Probiotics effects on the growth performance and meat quality of broiler chickens

Potential probiotics

Probiotic strains were obtained from the culture bank of LLP Research and Production Enterprise ‘Antigen’ (Almaty, Kazakhstan), which were stored at −80°C. Lactic acid bacteria (LAB) L. paracasei SH1 (GenBank Accession no. OQ411023) and E. faecalis SH6 (Genbank Accession no. OQ415517.1) were used in this study as the potential probiotic microorganisms isolated from fermented camel milk (shubat) and K. unispora Y 2.2 (Genbank Accession no. OP984721) isolated from fermented mare’s milk (koumiss). Strains were isolated from milk by using serial dilution and plating on selective media (Mithun et al. 2015): De Man Rogosa and Sharpe (MRS) media (TM Media, Titan Biotech, India) for LAB and Sabouraud media (TM Media, Titan Biotech, India) for yeasts. The strains were cultivated in a culture-media incubator at 30°C (for yeast) and 37°C (for LAB). The cells were harvested by centrifugation (3000g) for 15 min at 4°C after incubation, washed three times with phosphate-buffered saline (pH 7.2), and resuspended in the same buffer until use.

Experimental design

The present study was conducted at the accredited testing centre and Department of Microbiology, LLP Research and Production Enterprise ‘Antigen’ (Almaty, Kazakhstan). The experimental procedures were conducted by the guidelines of the Department of Microbiology, LLP ‘Research and Production Enterprise ‘Antigen’.

Animal material and diets

In total, 240 1-day-old Ross 308 chicks were randomly allotted to four treatment groups of three replicates (20 birds per replicate). The control groups were fed a basal diet (Table 1) without any probiotic and the three treatment groups were fed diets consisting of L. paracasei SH1, E. faecalis SH6 and K. unispora Y 2.2 at initial concentrations of 1.0 × 10⁹ colony-forming units (CFU)/mL, 5.0 × 10⁸ CFU/mL, and 2.5 × 10⁸ CFU/mL per bird (Groups Pro1, Pro2, and Pro3 respectively), for 42 days. The strains were gradually introduced to the water, mixing in portions in a water container, to produce these final concentrations. To determine and modify the number of probiotic bacteria in each water container, agar-plate count was performed every half an hour until the water ran out.

Management

During the experiment, all groups were kept in suitable conditions. Each replicate is housed in a separate battery cage (200 × 50 × 90 cm). Broiler chicks were kept on continuous light until the end of the first 7 days of life, after which they received 22 h of light per day. The room temperature was kept at 33°C for the initial 3 days and then adjusted to a range of 32–30°C from Day 4 to Day 7. Subsequently, it was gradually decreased by 23°C per week until it stabilised within the range of 22–24°C. The brooder battery came with two white fluorescent lamps. All of the birds were kept in the same sanitary, environmental, and management conditions. Feed was provided ad libitum, and freshwater was available at all times during the experiment. Drinkers and feeding troughs were cleaned daily.

Growth performance

To determine the bodyweight, the birds were individually weighed with an accuracy of 0.1 g at 1, 21, and 42 days of age. The feed intake of the birds, based on the pen they were in, was measured at 1, 21, and 42 days of the experiment. These measurements were used to calculate the average daily gain (ADG) and FCR. Throughout the experiment, the mortality rate of the Ross 308 broiler chicks was recorded daily, and it was calculated for each specific period of the study, namely, grower period (1–21 days), finisher period (22–42 days), and overall period (1–42 days).

Carcass yield

At the end of the experiment, randomly 13 chickens from each group were selected for slaughter to investigate the carcass characteristics. The liveweight (g) was recorded as the weight of each bird after undergoing the fasting process before slaughter at the designated facility. Following the slaughter, the feathers, head, feet, and giblets were removed, and the carcass weight (g) was measured, enabling the calculation of the carcass rate (%).

Proximate composition

Moisture content was determined by using the air oven-drying method of the AOAC (Official Method 990.19, AOAC 2016). The measurement was performed by drying the samples in a Binder ED-115 Oven (Binder, Germany) at 105°C overnight. The dish was then cooled in a desiccator and weighed and the process was repeated until constant weight was reached.

Kjeldahl method was used to determine protein content on the basis of the standard procedure of the AOAC (Official Method 973.48, AOAC 2016). This method is performed with an automated Kjeltec instrument UDK 142 (VELP, Italy) to determine protein content in meat samples.

