Feeding malic acid to chickens at slaughter age benets poultry production and microbial safety in regard to Campylobacter

Chicken has popular for consumption worldwide. However, chicken ocks suffer the Campylobacter infection during the rearing period, which allows chicken meat products act as a vehicle for transmitting this pathogen through the food chain and bring great health and economic burden to the public. Malic acid is a dicarboxylic organic acid with antimicrobial activity, the application of malic acid during food animal rearing period also found could improve the performance of animals and the quality of their products. This study applied malic acid to chicken ocks and evaluated its potential benets on the poultry production and microbial safety.


Results
In Experiment 1, AA broilers and partridge chicken were provided with the malic acid-supplemented drinking water for three weeks, although the Campylobacter carriages were found decreased by 0.91-0.98 log after the rst week of use (P < 0.05). However, this effect was not consistent, signi cant decontamination could not be found in the second and the third week of application. Thus, in Experiment 2, the malic acid-supplemented drinking water was used for ve days to ocks at slaughter age, the Campylobacter carriage was found decreased by 1.05-1.55 log (P < 0.05), suggesting an effective reduction effect exist. Malic acid has no adverse effects on chicken performance, including body weight, intestinal indices and micro ora. However, the meat quality of AA broilers was found to be promoted, the moisture increased by 5.12% − 5.92% (P < 0.05), and the fat decreased by 1.60% (P < 0.05). In Experiment 3, the malic acid-supplemented drinking water was provided to AA broilers which were suffering from respiratory disease. The results showed that the mortality rate of malic acid treated group was consistently lower than that of the control group during the experimental period, and the total mortality was decreased from 52% in the control group, to 32% in malic acid treated group.

Conclusions
Our results suggest that feeding malic acid to ocks could decrease the contamination of Campylobacter while also bene t chicken farming, and is worthy of application to promote safe development of the poultry production and its products.

Background
Poultry production is closely related to human life, and chicken meat has become popular for consumption worldwide, due to its characteristics such as high protein, ample micronutrients, low fat content, and comparatively low price when compared to beef or pork (1,2). However, chicken ocks suffer foodborne pathogen infections during the rearing period, which could then be transmitted through the farm-to-fork chain and pose a serious threat to food safety and human health. This issue has been one of the major problems that has beset chicken meat production, while the contamination of Campylobacter is particularly serious (2)(3)(4).
The chicken is generally recognized as the natural host for Campylobacter in the intestine without developing symptoms (5). During the slaughter processing, the chicken intestinal tract may leak or rupture and contaminate the meat, which provide a potential source of cross-contamination in the food chain (2)(3)(4). It is estimated that the handling and consumption of chicken meat-related products may account for 20-30% of human infection cases, while 50-80% may be attributed to the chicken reservoir as a whole (6). Recent studies showed that the decontamination of Campylobacter in chicken ocks would signi cantly reduce the risk of human campylobacteriosis(4).
Due to the increasing antibiotic resistance in pathogens, the use of antibiotics is limited in chicken production (7,8). Thus, nding alternative antimicrobial agents has recently become more and more important (3). Malic acid is a type of dicarboxylic organic acid which showed antimicrobial activity (9). Besides, malic acid is an intermediate in metabolic cycles of organisms for energy production, and could aid digestion and absorption by chelating various cations and enhancing the activities of some digestive enzymes (10). Malic acid has been permitted as GRAS (Generally Recognized As Safe), which does not has adverse effects for human intake or animal feeding when properly used. Attempts to provide malic acid to livestock were performed in previous studies, which showed its potential bene ts in promoting the performance of animals and their products (10)(11)(12).
The bactericidal effect of malic acid on Campylobacter has been demonstrated in laboratory culture and on raw meat samples (13,14). However, its antimicrobial e cacy during the chicken rearing period has never been investigated, and the potential effects of feeding malic acid to chickens on their performance and products still stay unclear. This study supplemented malic acid into the drinking water of the ocks that were found naturally Campylobacter-positive. The decontamination effect of malic acidsupplemented water and its in uence on chicken performance were evaluated in AA broilers and partridge chicken. During the slaughter of AA broilers, the intestine and meat samples were collected, and the in uence of this acidi ed drinking water on chicken products was analyzed. In another ock of AA broilers, which were suffering from respiratory disease. The malic acid-supplemented drinking water was provided to evaluate its potential role in helping the ocks resist disease. The obtained results provide a reference for the further practical application of malic acid to promote safe poultry production.

