1. IntroductionOver the past few decades, a variety of additives with antimicrobial and/or growth-promoting effects have been widely used in poultry feed during commercial grow-out to enhance the economic benefits [1]. Pathogens develop resistance to antibiotics at a rate much faster than the discovery of new antibiotics, and resistant microorganisms can spread between food-producing animals and humans [2]. Antibiotic resistance and residues in broiler production have been taken as one of the biggest challenges of human health [3,4,5]. As the voice of antibiotic-free food is gradually increasing, it is a global trend in animal production to reduce or ban the use of antibiotics for growth in feed [6,7,8,9]. Nowadays, researchers have focused on identifying functional feed additives as alternatives to synthetic antibiotics in broiler production, such as probiotics, prebiotics, synbiotic, organic acid, bioactive peptide, emulsifiers, enzymes, and some bioactive substances [10].The addition of acidifier in the diet of broilers could minimize inflammation-induced damage, prevent mortalities from excessive intestinal lesions and oxidative stress, and improve growth performance [11,12]. Antibiotics promote abdominal fat accumulation in broilers [13], while dietary supplementation with acidifiers reduced abdominal fat mass deposition and improved the meat quality of broiler chickens besides growth performance and intestinal health [11,14]. Acidified drinking water can compensate for gastric acidity, control pathogenic bacteria, and improve production parameters in broilers [15,16]. The acidifier, benzoic acid, was found as a substitute for antibiotics to improve growth performance, antioxidation, nutrient digestion and gut microbiota of yellow-feathered broilers [17]. Humic acid was demonstrated to be valuable in improving the production and health safeguarding of livestock and poultry [18]. Organic acid mixture could control Salmonella typhimurium infection and maintain efficient growth performance of broilers [19]. Contaminated poultry meat is considered to be the main source of human infection with Campylobacter spp. The acidification of drinking water through the addition of organic acids has been reported to decrease the risk of Campylobacter spp. colonization in broiler flocks [20,21]. In-feed inclusion of medium chain fatty acids, together with vaccination of a live attenuated coccidiosis vaccine, reduced necrotic enteritis in broilers [22]. A microencapsulated blend of organic acids and essential oils could improve the quality and shelf life of poultry meat [23]. Therefore, acidifiers are promising alternatives to antibiotics for growth performance, antioxidant status, and intestinal health of broilers.As an intermediate of the tricarboxylic acid (TCA) cycle, malic acid is one of the most promising and renewable chemicals. L-malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Owing to the abundance of malic acid, tomato seed flour was used as a value-added food ingredient for its radical scavenging, anti-inflammatory and gut microbiota modulating properties [24]. Dietary supplementation of malic acid showed no significant effect on the growth of broilers [25]. However, the effects of antioxidant capacity of malic acid on the cecal microbial community and meat quality of broilers remain largely unknown. 2. Materials and Methods 2.1. Animals and Experimental DesignThe bio-fermented malic acid (BFMA) was produced in Anhui Xuelang Biotechnology Co., Ltd. (Bangbu, China) by the fermentation of aureobasidium pullulans CCTCC NO:M 2,012,223 with the substrate containing glucose, citric acid, and corn starch. The BFMA was an unrefined malic acid product designed for the feed additive, the middle product during the production of pure malic acid from the fermentation products. The BFMA contained 20% L-malic acid and 80% carrier (1% bacterial protein, 2% polysaccharide, 2% fermentation residue, and 75% zeolite powder) [26]. The carrier was used in the present study to balance the dose of BFMA among experimental groups. A total of 288 one-day-old Arbor Acres (AA) male broiler chicks were randomly divided into 4 treatments; each treatment had 6 replicates. Each replicate had 12 chicks, raised in a wire floor cage (110 × 100 × 55 cm). The control group was fed a corn–soybean meal basal diet containing 12 g/kg carrier of malic acid, and the other three treatment groups were fed the basal diets with the carrier being substituted with 4, 8, and 12 g/kg BFMA, respectively. The feeding trial lasted for 42 days, and was divided into 3 stages: days 0–14 (starter period), days 15–28 (grower period), and days 29–42 (finisher period). Accordingly, three basal diets were formulated to meet the nutrient requirements of AMINOChick®2.0 and Chinese Feeding Standard of Chicken (NY/T, 33-2018). The feed was made into granules using a cold pelleting process before being given to the broilers. Ingredient and calculated nutrient compositions of basal diets are listed in Table 1. 2.2. Birds Management, Data Record, and Sample Collection