Fat content was determined by the Soxhlet extraction method, by using the AOAC Official Method 960.39 (AOAC 2016). Instead, fat content was measured by weight loss of the sample or by the weight of the fat removed.

The ash content of the samples was determined using the dry-ashing method of the AOAC (Official Method 999.11, AOAC 2016). Ashing of the meat sample (10 g) was performed in a SNOL 8,2/1100 LSM (AB ‘Umega’, Lithuania) muffle furnace at 550°C. Before ashing, the crucible was dried in an oven at 105°C for 3 h.

The amino acid content of the meat was determined by chromatography method of the AOAC (Official Method 982.30, AOAC 2016).

Sensory characteristics

Meat samples were subjected to boiling in water until their internal temperature reached 70°C for assessment. The temperature verification was conducted through the placement of a thermometer within the central geometric region of the meat specimen. A sensory panel of 10 experienced evaluators investigated the sensory qualities after the slaughter. The evaluation was conducted utilising a nine-point rating scale to gauge various quality aspects, including colour (ranging from 1 (very light/pale) to 9 (reddish-black)), flavour (ranging from 1 (very poor) to 9 (excellent)), tenderness (ranging from 1 (tough) to 9 (very soft/tender)), taste (ranging from 1 (low) to 9 (high)), and juiciness (ranging from 1 (very dry) to 9 (succulent)) (Standards of Kazakhstan 2014).

pH measurement

The pH of all broiler breast muscles was measured in triplicate by using an insertion-type pH meter (Seven Compact pH/Ion meter S220-Std-Kit) 24 and 192 h postmortem after slaughter.

Drip-loss measurement

Drip loss was measured in breast fillets, beginning 24 and 192 h after slaughter. The breast filets were hung on hooks through their thickest part. To reduce evaporation, the filets were packed in plastic bags and sealed with rubber bands, with the hooks extending out of the bags. The filets in bags were then hung from a grate for 24 h in a 4°C incubator. The samples were weighed before and after hanging. Drip loss (DL) was calculated as the difference in weight, corrected for mass, and expressed as a percentage:

where DL is drip loss (%), m₁ is mass before hanging, and m₂ is mass after hanging (Albrecht et al. 2017).

Cook-loss measurement

For cook-loss measurements, about 80 g of fillet sample was vacuum-packed and cooked at 80°C to a core temperature of 75°C. During cooking, the core temperature was recorded using a portable needle-tipped thermometer (GT-309, Giltron, New Taipei, Taiwan). The samples were then equilibrated to room temperature and re-weighed. Cook loss (CL) was determined by calculating the weight loss during cooking as a percentage of the weight before cooking and was expressed as follows (Lee et al. 2022):

where CL is cook loss (%), m₁ is mass final weight, and m₂ is mass initial weight.

Microbiological analyses

Microbiological analysis was performed 24 and 192 h after slaughter to determine the total number of viable cells (TVC) as well as the amount of Pseudomonas spp. (Pse), a specific microorganism that causes spoilage of fresh, in aerobically packed poultry. For the microbiological analysis, 25 g of surface meat tissue measuring 4 × 7 × 0.5 cm was taken aseptically with a sterile scalpel. The sample was transferred to a sterile filtered gastric bag and filled with 225 mL of physiological solution–peptone diluent (0.85% NaCl with 0.1% peptone). The samples were stirred for 60 s and 10-fold dilutions of the homogenate were prepared in the saline–peptone diluent. TVC was determined using a pour-plate method on a countable amount of agar in the plate. The plates were incubated at 30°C for 72 h. Pseudomonas spp. (Pse) was identified by plating on Pseudomonas agar plates with a cetrimide–fucidin–cephalosporin selective additive. The plates were incubated at 25°C for 48 h.

Statistical analyses

The statistical analysis of all the data was conducted using the one-way ANOVA and principal-component analysis (PCA, using Pearson (n–1)) procedures provided by the XLSTAT-Student, ver. 2023 (Lumivero, USA, https://www.xlstat.com/). Subsequently, the Fisher’s test was applied to identify significant (P < 0.05) differences whenever they were observed. The determination of significance was based on a threshold of P < 0.05. The data are presented as the means ± standard errors of the mean (s.e.m.).

