Effects of Dietary Probiotic (Bacillus subtilis) Supplementation on Carcass Traits, Meat Quality, Amino Acid, and Fatty Acid Profile of Broiler Chickens

The aim of the present study was to evaluate the effects of dietary supplementation with or without Bacillus subtilis (B. subtilis) on carcass traits, meat quality, amino acids, and fatty acids of broiler chickens. In total, 160 1-day-old Arbor Acres male broiler chicks were divided into two groups with eight replicates of 10 chicks each. Chickens received basal diets without (CN group) or with 500 mg/kg B. subtilis (BS group) for 42 days. Eight chickens from each group were slaughtered at the end of the trial, and carcass traits, meat quality, chemical composition, amino acid, and fatty acid profile of meat were measured. The results showed that the breast muscle (%) was higher in BS than in CN (p < 0.05), while abdominal fat decreased (p < 0.05). The pH24h of thigh muscle was increased (p < 0.05) when supplemented with BS; however, drip loss, cooking loss of breast muscle, and shear force of thigh muscle decreased (p < 0.05). Lysine (Lys), methionine (Met), glutamic acid (Glu), and total essential amino acid (EAA) in breast muscle and Glu in thigh muscle were greater in BS than in CN (p < 0.05). C16:1, C18:1n9c, and MUFA in breast muscle and thigh muscle were greater in BS than in CN (p < 0.05). In conclusion, dietary supplementation with B. subtilis could improve the carcass traits and meat quality of broilers, which is beneficial for the consumers due to the improved fatty acid profile and amino acid composition.


INTRODUCTION
The widespread use of antibiotics has led to the emergence of resistant bacteria and drug residues in animal products, which directly or indirectly endangers human's health and environmental safety (1,2). Nowadays, with the development of molecular biology, genetic improvements in broiler production have improved body size and growth rate of broilers, resulting in a higher yield of meat in broilers (3). However, the meat quality was decreased drastically with the rapid growth of broilers. Thus, in order to satisfy people's growing demand for high-quality meat, the improvement of meat quality becomes a current hot topic in the field of animal nutrition. Previous studies have demonstrated that probiotics can play a role in improving the microbial balance and intestinal environment (4)(5)(6)(7) and are believed to promote the growth performance (8) and meat quality of broilers by multiple ways (9,10). It is important to note that in all of these studies, Bacillus species, including Bacillus subtilis (B. subtilis), Bacillus cereus (B. cereus), Bacillus clausii (B. clausii), and Bacillus coagulans (B. coagulans), have been identified as effective probiotics to promote animal growth, maintain intestinal barrier function, and promote meat quality of broilers (9,(11)(12)(13)(14)(15)(16).
The adverse environment such as low pH value, high temperature, and relatively greater bile salt within gastrointestinal tract is a severe challenge for probiotics survival (17). B. subtilis is a spore gram-positive aerobic bacterium that has a better heat stability and higher acid tolerance, making it a potential feed additive in animal production (18,19). Previous studies have demonstrated that the properties of probiotic bacteria vary from strain to strain (20). With respect to B. subtilis, multiple strains, including B. subtilis DSM 17299 (19), B. subtilis fmbJ (12), B. subtilis RX7 and B2A (13), B. subtilis C-3102 (21), B. subtilis 29784 (22), B. subtilis BYS2 and BG5 (23), B. subtilis 1781 and 747 (24), and B. subtilis DSM 32315 (25)(26)(27)(28), have been gaining interest as they have been indicated to improve the growth performance, feed efficiency, antioxidant capability, immune function, and intestinal microflora balance of broilers. B. subtilis DSM 32315, a unique naturally occurring strain, has shown to improve growth performance and intestinal microflora balance of broilers (25)(26)(27)(28)(29). For example, Ma et al. (25) showed that dietary supplementation with B. subtilis DSM 32315 improved growth performance, intestinal structure, and the manipulation of cecal microbial composition of broilers. Sokale et al. (26) indicated that dietary supplementation with B. subtilis DSM 32315 can maintain the intestinal structural integrity and ameliorate the pathology and performance detriments associated with necrotic enteritis challenge. However, previous studies about the application of B. subtilis DSM 32315 in poultry mainly concentrated in growth performance and intestinal health; the effect of dietary supplementation with B. subtilis DSM 32315 on carcass traits and meat quality of broiler chickens has not been investigated in detail. Thus, the present study aims to investigate the effects of B. subtilis DSM 32315 supplementation on carcass traits, meat quality, amino acid (AA), and fatty acid profile of broiler chickens to provide more references for the application of B. subtilis in poultry production.