The feeding trial was carried out at the Nankou Test Base of the Chinese Academy of Agricultural Sciences (Beijing, China). The chicks were housed in three-layer cages and had free access to feed and water through individual feeders and drinkers in each cage. The experimental house was kept in 23 h of light and 1 h of darkness every day. The temperature was controlled at 34–35 °C in the first week, then decreased by 2 °C every week, and finally controlled between 24–26 °C. The relative humidity was 45–55%. According to the routine immunization program and feeding management, the health status of the birds was observed daily. Chicks were vaccinated with inactivated Newcastle disease vaccine on days 7 and 21, and inactivated infectious bursal disease vaccine on days 14 and 28. During the trial, the mortality of birds and their body weight were recorded daily. Feed intake (FI) and BW of each replicate were recorded every week. Average daily feed intake (ADFI), average daily gain (ADG), feed conversion ratio (FCR), and death and culling ratio (DCR) were calculated according to the record.

At day 21 and 42 of the trial, the birds were weighed after 8 h fasting. One bird was randomly selected from each replicate, and 10 mL of nonanticoagulated blood was collected through the wing vein, placed at an incline for 30 min at room temperature, centrifuged at 3000 rpm for 15 min to obtain serum, and stored at −20 °C for use in the determination of biochemical indexes. On day 42, the broilers from which blood samples had been collected were then slaughtered for the measurement of carcass traits and meat quality. The cecal digesta were collected and stored at −80 °C for microbiological analysis.

2.3. Carcass Traits and Meat Quality

After being slaughtered at the age of 42 days, the carcass, evisceration, breast muscle, leg muscle, and abdominal fat of broilers were weighed, and the carcass rate, evisceration rate, complete evisceration rate, chest muscle rate, leg muscle rate and abdominal fat rate were calculated. The formulas are as follows:

Dressing = (carcass weight/live weight) × 100%

Evisceration rate = (eviscerated weight/live weight) × 100%

Breast muscle rate = (pectoral muscle weight/evisceration weight) × 100%

Leg muscle rate = (leg muscle weight/evisceration weight) × 100%

Abdominal fat rate = (abdominal fat weight/evisceration weight) × 100%

The right pectoralis major muscle of each broiler was taken after slaughter, and the color (L*, brightness; a*, Redness; b*, Yellowness) was measured immediately with a fully automatic colorimeter (CR-410, Konica Minolta, Tokyo, Japan) with a whiteboard for reference. Using a portable pH meter, the pH probe (pH-star, DK2730, Herlev, Denmark) was inserted into the meat for measurement; each piece of meat was measured at 3 different positions, and the average value was taken as the final result, which was recorded as pH45 min. Then, the meat was placed in a refrigerator at 4 °C for 24 h; subsequently it was taken out, each piece of meat was measured at 3 different locations, and the average value was taken as the final result, which was recorded as pH24 h. After slaughter, the middle part of the left pectoralis major muscle was taken, trimmed into a length × width × thickness of 30 mm × 15 mm × 5 mm, and weighed (W1). Then one end of the meat sample was caught with an iron wire so that the muscle fibers were vertically upward. This was placed into an air-filled plastic bag with no contact with the bag wall, and the bag mouth was tied. The meat sample was hung in a refrigerator at 4 °C for 24 h, then taken out and dried with filter paper, the meat was weighed (W2), and its dripping water loss was calculated as (W1 − W2)/W1.