Ethics approval and consent to participate

The study adhered to the principles of good laboratory practice outlined in the State Standard of the Republic of Kazakhstan, along with the main provisions of ST RK 1613-2017. Ethical approval for the study was granted by the Local Committee on Animal Research Ethics of the LLP Research and Production Enterprise ‘Antigen’ under Protocol number 39, dated 28 August 2022, in Almaty, Kazakhstan.

Growth performance and carcass rate

Rahmani Alizadeh et al. (2023) showed that probiotics isolated from camel milk had a good effect on broiler growth rate and FCR. The bodyweights of Ross 308 chicks on Days 21 and 42 were comparable to the findings of Martínez and Valdivié (2021). The literature data on the effect of probiotics on the bodyweight and carcass parameters yield inconsistent results. Kalavathy et al. (2003) discovered that probiotic-fed chicks acquired considerably more weight from Day 22 to Day 42, although there was no difference between groups for the first 21 days. Rehman et al. (2020) observed considerable weight growth during the first 21 days, but no significant difference over the entire trial period (0–35 days). They also found that probiotics did not influence carcass properties. In line with our findings, Liu et al. (2017) found that the experimental group receiving 1 mL probiotics achieved a statistically significant improvement in daily weight gain at 4 and 6 weeks of age. However, Lee et al. (2010) found that dietary probiotics had little or no influence on broiler chicken growth performance. These discrepancies in outcomes were caused by probiotic strains, administration dosage, preparation methods, bird age, meat composition, and hygiene state (Zhang et al. 2012).

Proximate composition

Zhou et al. (2010) reported that the application of probiotics had no discernible impact on the levels of moisture, protein, crude fat, and crude ash in the musculature of chicken breast and thigh meat. In contrast, Aziz et al. (2020) found a noteworthy augmentation in the protein content within the breast and thigh meat of broiler chickens. An independent investigation undertaken by Abdulla et al. (2017) unveiled a significant reduction in the fat composition of broiler chicken breast muscles when a probiotic formulation containing Bacillus subtilis was used. The substantial reduction in adipose tissue content in poultry meat contributes positively to the nutritional value of the meat, aligning with the preferences of a majority of global consumers. Conversely, an increased fat content augments the sensory attributes encompassing meat taste and tenderness, as emphasised by Chartrin et al. (2006). In another study, Ivanovic (2012) showed a marked increase in moisture content, coupled with a notable decline in fat and protein constituents, in the drumstick meat harvested from 42-day-old Arbor Acres broilers fed diverse probiotics, including Lactiplantibacillus plantarum, E. faecium, Bacillus cereus IP5832, or a combination of Bacillus CH200 and Bacillus CH201. Similar results were obtained by Sharipova et al. (2017), who also observed a decrease in moisture content when the animals were fed with probiotics, which was explained by probiotics stimulating protein synthesis that results in a decrease in moisture content as a percentage of the overall weight. Consistent with our findings, the use of probiotics led to an increase in the crude protein content of the meat. High moisture content indicates low quality of meat (Freitas et al. 2018). Consequently, probiotics, by lowering the moisture content, can improve meat quality.

Meat amino acid composition

The quality and flavour of broiler chicken meat are greatly influenced by the amino acid profile of the meat. Whereas specific amino acids that contribute to flavour, such as alanine, glycine, glutamine, and serine, particularly glutamine, significantly influence the overall flavour of meat, essential amino acids have a significant impact on the quality of muscle proteins (Tang et al. 2021). Although limited research has been conducted on the influence of probiotics on the amino acid composition of chicken meat, several notable studies have examined this relationship (Guldas 2016; Popova 2017). Comparing control and experimental groups, it was found that the control group had greater levels of the majority of essential amino acids, such as lysine, leucine, threonine, valine, isoleucine, and phenylalanine. Similarly, Liu et al. (2017) demonstrated that adding probiotic Lactobacillus johnsonii to broiler chicken meals at low and high levels had no discernible effect on the meat amino acid composition. Sharipova et al. (2017) did not find any beneficial effects of B. subtilis supplementation on the amino acid content of broiler chicken meat. However, the results of the present study showed that the contents of a few amino acids were the highest in the experimental group. In addition, the means of valine, leucine/isoleucine, arginine and alanine were the highest in the group with diet of 0.5 mL of probiotics per bird. Intriguingly, chickens fed a diet containing a low dose of probiotics showed significantly higher concentrations of most amino acids than in the control group. Our results are consistent with those made by Podolian (2017), who noted that different dietary doses of a probiotic product made up of Lactobacillus bulgaricus and E. faecium resulted in appreciable increases in the amounts of lysine, valine, methionine, and phenylalanine. Contrary to the findings of our investigation, Sharipova et al. (2017) did not find any beneficial effects of B. subtilis supplementation on the amino acid content of broiler chicken meat. These collective findings underscore the intricate relationship between probiotics and amino acid composition in broiler chicken meat, suggesting that various factors including probiotic type and dietary application level play a role in influencing these outcomes.