Results
The reduction effect of malic acid-supplemented drinking water on Campylobacter was not consistent for a long period of use In Experiment 1, the malic acid-supplemented drinking water was provided to ocks continuously for three weeks. Before the experiment, the Campylobacter carriages in the control group and malic acid treated group were similar both in the partridge chickens and AA broilers (p > 0.05). After one week treatment by acidi ed drinking water, the Campylobacter carriages were found to be signi cantly decreased by 0.98 log and 0.91 log in the partridge chickens and AA broilers, respectively, compared to the control group (p < 0.05). After two weeks of malic acid treatment, the average amounts of Campylobacter in the acid treated groups were slightly higher than the control group for both the partridge chickens and AA broilers, but no signi cant differences was observed (p > 0.05). Similar results were also observed in the third week, suggesting that the decontamination effect of the malic acidsupplemented water was not consistent, which is decreased with extended of the time of use (Fig. 1).
The use of malic acid-supplemented drinking water for 5 days before slaughter could reduce the contamination of Campylobacter in ocks For Experiment 2, the acidi ed drinking water was given for ve days to ocks at slaughter age. Before the experiment, similar carriages of Campylobacter, approximately 4 log cfu (colony forming units) /g were detected in all groups (p > 0.05). After ve days treatment, for the partridge chicken, the Campylobacter carriage detected in the cloaca of the control group was 3.54 log cfu/g, which was similar to the data before the experiment. However, in the malic acid treated group, the Campylobacter carriage was signi cantly decreased to 1.98 log cfu/g, and a 1.55 log reduction was found when compared to the control group (p < 0.05) (Fig. 2a). In the AA broilers, the Campylobacter carriage in the cloaca of the control group was increased to 5.19 log cfu/g during the ve days experimental period, while the carriage of the malic acid treated group was decreased to 4.14 log cfu/g and a 1.05 log reduction was found (p < 0.05) (Fig. 2b). In AA broilers caeca samples, the Campylobacter contamination load of the control group was 9.96 log cfu/g, while the corresponding colonization level in the malic acid treated group was only 8.40 log cfu/g with a 1.56 log reduction (p < 0.05) (Fig. 2c).
The treatment of malic acid-supplemented water does not in uence the chicken performance, intestinal indices and micro ora In Experiment 2, the average weight and micro ora of ocks in different groups were similar before the experiment (p > 0.05). For the partridge chickens, the average weight of the malic acid treated group was approximately 0.81 kg, which was slightly higher than that of the control group after treating by the malic acid-supplemented water for ve days (p > 0.05). Similar results were also observed in AA broilers, where the average weights of the malic acid treated group and control group were approximately 1.52 kg and 1.50 kg, respectively (p > 0.05) ( Table 1). For the intestinal micro ora, in the cloaca of partridge chickens, they were slightly decreased to 11.66 log cfu/g in malic acid group after ve days treatment, when compared to 11.72 log cfu/g in the control group(p > 0.05). In AA broilers, the detected micro ora in the malic acid treated group and control group were 11.79 and 11.51 log cfu/g, respectively (p > 0.05), suggesting malic acid does not affect the body weight and micro ora of the ocks (Table 1). The potential effect of malic acid-supplemented water on the intestines of chickens was evaluated in AA broilers. As shown in Table 2, after ve days experimental period, the average weight of the intestine was approximately 25.1 g, the length of small intestine was approximately 112.6 cm, the caecum was approximately 11.0 cm, and the pH of the caecal content was approximately 7.26 in the control group.
Meanwhile, in the malic acid treated group, the weight of the intestines was approximately 26.7 g, the length of small intestine was approximately 110.9 cm, the caecum was approximately 12.1 cm, and the pH of the caecal content was approximately 7.03. No signi cant differences observed between the treatment group and control group (p > 0.05). Drinking malic acid-supplemented water bene ts the quality of chicken meat In Experiment 2, the meat composition of AA broilers was evaluated by measuring the contents of moisture, crude protein, ash and fat. As shown in Table 3, there were no signi cant difference of the protein and ash contents in groups supplemented with and without malic acid (p > 0.05), suggesting that malic acid has no in uence on the nutrition of chicken meat. Compared to the control group, the moisture content in the malic acid treated group was increased by 5.12% in the thigh meat portion, and 5.92% in breast meat portion, respectively (P < 0.05), indicating the juiciness of meat was improved. The fat content in the malic acid treated group was decreased by 1.60% in the thigh meat compared to the control group (p < 0.05), which contributes to the taste sensory of the meat. * Asterisks indicate signi cant difference (P < 0.05) was found in the malic acid treated group when compared to the control group Malic acid-supplemented drinking water decreased the mortality of the chicken ocks In Experiment 3, the acidi ed water was provided to another chicken ock which suffers from respiratory disease, for three weeks of use. In the rst week, none broilers were dead in both the control and malic acid treated group. During the second week, the mortality rate of control group was 36%, while it decreased to 24% in the malic acid treated group. On week 3, the mortality rates of the control group and malic acid treated group were 16% and 8%, respectively. In total, 13 of broilers were found dead in the control group, and the total mortality rate was 52%, while only 8 broilers were dead in the malic acid treated group with a total mortality rate of 32% (Table 4).