2.4. Immune and Antioxidant Indicators of Serum and Breast MuscleSerum antioxidant indicators include glutathione peroxidase (GSH-Px), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA) and total antioxidant capacity (T-AOC). Serum immune capacity indicators include immunoglobulin A (IgA), immunoglobulin M (IgM) and immunoglobulin G (IgG). Breast antioxidant indicators include GSH-Px, GSH, SOD, MDA, T-AOC, and lactic acid. The assay was performed with the colorimetric method using an automatic biochemical analyzer (Model 7160, Hitachi Group, Tokyo, Japan), following the instructions of kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Information regarding the commercial kits is listed in Supplemental Table S1. 2.5. Determination of Inosinic Acid in Chicken

Inosinic acid (inosine monophosphate, IMP) is the major umami compound in chicken, having an important function in meat flavor formation. An Agilent TC-C18 chromatographic column (5 μm, φ4.6 mm × 250 mm) was used in the detection of high performance liquid chromatography (HPLC). The mobile phase was 0.05 mol/L pH 6.5 sodium dihydrogen phosphate buffer solution. The flow rate was 1 mL/min, and the column temperature was 30 °C. The UV detection wavelength was 254 nm.

2.6. DNA Isolation, PCR Amplification, and Miseq SequencingThe total genomic DNA of cecal digesta was extracted using the Qiagen QIAamp DNA Kit in accordance with the manufacturer’s guidelines. The concentration of DNA samples was measured using a Nano-Drop 3000 spectrophotometer. The 16S rRNA gene was amplificated by polymerase chain reaction (PCR) using specific primers for the V3 region according to our previous report [27]. All PCR products were isolated using 2% agarose gels, and purified with a DNA gel extraction kit (Axygen, Hangzhou, China). Before sequencing, PCR products were mixed with the same proportion based on their DNA concentration determined by a QuantiFluor™-ST fluorescent quantitative system (Promega, Madison, WI, USA). The construction of PE amplicon libraries and sequencing were conducted on the Illumina Miseq platform (Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China). 2.7. Sequence AnalysisBefore analysis, sequences were filtered based on their quality using QIIME (v1.9.1) with the following criteria: (1) the 300 bp reads truncated at any site with an average quality score http://rdp.cme.msu.edu/ (accessed on 7 April 2022)) against the SILVA ribosomal RNA gene database with 70% as the confidence threshold. 2.8. Ecological and Statistical Analyses

The abundance of communities and sequencing data of each sample were assessed based on rarefaction curves. Alpha-diversity was analyzed using Mothur software based on the number of actually measured OTUs (Sobs), including community diversity (Shannon, Simpson), community richness (Chao, Ace), and sequencing depth (Good’s coverage). Analyses of beta diversity were performed, including partial least squares discriminant analysis (PLS-DA), principal coordinate analysis (PCoA), and nonmetric multidimensional scaling (NMDS). Differences between populations at phylum and genus levels were analyzed using a one-way ANOVA. p < 0.05 was considered statistically significant.

2.9. Statistical Analysis

The experimental data were analyzed using the one-way ANOVA program of SPSS Version 19.0 (SPSS, Chicago, IL, USA) and multiple comparisons were conducted using Tukey’s Test. Means were considered significantly different at p < 0.05, and a trend of change at 0.05 ≤ p < 0.10.