pH, DL, CL

According to the obtained results, after 1 and 24 h of broiler meat storage, there was a substantial pH change between the control and probiotic groups. However, there was no difference by the end of storage at 192 h. No difference in DL was noticed after 24 h of storage. However, after 192 h, DL was significantly higher in the Pro2 group. No difference was observed in CL among the studied groups. Zheng et al. (2015) found that the pH of chicken breast meat in the groups treated with E. faecium was higher, resulting in a reduction in DL and cooking loss. DL and cooking loss are important in determining meat quality because some nutrients are rapidly lost owing to water loss during exudation (Bai et al. 2017), and DL is a growing problem in the meat industry, affecting both meat yield and quality (Abdullah et al. 2014). Furthermore, Xiong et al. (2015) found that pH was significantly and adversely linked with DL in pig meat after 24 h. However, the results of our current study indicated that pH was insignificantly connected with DL. pH results are consistent with Mohammed et al. (2021), who found that supplementing probiotic B. subtilis decreased the pH of meat 0.5 and 5 h after slaughter. Ivanovic (2012) reported meat pH increase with supplementation of B. cereus, whereas supplementation of E. faecium decreased the pH of meat. Dietary probiotics were beneficial for meat quality by improving the pH, tenderness, and colour (Pelicano et al. 2003).

Microbiology

Pseudomonas spp. have been identified as a possible spoiling factor in poultry meat (Rouger et al. 2017). According to Carrizosa et al. (2017), spoilage and off-flavour can result from the breakdown of amino acids when the concentration of putrefactive bacteria exceeds 8 log CFU/g. Furthermore, Pseudomonas spp. demonstrated a microbiological rejection level of 7.5 log CFU/g (Bruckner et al. 2012). In the present study, it was observed that after 192 h, all samples belonging to both the control and experimental groups did not reach the microbiological rejection limit. The higher level of moisture, drip loss, and pH value suggested by the Pro1 and Pro3 groups in comparison with the Pro2 and control groups could explain the differences in microbial count development between the control and supplemented groups. However, Lee et al. (2017) identified that after 120 h of bacterial growth in chicken meat increased over 8.3 log CFU/g, and Pseudomonas spp. was the most common genus responsible for the spoiling of chicken meat. The maximum acceptable level for the TVC in fresh meat, as indicated by Lázaro et al. (2015), is 7 log CFU/g. In our study, we observed that the control group exhibited a significantly higher TVC level than did the experimental groups, with an average of 6.8 log CFU/g over 192 h. These findings align with the results of Chmiel et al. (2022), who also reported an increase in TVC to approximately 6.5 log CFU/g on Day 9.

Sensory characteristics

The findings derived from our experimental study suggest that the addition of probiotics containing L. paracasei SH1, E. faecalis SH6, and K. unispora Y 2.2 in the water did not provide any statistically significant impact on the texture and preference of breast muscles obtained from Ross 308 broiler chickens at 42 days of age. According to Kabir (2009), probiotics have been found to have a positive impact on enhancing the sensory attributes of cooked broiler meat. In a study conducted by Alfaig et al. (2013), it was observed that the addition of probiotics to broiler cooked meat resulted in enhanced juiciness and tenderness. In contrast to the findings of Loddi et al. (2000), no statistically significant impact on various sensory attributes such as aroma intensity, flavour, tenderness, juiciness, acceptability, and overall appearance of breast muscles in 42-day-old Ross chickens was observed when administered with probiotics containing E. faecium, avoparcin probiotic, or a combination of probiotics and antibiotics, as compared with the control group. In a separate investigation conducted by Abdulla et al. (2017), it was shown that the treatment of probiotics containing B. subtilis did not have any statistically significant impact on the tenderness of breast muscles in 42-day-old Cobb 500 chickens.