Discussion
Campylobacter is one of the leading causes of food-borne gastroenteritis in humans worldwide (2)(3)(4), which accounts for approximately 96 million cases of human illness per year on a global scale (15). Poultry is the most common species associated with human Campylobacter illness, and most chicken ocks became Campylobacter-positive when reached slaughter age, making it an important reservoir for human infection. After Campylobacter infect humans, the clinical symptoms could include mild abdominal pain, headaches, fever, vomiting and sever watery and bloody diarrhea, and the infection can sometimes lead to serious sequelae such as Guillain-Barré syndrome, Miller Fisher syndrome and reactive arthritis(15), although most cases were self-limiting. Still, a number of patients required medication and hospitalization treatment, which brings great health and economic burden to the public (3,4,15). Therefore, the control of Campylobacter colonization in chickens at the farm level should be meaningful work.
Malic acid can be industrially produced at present and has the advantages of no pollution/residue, lack of toxicity and easy of application (16). In previous studies, it was found that malic acid could cause a 6 log reduction of Campylobacter in laboratory broth and a 4 log reduction in chicken juice after 24 h of exposure at 4 °C (13). The contamination of Campylobacter on chicken legs was also observed to decrease 1.18 log after treatment with malic acid solution at 4 °C for 8 days (14). These results foreshadow the promising application of malic acid in the poultry industries, but the antimicrobial effect of malic acid towards chicken feeding still stayed unclear. Malic acid is commonly recognized as a mild acid and has wide application in the food industry (16), which makes it possible to be applied in animal feeding. Our pre-experiment found that the effect of malic acid against the growth of Campylobacter in vitro was obvious (Supplementary Figure S1). Besides, an in vivo study showed that malic acid had a more stable effect to control the Campylobacter contamination when compared to other acids (Supplementary Figure S2). The minimum inhibitory concentration of malic acid against Campylobacter was also found to be lower than for other acids (17), suggesting that a more effective and economical potency may exist.
Organic acids exploit their antimicrobial activity in the undissociated form, which is closely related to the pH of the medium (18). Previous studies have indicated that the well bactericidal effect of organic acids on Campylobacter strains shows up at a pH of 4.0 (19), and thus we decided to adjust the pH to 4.0 using malic acid in this study. There is no evidence of the vertical transmission of Campylobacter in chicken ocks, while the cross contamination from the environment seems to be an important infection source (4,20). Malic acid could be supplemented into the feed or drinking water of the broilers. The dry conditions of the feed is lethal to Campylobacter, which is thus recognized as not being a potential source for contamination (21). Meanwhile, water is an important vehicle for spreading Campylobacter that is prominent in chicken ocks (4,21,22). Thus, malic acid was added into the drinking water in our study.
Although most studies reported the effectiveness of using organic acid to control the contamination of pathogenic bacteria during animal rearing (17,(22)(23)(24), some results showed its limited or variable effect which could not be ignored (25)(26)(27). The bacteriostatic or bactericidal effect of the organic acid may depend on the manner of use (concentration, vehicle, and duration) and is also related to the status of the host. In our study, we found that supplementation of malic acid into the drinking water for the chicken ocks at slaughter age was effective in two chicken lines, the Campylobacter load in the cloaca was found to be decreased by 1.05-1.55 log cfu/g, while in the caeca was decreased by 1.56 log cfu/g. However, in a long period daily rearing for three weeks, the effect of malic acid was not consistent, with signi cant decontamination only found in the rst week of application. Our present study suggests that application of acidi ed drinking water to the broilers at slaughter age could effective limited the Campylobacter infection load. Limitation of its use to the last week before slaughter also has other advantages, such as cost savings, thus making it more economical. In addition, Campylobacter infection of the broilers at slaughter age are epidemiologically more relevant to human infection, thus making the decontamination more meaningful. Nevertheless, the reason why a long period of application of acidi ed drinking water has a limited decontamination effect on Campylobacter still requires further investigation.
Malic acid is a avoring agent, and it is also an intermediate in metabolic cycles, including the tricarboxylic acid (TCA) cycle and the glyoxylic acid cycle (GAC). Feeding diets with malic acid was found to increase the weight gain and feed consumption in Japanese quails (11). For dairy cows, the feed e ciency was improved, the milk yield was increased and the fat content in the milk was subtly changed (12). The growth and feed utilization were found to be improved in tilapia, but an excess supply would compromise the bene cial effect (10). Despite that inconsistent ndings were reported (28), these results showed the potential bene ts of feeding malic acid to animals when applied with suitable nutritional and managerial measures. In our study, the broiler body weight as well as intestinal indices, including length, weight, pH, and micro ora were not in uenced by the malic acid supplementation, indicating that the chicken performance was not affected at least. The bacteriostatic effect was found on Campylobacter, while not on the normal intestinal micro ora, which may be attributed to the acidintolerance characteristics of the pathogens when compared to the gut micro ora (especially for the probiotics) (29). The propagation of the pathogens was inhibited, which will in turn promote the growth of micro ora as it reduces microbial competition for nutrients (22,30).
A previous study showed that feeding malic acid to cows could in uence the composition of milk (12), and thus its potential effect on chicken meat was analyzed. Our results showed that the protein content of the chicken meat was not in uenced by the malic acid treatment, suggesting that the nutrition was maintained. Compared to the control group, the moisture was increased by 5.12% and 5.92% in thigh and breast meat, respectively, while the fat content was decreased by 1.60% in thigh meat. This contributes to the tenderness, juiciness and taste sensory of the meat and promotes the acceptability and preference of the customers (31,32). The changes in the chicken meat composition may be attributed to the in uence of malic acid on the sense of chicken taste, and could affect the feed intake, nutrient digestion and conversion. Organic acids could exert bene cial effects on disease resistance, which was observed in tilapia (33,34). In this study, the malic acid-supplemented drinking water was provided to one ock, which suffers from respiratory disease, and after three weeks of treatment with acidi ed water, the mortality was found to be decreased from 52-32%, which is also an additional bene t for chicken rearing.
This study showed that the malic acid-supplemented drinking water could bene t chicken production while also improving food safety. Our study only applied malic acid, and many improvements could be considered in the subsequent research, such as the use of malic acid in combination with prebiotics or bacteriophages (35) as well as with nutritional, managerial and biosecurity measures (30). Additionally, the bactericidal effect of malic acid was observed as not only restricted to Campylobacter but also on other food-borne pathogens (9), which foreshadows its promising application improving the safety of poultry production.