4. DiscussionL-malic acid mainly acts as an antioxidant ingredient in the additives of the feed and food industries. Dietary supplementation of malic acid could reduce nitrogen emission pollution, and improve the performance and product quality of calves, cows, and lambs [28,29,30,31,32]. Although adding malic acid to the diet of broilers has no significant effect on growth performance [25], malic acid, an intermediate of the tricarboxylic acid (TCA) cycle, is hypothesized to influence the energy metabolism of muscle, whose effects on body health and meat quality of broilers are worthy of being disclosed. The L-malic acid product used in the present study was a mixture obtained from biological fermentation. The potential of organic acids as an in-feed antibiotic replacement to improve the intestinal health, feed efficiency and growth performance of broilers has been demonstrated [33,34,35]. It was confirmed again in this study that dietary supplementation with the organic acid, BFMA, improved the growth performance of broilers, including increasing ADFI, ADG, and BW, and decreasing FCR and DCR. Besides the benefits on growth performance, supplementation of mixed organic acids increased pH24 h of the breast and thigh muscles, and the redness in thigh meat was also improved [36]. Organic acids prevent meat quality deterioration without leaving any chemical residues [37]. The combination of humic acid and organic acids improves the sensory attributes of cooked breast meat of broilers [38]. In the current study, dietary BFMA also increased the pH24 h value and water-holding capacity of breast muscle of broilers without negative effects on carcass traits. Therefore, we conclude that BFMA, like other acidifiers, could improve the growth performance and meat quality of broilers.Cells generate reactive oxygen species upon stress and accumulate the by-products of oxidation reactions, which could be eliminated by several antioxidant factors, mainly including GSH, SOD, and catalase [39]. The health status of broilers was improved by dietary supplementation with mixed organic acid through enhancing the immune function, the antioxidative characteristics and the tight junction proteins expression of the intestine [40]. Organic acids improved the welfare, health status, and cecal bacteria composition of broilers, but did not alter blood levels of antioxidant indices or liver function indicators [41]. MDA only slightly decreased with an increase in the level of formic plus lactic acid [42]. In the current study, dietary supplementation with BFMA significantly increased the serum immunity of broilers but did not influence serum antioxidant capacity. Antioxidants added in diets can minimize the negative effects of oxidative stress on the quality of broiler meat, and augment its water-holding and textural properties [43]. Judging from the indexes of GSH, GSH-Px, SOD, T-AOC, and IMP in the breast muscle of broilers, dietary supplementation with BFMA in the present study significantly enhanced the antioxidant capacity of breast muscle. Therefore, we deduced that BFMA probably increased the growth performance and body health of the broilers by reinforcing immunity, and decreased water loss of breast muscle via its antioxidant capacity against oxidative stress. Malic acid is one of the intermediates of the TCA cycle. Feeding tricarboxylic acid cycle intermediates improves lactate consumption in Chinese hamster ovary cell cultures [44]. In the current study, BFMA in the diets of broilers increased the pH24 h value of breast muscle to a certain extent after slaughter at the age of 42 days. Therefore, it could be deducted that the decreased water loss of breast muscle of broilers was probably realized through reducing lactate accumulates and enhancing antioxidant capacity.Intestinal microbiota could optimize the metabolic health of the host; how gut microbiota and their metabolites may link to the metabolism of the host or to the pathogenesis of metabolic disorders is a nascent and promising research field [45]. Besides performance, the chyme pH and microbiota of broilers were affected by acidifiers, 2-hydroxy-4-(methylthio) butanoic acid and DL-methionine [46]. The inclusion of benzoic acid in diets was effective in improving antioxidant capacity, nutrient digestion, and gut microbiota composition [17]. Dietary supplementation of a mixture of a free butyrate acidifier and gluconic acid improved gut health, including its morphological structure and microbiome activities, in chickens [47]. As an organic acid, addition of 2-hydroxy-4-methylthiobutyric acid in drinking water shows excellent bacteriostasis to benefit intestinal development and gut microbiota, and