Birds, Experimental Design, Diets, and Management
The experimental procedures involving animals were reviewed and approved by the animal welfare committee of the Guizhou Normal University (Guiyang, China). A total of 160 1-dayold Arbor Acres (AA) male broiler chicks with similar initial body weights were randomly divided into two groups with eight replicates of 10 chicks each. Chickens received basal diets without (CN group) or with 500 mg/kg B. subtilis (BS group, B. subtilis DSM 32315, 2.0 × 10 9 spores/g, Evonik Nutrition & Care GmbH, Germany) throughout the trial period (25). The basal diets (  Approximately 5.0 g of muscles from the medial pectoralis major and thigh were sampled for chemical composition measurement. The remaining muscles from each bird were kept at 4 • C for the analysis of meat quality.

Meat Quality Assessment
Meat quality was assessed by determining pH, meat color, drip loss, shear force (N), and cooking loss of the breast and thigh muscle. The pH of breast and thigh muscle was measured at 24 h (pH 24h ) postmortem by insertion of a handheld pH meter (Russell CD700, Russell pH Limited, Germany). Meat color, including lightness (L * ), redness (a * ), and yellowness (b * ), was measured using Chroma Meter (Opto-Star Lab, Matthaus, Germany) according to the manufacturer's instructions. The measurements of drip loss from 0 to 24 h were conducted as described by Zhou et al. (16). Briefly, ∼30 g of muscle sample was weighed and put into zip-lock plastic bags and stored in a chilling room at 4 • C for 24 h and then reweighed after wiping out the surface moisture, and the drip loss was calculated as: drip loss (%) = [(initial weight -final weight)/final weight] × 100. The measurements of shear force and cooking loss were conducted as described by Cai et al. (3) with appropriate modification. Approximately 20 g of a sample of muscle sample was weighted and heated in a water bath to a final internal temperature of 75 • C. After cooling to room temperature, the muscle sample was reweighed after wiping out the surface moisture and the cooking loss was calculated as: cooking loss (%) = [(initial weight -final weight)/final weight] × 100. The sample used for the measurement of cooking loss was then used for the measurement of shear force. The sample was cut into 2 cm (length) × 1 cm (width) × 0.5 cm (height) along the direction of the muscle fibers, and the shear force was measured using a digital texture analyzer (Model 2000D, G-R, US).

Chemical Composition Analysis
Muscle samples were cut into slices, dried in a vacuum-freeze dryer, and then ground into powder. The moisture, crude ash, crude protein (CP), and extracted fat (EE) were measured according to the methods described by AOAC [2000; (30)].
To measure the AA content in breast and thigh muscle, the samples were pretreated according to AOAC [2000; (30)]. Briefly, ∼200 mg of fresh muscle sample was accurately weighed into the hydrolysis tube (about 15 ml), 10 ml of hydrochloric acid (6 mol/L) was added, and the mixed sample was filled with nitrogen, sealed, and put in a constant temperature drying box at 110 • C for hydrolysis for 24 h; after cooling and mixing well, 100 µl of mixed sample was taken out and dried at 55 • C and then 1,000 µl of AA sample diluent was added and mixed well; 1 ml of mixed sample was filtered used a needle filter (diameter 0.2 µm) and then analyzed using an automatic AA analyzer (L-8800; Hitachi, Tokyo, Japan) according to the manufacturer's instructions, and the contents of AA were expressed as mg/g wet tissue.
To measure the fatty-acid profiles in breast and thigh muscle, the samples were pretreated according to AOAC [2000; (30)]. Briefly, ∼50-200 mg of freeze-dried powder samples was accurately weighed into a 10-ml glass tube, and 3 ml of chloroform-methanol-water (volume ratio: 8:4:3) mixture was added. The mixture was shaken in a whirlpool for 1 min, followed by ultrasound for 5 min in an ice bath, repeated three to five times, left for 2 h, and centrifuged at 1,500 rpm for 15 min. The chloroform layer was transferred to another 10-ml glass centrifuge tube and dried in a vacuum drying oven to obtain the mixture of fatty acid and glyceryl ester; 2 ml of KOH-CH 3 OH (0.5 mol/L) was added and placed in a slightly boiling water bath for 10 min; after cooling for 3 min, 2 ml of BF 3 -CH 3 OH solution (10%) was added and then placed in a water bath at 80 • C to heat reflux for 2 min, cooled to room temperature, added 1 ml n-hexane and 3 ml of saturated NaCl solution, fully mixed, and centrifuged at room temperature at 2,000 rpm for 5 min, and then the upper n-hexane layer was absorbed into the gas phase vial for the fatty-acid profiles analyzed using an automatic fatty acid analyzer (Shimadzu GC-2010 plus, Japan) according to the manufacturer's instructions.

Statistical Analysis
All the data were subjected to the paired t-test using SPSS 21.0 programs (SPSS, Inc., Chicago, IL, USA). Results are expressed as the mean ± standard deviation (SD). Probability values < 0.05 were taken to indicate statistical significance.