Conclusion
This study supplemented malic acid in drinking water of chicken ocks to evaluate its antimicrobial effect to the broiler chicken. The signi cant reduction of Campylobacter contamination loads was observed in the rst week and decreased with extended of time supplementary. Thus, providing the acidi ed drinking water to ocks at slaughter age for ve days showed to be an effective decontamination method. Malic acid has no adverse effect on chicken performance, including the body weight, intestinal indices, and micro ora. Meanwhile, it could bene t the quality of chicken meat, by increasing the moisture and decreasing the fat content. In addition, malic acid was found to decrease the mortality of broiler suffering from disease. Our present results suggest that the application of malic acids to the ocks at slaughter age is a feasible and effective way to control Campylobacter contamination while also bene ting poultry production.

Animals and treatments
The AA broilers were provided by Jiangsu Jinghai Poultry Industry Group Co., Ltd, and raised in oor pens with dimensions of approximate 8 m 2 per group. The partridge chickens were provided by poultry farms in Nantong city, Jiangsu province, and raised in cages with dimensions of approximate 3.5 m × 1.75 m per group. For all chicken houses, the temperature was maintained around 21 -28 ℃, and humidity was around 50 % -60 %. The ocks were supplied with commercial feed with corn, wheat, soybean meal as the main ingredients, the detailed composition of the feed was listed in Supplementary Table S1. Normal water, or acidi ed water prepared by supplementation of L-malic acid to a nal pH value of approximate 4.0, were providing to the ocks for drinking. Chicken had free access to water and commercial feed at all the time, the feed and drinking water were refreshed every day.