the subsequent performance of broilers [48]. Moreover, the tibia mass of broilers was improved by acidification of drinking water through the alterations of intestinal barrier and microbiota [16]. In the current study, the diversity of cecal microbiota of broilers was also significantly changed by dietary supplementation with BFMA, which was probably responsible for the enhanced immunity and performance.The abundance of o_RF39 in the gut was significantly decreased in rats with ulcerative colitis and severe colon tissue injury [49]. Oscillospiraceae in murine gut microbiota were found to be related to the development of rheumatoid arthritis and hyperlipidemia [50,51]. Helicobacter organisms are recognized as important pathogenic agents in colitic diseases of rodents and primates, potentially causing inflammatory bowel disease [52]. An abundance of Tuzzerella negatively affected the activity of methanogens [53]. The relative abundance of Oscillospira widely present in the animal and human intestines was positively associated with osteoporosis [54]. With the function of amino acid metabolism, o_Rhodospirillales is widely distributed in the environment with organic matter pollution. In the present study, dietary addition of BFMA significantly decreased the amount of norank_f_norank_o_RF39, norank_f_Oscillospiraceae, Helicobacter, Tuzzerella, norank_f_norank_o_Rhodospirillales, and Oscillospira in the cecum of broilers. This result indicated that the growth or reproduction of these harmful bacteria was effectively suppressed by BFMA.Barnesiella intestinihominis could facilitate therapeutic immunomodulatory effects [55] and enable clearance of intestinal vancomycin-resistant Enterococcus colonization [56]. Conversion of dietary inositol into propionate and acetate by commensal Anaerostipes benefited host health [57]. Merdibacter was demonstrated to be a beneficial bacterium for gut health in laying hens [58]. The Caproiciproducens mainly degraded the proteins and carbohydrates from food residues to produce caproic acids through a chain elongation procedure [59]. The increase of Gordonibacter in gut microbiota of mice may reduce intestinal permeability and inflammatory cytokine levels, maintaining a healthy intestinal microenvironment [60]. Roseburia, a butyrate-producing genus, is considered a marker of gut health [61]. Members of the genus Rhodococcus, with versatile abilities to degrade various organic compounds, are able to generate triacylglycerols [62,63]. Intestinal Bacteroidales can play important roles in the bioactivities of sulfated polysaccharides [64]. In the current study, the abundances of Barnesiella, Anaerostipes, Merdibacter, Caproiciproducens, Gordonibacter, Roseburia, Rhodococcus, and unclassified_o_Bacteroidales in the cecum of broilers were significantly increased by the appropriate addition of BFMA in diets. Therefore, we deduced that these beneficial bacteria were enhanced to maintain the gut health of broilers.Bifidobacterium has a diverse host range and shows several beneficial properties to the host [65]. Victivallis, a short-chain fatty acids (SCFAs) producer, increased significantly in irritable bowel syndrome patients with a starch- and sucrose-reduced dietary intervention [66]. Genus ASF356, also a SCFAs producing bacterium, increased along with the programming of hypertension prevention by maternal resveratrol supplementation [67] and decreased following Gastritis in mice [68]. Achromobacter was assumed to be an opportunistic pathogen [69]. In this study, dietary supplementation with BFMA decreased the abundance of Bifidobacterium, Victivallis, and ASF356, and increased that of Achromobacter. These adverse effects require attention when applying BFMA in broiler production. 5. Conclusions

As one of the intermediates of the TCA cycle, BFMA increases the water-holding capacity of breast muscle of broilers, probably through reducing lactate accumulates and enhancing antioxidant capacity. In addition, supplementation of BFMA in diets could improve performance at early stage and body health of broilers, probably by reinforcing immunity and perfecting the cecal microbiota structure. To be specific, harmful bacteria, including norank_f_norank_o_RF39, norank_f_Oscillospiraceae, Helicobacter, Tuzzerella, norank_f_norank_o_Rhodospirillales, and Oscillospira, were suppressed by BFMA. The probiotics, Barnesiella, Anaerostipes, Merdibacter, Caproiciproducens, Gordonibacter, Roseburia, Rhodococcus, and unclassified_o_Bacteroidales, were increased by BFMA. Meanwhile, it is alarming that BFMA decreased the abundance of probiotics Bifidobacterium, Victivallis, and ASF356, and increased the noxious bacterium Achromobacter.