Carcass Traits
The effects of B. subtilis supplementation on carcass traits of broiler chickens are presented in Table 2. Compared to the control group, diet supplementation with B. subtilis improved the breast muscle percentage (27.14 vs. 25.04%; p < 0.05), but decreased the abdominal fat percentage (1.52 vs. 1.74%; p < 0.05). No influences were observed in carcass weight, dressing percentage, semi-eviscerated percentage, eviscerated percentage, and leg muscle percentage (P > 0.10) among the treatments.

Meat Quality
The effects of B. subtilis supplementation on meat quality of broiler chickens are presented in Table 3. Supplementation with B. subtilis improved the pH 24h value (5.97 vs. 5.73; p < 0.05), but decreased the shear force (15.19 vs. 18.04; p < 0.05) in the thigh muscle compared to the control group. For the breast muscle, dietary supplementation with B. subtilis significantly increased  in the breast muscle compared to the control group. It seems that the thigh muscle had a lower Lightness (L * ), Yellowness (b * ), drip loss, cooking loss, and shear force (p < 0.05), but had a higher Redness (a * ) (P < 0.05) than breast muscle.

Meat Chemical Composition
The effects of B. subtilis supplementation on meat chemical composition (moisture, crude protein, ether extract, and crude ash) of broiler chickens are presented in Table 4. Dietary supplementation with B. subtilis had no effect on chemical composition of both breast and thigh muscles of broilers, and only had a tendency (p = 0.093) to increase ether extract content in the thigh muscle. In addition, the content of crude protein in breast muscle was higher, and ether extract was much lower than thigh muscle (P < 0.05).

Meat Amino Acid Composition
The AA contents in breast muscle and thigh muscle are presented in

