normal. There is some B. Engström. 1992. Factors affecting the incidence of necrotic
evidence that the metabolic degradation or intestinal enteritis, caecal carriage of Clostridium perfringens and bird per-
absorption of lactylates may be delayed in comparison formance in broiler chicks. Acta Vet. Scand. 33:369–378.
to lactic acid or FFA (Phillips et al., 1981). This de- Hofacre, C. L., T. Beacorn, S. Collett, and G. Mathis. 2003. Using
competitive exclusion, mannan-oligosaccharide and other intesti-
lay may be sufficient to allow the lactylate to have a nal products to control necrotic enteritis. J. Appl. Poult. Res.
stronger effect on bacterial infection in the intestinal 12:60–64.
tract than either FFA or lactic acid alone. But even Hofshagen, M., and M. Kaldhusdal. 1992. Barley inclusion and
when it does hydrolyze, both of the molecules released avoparcin supplementation in broiler diets. 1. Effect on small

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intestinal bacterial flora and performance. Poult. Sci. 71:959–
(lactic acid and MCFA) still have some antibacterial 969.
activity. The combination between the strong antimi- Jansman, A. J. M., C. M. F. Wagenaars, A. Schonewille, and H.
crobial lactylate, its bacteriostatic products in the form Snel. 2006. Control of Clostridium and Campylobacter infections
of MCFA and lactic acid, and its slow metabolization in poultry via natural antimicrobial feed components. (In Dutch).
Pages 19–26 in Nutrition and Intestinal Health. Series of Re-
is the most probable explanation for the observed ef- search Reports 6. Product Board of Animal Feed, Wageningen,
fects on clostridial lesion scores and mortality in broil- the Netherlands.
ers seen in experiments 1 and 2. Still, more attention Kabara, J. J., and D. L. Marshall. 2005. Medium-chain fatty acids
and esters. Pages 327–360 in Antimicrobials in Food. CRC Press,
should be paid to the molecule and its physiological Boca Raton, FL.
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prove results in experiment 3. Reduced incidence of Clostridium perfringens-associated lesions
It can be concluded from this study that higher con- and improved performance in broiler chickens treated with nor-
mal intestinal flora of adult fowl. Avian Dis. 45:149–156.
centrations of LauL in broiler diets have a potential to Løvland, A., and M. Kaldhusdal. 2001. Severely impaired production
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