Experimental design
Three experiments were carried out in this study. In each experiment, chickens with less individual differences were randomly divided into two groups and reared in separate areas. The experimental group was provided the malic acid supplemented water, while the control group was given the non-supplemented water. For experiment 1 and 2, the cloacal swabs were sampled for each ock before conducting the experiments to ensure Campylobacter-positive. Experiment 1. Two ocks (2-week-old AA broilers and partridge chickens) were selected for evaluating the antimicrobial effect of the malic acid-supplemented drinking water on Campylobacter during rearing period. In each ock, the chickens were divided into two groups (20 chickens per group), the experimental group and control group were given the acidi ed water and non-supplemented water continuously for three weeks, respectively. The cloacal swab samples of the chickens were collected before the experiment and one, two and three weeks later after giving the speci c water for counting the contamination load of Campylobacter. Experiment 2. Another two ocks (5-week-old AA broilers and 10-week-old partridge chicken) were selected for evaluating the application of the acidi ed water to the ocks at slaughter age. In each ock, the experimental group and control group (20 chickens per group) were given the acidi ed water and nonsupplemented water for ve days. The body weights and cloacal swab samples of each chicken were recorded before conducting the experiment and ve days later. The AA broilers were sent to slaughter house after the experimental period, the broilers were electrically stunned and killed by neck cutting, after defeathering process, the intestine and meat samples were collected and analyzed. Experiment 3. For evaluate the effect of acidi ed water in helping the ocks to improve disease resistance, another chicken ock (AA broilers approximate 3-week-old) which were suffering from the respiratory disease was selected. 50 chickens were picked out from this sick ock, divided into two groups by giving the malic acid supplemented or normal water for three weeks, respectively. The mortality rates of the malic acid treated group and the control group was recorded after one, two and three week treatment.

Analysis of the chicken performance
The body weights of the chickens were recorded before and after giving the acidi ed water to analyze its in uence on the chicken weight gain, the cloacal swabs were collected to analyze the intestinal micro ora and Campylobacter carriage. During the slaughter of AA broilers (experiment 2), the intestines of the chicken were collected, the total weight of the intestine samples were recorded, then separated and ligated at the subsection to compare the length of the intestinal components. The caecal contents were squeezed into 10 mL tubes, diluted (1:8) with distilled water, vortexed, and read using a pH meter as described previously (23).

Enumeration of Campylobacter and micro ora from cloacal swabs
The swabs were moistened in buffered peptone water (BPW) and weighed both before and after taking the fecal samples. The number of Campylobacter and micro ora presented in the feces were than calculated as described previously with some modi cations (26). The cloacal swab with sample was immersed in 1 mL of phosphate buffer saline (PBS) for 20 min and shaken several times. The swab was then removed from the solution and appropriately diluted, with 100 μL of the diluent spread onto the CCDA agar containing rifampicin, polymyxin B, trimethoprim, cycloheximide, vancomycin, amphotericin B and cefoperazone and incubated at 42°C under a microaerophilic atmosphere for 36 h to count the Campylobacter. For enumeration of the intestinal micro ora of the chicken, 100 μL of the appropriate diluent prepared above was spread on the plate count agar (PCA), the plate was incubated at 37°C for 24 h for colony count.
Determination of Campylobacter colonization in the caecum Colonization assay was performed as described in a previous study (36). The caecum of the chicken was collected during the slaughter (experiment 2), and the lumenal contents were gently extruded. The tissue was weighed, homogenized, serially diluted and plated on selective CCDA agar to count the Campylobacter as described above.

Analysis of the proximate composition of chicken meat
The chicken meat samples were collected during slaughter (experiment 2), approximately 10 g of breast and thigh meat samples were collected and stored at −70°C. The samples were thawed overnight at 4°C, and ground when used for subsequent analysis. The moisture present in the samples was determined by drying meat samples at 105 °C until a constant weight was achieved. Crude proteins were determined according to the Kjeldahl method from the amount of ammonia ions neutralized by sodium hydroxide.
Crude fat was determined by Soxhlet extraction procedure with petroleum ether. Crude fat was determined by burning the samples in the mu e furnace at 550 °C for 12 h. All procedures were performed according to the method described by the Association of O cial Agricultural Chemists (AOAC) (37).

Statistical analysis
Statistical analyses were performed by GraphPad Prism software 8.0 (San Diego, CA, USA). Data were expressed as the mean ± standard deviation (SD). Normality was tested with the Kolmogorov-Smirnov test or with the D'Agostino-Pearson test to assure Gaussian distribution of values. Statistical signi cance between 2 groups was analyzed using unpaired t-test (Welch's correction was used if there were unequal variances) when normality test passed, otherwise using Mann-Whitney U test applying 95% con dence intervals, and P < 0.05 was considered statistically signi cant. Campylobacter carriage in the cloaca of chickens after a long period of use of malic acid. Two-week-old partridge chickens (a) and AA broilers (b) were provided with the malic acid-supplemented drinking water for 3 weeks, and the cloacal swab samples were collected at designated time to determine the