Meat Fatty Acid Composition
The fatty acid contents in breast muscle and thigh muscle are presented in   (11) reported that dietary supplementation with B. subtilis did not affect the breast muscle weight of broilers exposed to chronic heat stress, and Park and Kim (13) reported that dietary supplementation with B. subtilis RX7 or B2A did not affect the abdominal fat of broilers challenged with Salmonella typhimurium. This is probably because the beneficial effects of B. subtilis in broilers were markedly strain-dependent (7,32). Abdominal fat is an important index used to measure lipid deposition in broilers (33,34). Excessive fat deposition is undesirable, because it degrades meat quality, decreases feed efficiency, and increases production and health costs (35,36). The lower abdominal fat in broiler chickens supplemented with B. subtilis in the present study indicated that B. subtilis can lead to a higher economic efficiency since the public tends to favor low-fat-content chicken. The pH value, meat color (lightness, redness, and yellowness), drip loss, cooking loss, and shear force are commonly used indices for evaluating meat quality (37). Previous studies have reported that dietary supplementation with B. subtilis could improve the carcass traits and meat quality of broilers by improving the pH, tenderness, and color (11)(12)(13). pH is related to shelf life, color, and water-holding capacity of the meat, which are determined mostly by postmortem conversion of muscle glycogen to lactic acid, and often used as an important indicator to evaluate meat quality (38,39). The higher pH 24h in the muscle of broilers fed with B. subtilis indicated a better shelf life, color, and water-holding capacity of meat. Similarly, Cramer et al. (11) also showed that dietary supplementation with B. subtilis increased the pH 24h of breast muscle. In contrast, Park and Kim (13) and Bai et al. (12) reported that dietary supplementation with B. subtilis had no effect on the pH value of meat. B. subtilis supplementation did not affect the meat color (a * , b * , and L * ) in the present study, which is consistent with Jeong and Kim (21) and Cramer et al. (11), who also reported that B. subtilis had no effect on the meat color. However, in contrast, Bai et al. (12) reported that B. subtilis could decrease L * and b * , and increase a * . Drip loss and cooking loss could reflect the water-holding capacity and are related to nutrition, flavor, and juiciness, because some nutrients are easily lost during exudation by water loss (40,41). In the present study, dietary supplementation with B. subtilis decreased the drop loss and cooking loss of the breast muscle after storage for 1 day, which suggested the increase of waterholding capacity of meat. The results of our study support the previous research, which concluded that dietary supplementation with B. subtilis RX7 or B2A in broiler diets increased the drip loss of breast meat (13). However, the detailed reasons for B. subtilis decreased the drip loss and cooking loss was unclear. Tenderness is considered as the most important trait for consumers, which can be represented by shear force (42). It was clear from our study that the administration of B. subtilis had a beneficial effect on meat tenderness with a decreased shear force. Inconsistent results between studies have been reported for the impact of B. subtilis supplementation on meat tenderness. Similarly to the present study, Bai et al. (43) reported that the administration of B. subtilis in the diets could decrease the shear force and thus improve muscle tenderness, whereas Cramer et al. (11) and Abdulla et al. (44) had found that dietary supplementation with B. subtilis had no difference in shear force. Therefore, the present study indicated that dietary supplementation with B. subtilis could improve the meat quality of broilers by improving the pH and tenderness and by decreasing the drop loss and cooking loss.
In the current study, B. subtilis treatment did not affect the routine nutrients (moisture, crude protein, ether extract, and crude ash) in both breast and thigh muscles compared to the control group, but it showed that dietary supplementation with B. subtilis had a tendency to improve the ether extract content in the thigh muscle. Similarly, Zhou et al. (16) indicated that dietary probiotic Bacillus coagulans supplementation did not affect moisture, crude protein, ether extract, and crude ash content in the muscles of Guangxi Yellow chicken. In contrast, Abdulla et al. (44) showed that dietary supplementation with B. subtilis significantly decreased the fat content in breast and thigh muscles of broilers. Intramuscular fat content is an important sensory characteristic of meats and is related to tenderness, succulence, and flavor of meats (45). The higher fat content of thigh muscle from B. subtilis treatment in the present study suggested better tenderness, succulence, and flavor of meats.
The AA content in meat is important to evaluate the quality and flavor of meat. EAA determines the muscle protein quality, and flavor amino acid (FAA), such as alanine (Ala), glycine (Gly), Glu, aspartic acid (Asp), and serine (Ser), especially Glu, can greatly contribute to the taste of meat (38,46). In the breast muscle, Met, Lys, Glu, total EAA, and total AA were increased by B. subtilis treatment in the present study. In the thigh muscle, only Glu content was increased by B. subtilis treatment. The reasons for this phenomenon might be due to the higher content of total AA in the breast muscle that made it more sensitive to FAAs. The results from Guo et al. (37) and Chen et al. (47) also support this opinion, who reported that breast muscle contained higher levels of total AAs than those of thigh muscle in broiler chickens. Lys and Met are two kinds of important EAA in meat of broilers. The higher Lys and Met in the meat of broilers fed with B. subtilis meant that B. subtilis had a beneficial effect on improving the protein quality and nutrition value of muscle. Glu is one of the most important FAA, which is the primary flavor molecule and functions in meat freshness and buffering salty and sour tastes (48). The Glu content in breast muscle and thigh muscle both increased due to B. subtilis treatment, indicating that B. subtilis has a role in promoting the meat flavor. In a word, B. subtilis could improve the meat protein quality as well as meat flavor of broilers.
It is well-known that fatty acids, including MUFA, polyunsaturated fatty acids (PUFA), and SFA, are an important indicator to evaluate the quality and nutritional value of meat and they also are the basis for the characteristic flavor of meat (49). The major fatty acids in chicken meat are C16:0, stearic acid (C18:0), C18:1n9c, and linoleic acid (C18:2n6c) (50). It is generally accepted that higher dietary intakes of SFA, especially C16:0 and myristic acid (C14:0), are positively correlated with increased risk of coronary heart disease, due to their ability to raise the total cholesterol and low-density lipoprotein (51,52). In the present study, B. subtilis treatment significantly decreased C14:0, C16:0, and C20:0 in the breast muscle, and significantly decreased C16:0 and total SFA in the thigh muscle, which indicated that the muscles from broilers treated with B. subtilis might be more beneficial to human health. The UFA not only is the precursor substance to meat flavor, but also plays a wide range of roles in promoting growth, scavenging free radicals, and regulating lipid metabolism (49). In the present study, B. subtilis treatment significantly increased C16:1, C18:1n9c, C20:3n6, and total MUFA in the breast muscle, and significantly increased C16:1, C18:1n9c, and total MUFA in the thigh muscle. C18:1n9c is the most abundant UFA, which is suggested to be positively associated with the softness of fat and the sensory quality of meat; thus, rich oleic acid content can make meat tasty (51,52). The higher C18:1n9c content in muscles from broilers treated with B. subtilis meant a better meat quality. C18:2n6c was reported to cause a negative flavor in meat (53). B. subtilis treatment had no effect on C18:2n6c content in both breast and thigh muscles, which meant that there was no adverse effect on meat flavor when broilers are supplemented with B. subtilis. In summary, B. subtilis could improve the composition of muscle fatty acids by decreasing the SFA content and increasing the UFA content, thus improving the meat quality as well as meat flavor.

CONCLUSIONS
In conclusion, dietary supplementation with B. subtilis could improve the carcass traits and meat quality of broilers by improving the pH and tenderness, decreasing the drip loss and cooking loss, and adjusting the composition of AAs and fatty acids.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The experimental procedures involving animals were reviewed and approved by the Animal Welfare Committee of the Guizhou Normal University (Guiyang, China).