Evaluating the efficacy of postbiotics derived from Lactobacillus spp. on growth performance in broilerABSTRACT
Postbiotics are increasingly recognized as functional feed additives with the potential to improve poultry health and growth performance; however, their efficacy depends largely on their bioactive composition. This study evaluated the effects of postbiotics derived from Lacticaseibacillus rhamnosus cultivated in different culture media on the growth performance, carcass characteristics, and gut microbiota of broiler chickens. Three distinct postbiotic products-postbiotic 1(P1), postbiotic 2(P2), and postbiotic 3(P3)-were produced and characterized based on their physicochemical and bioactive properties. While L-lactic acid levels were similar among all postbiotics, P1 exhibited the highest acetic acid, antioxidant activity(DPPH), and total phenolic content. In contrast, P2 showed the highest levels of total lipid, total protein, and butyric acid. A total of 288 Ross 308 broiler chickens were allocated to experimental groups with six replicate pens per treatment and 12 birds per pen. The postbiotics (P1,P2 and P3) were incorporated into the basal diet at a level of 0.2%. Postbiotic supplementation significantly increased body weight on days 21 and 42. At the end of the 42-day feeding period, all postbiotic-treated groups demonstrated improved body weight gain and reduced feed conversion ratio. Additionally, carcass yield was significantly higher in birds receiving P2 and P3. A notable increase in relative duodenum weight was observed in the P2 group. Metagenomic analysis revealed enhanced diversity of beneficial gut microbiota, particularly in the P2 group. Overall, postbiotics produced using different growth media formulations of L. rhamnosus positively influenced broiler growth performance and intestinal health.
Index terms:
Postbiotics; broiler chickens; feed conversion ratio; lactic acid bacteria
RESUMO
Os pós-bióticos são considerados aditivos alimentares potenciais para melhorar a saúde e o desempenho de crescimento das aves. Este estudo avaliou os efeitos de pós-bióticos obtidos de Lacticase ibacillus rhamnosus cultivado em diferentes meios de cultura sobre o desempenho de crescimento, características da carcaça e microbiota intestinal de frangos de corte. Três produtos pós-bióticos diferentes (P1, P2, P3) foram produzidos e caracterizados de acordo com suas propriedades físico-químicas e bioativas. Os níveis de ácido L-láctico foram semelhantes em todos os pós-bióticos. P1 apresentou os maiores teores de ácido acético, atividade antioxidante e fenólicos totais; P2 apresentou os maiores teores de lipídios totais, proteínas e ácido butírico. Um total de 288 frangos de corte Ross 308 (6 galpões com repetições, 12 aves em cada) foram divididos em grupos experimentais. Os pós-bióticos foram incluídos na dieta basal na proporção de 0,2%. A suplementação com pós-bióticos aumentou significativamente o peso corporal nos dias 21 e 42. Ao final do período de alimentação de 42 dias, observou-se melhora no ganho de peso e redução na taxa de conversão alimentar em todos os grupos tratados com pós-bióticos. O rendimento de carcaça foi significativamente maior nas aves que receberam P2 e P3. Um aumento acentuado no peso do duodeno foi observado no grupo P2. A análise metagenômica revelou um aumento na diversidade da microbiota intestinal benéfica no grupo P2. Os pós-bióticos produzidos a partir de L. rhamnosus afetaram positivamente o desempenho de crescimento e a saúde intestinal de frangos de corte.
Termos para indexação:
Pós-bióticos; frangos de corte; taxa de conversão alimentar; bactérias do ácido lático.
Introductıon
Global demand for safe and sustainable poultry products has necessitated innovative approaches to animal nutrition and health management. Growing concerns about antibiotic resistance have made the development of alternative feed additives a priority in animal production systems. In this context, postbiotics stand out as a promising solution due to their ability to promote gut health, enhance nutrient absorption, and support overall animal performance. While postbiotics act through mechanisms similar to probiotics, they differ in that they are composed of non-living organisms, making them easier to use as feed supplements (Thanh et al., 2009).
Postbiotics not only improve host health but also exert antimicrobial, antioxidant, immunomodulatory, anti-inflammatory, antiproliferative, hypocholesterolemic, and hepatoprotective effects (Aguilar-Toalá et al., 2018). Research has shown that postbiotics can enhance the growth performance of broilers and laying hens while improving feed efficiency (Loh et al., 2010; Kareem et al., 2016; Atan Çırpıcı & Kırkpınar, 2025). For instance, significant weight gain and reduced lesion scores were observed in postbiotic-treated broilers in a study by Johnson et al. (2019). Moreover, postbiotics can reduce the environmental impact of poultry production by mitigating avian diseases and mortality (Zhang & Kim, 2014).
Agricultural residues and food industry waste, such as fruit and vegetable by-products, serve as promising sources of prebiotics that promote the growth of probiotic bacteria and yield bioactive compounds like fiber. This research focused on utilizing agricultural by-products and food industry waste. Ingredients such as sesame husks, whey, molasses, and alfalfa, which are rich in bioactive compounds, provide a sustainable approach that simultaneously supports the growth of probiotic bacteria and minimizes environmental impacts. For example, sesame husks, often discarded into sewage systems and posing environmental risks, contain valuable bioactive compounds that make them suitable for animal feed. Sesame seeds, grown globally for oil and protein, consist of 55% lipids and 20% protein. Defatted sesame meal contains about 50% protein, while sesame seed hulls are rich in oxalic acid and fiber (Shahidi, Liyana-Pathirana, & Wall, 2006).
In addition to sesame peels, ingredients such as alfalfa, molasses, and whey, which possess nutritional and prebiotic properties, create an ideal environment for postbiotic production. Alfalfa, a high-yielding perennial legume cultivated worldwide, is rich in nutrients and bioactive compounds. Molasses, a by-product of sugar production, typically contains about 50% (w/w) of total sugars. Whey, a greenish-yellow liquid by-product of cheese production, and permeate, the liquid remaining after the ultrafiltration of milk or whey, are significant environmental pollutants due to their high biological oxygen demand and chemical oxygen demand. Their disposal is challenging for the dairy industry, requiring extensive pre-treatment processes that increase operational costs (Shahidi, Liyana-Pathirana, & Wall, 2006). Despite limited applications, whey and permeate are utilized in the production of protein concentrates, lactose, powders, and animal feed. Their high lactose content and rich nutrient profile make them excellent substrates for fermentation microorganisms (Pescuma, de Valdez, & Mozzi, 2015).
Compared to probiotics, postbiotics offer advantages such as a longer shelf life, ease of use, and greater safety. With physiological benefits including antioxidant, anti-inflammatory, and immunomodulatory effects, postbiotics are emerging as an effective feed additive that promotes animal health and contributes to environmental sustainability (Barros et al., 2020).
In this study, formulations containing sesame husks, whey, molasses, and alfalfa were fermented with lactic acid bacteria to obtain postbiotics. The effects of these postbiotics on the growth performance and overall welfare of chickens were evaluated. This research aims to provide a natural alternative to antibiotics in feed additives and contribute to sustainable poultry production practices. By exploring sustainable fermentation substrates and antibiotic-free feeding strategies, this study contributes to environmentally friendly and health-promoting practices in poultry production. This study aimed to investigate the effects of three postbiotics obtained by fermentation of L. rhamnosus in culture media composed of different components on the growth performance, feed efficiency, and intestinal health of broiler chickens.
Materıal and Methods
Production of bacterial postbiotics at biofermenter scale
In postbiotic production, sesame hulls, whey, molasses and inulin were used in the preparation of the medium. Waste products such as whey, sesame hulls and molasses were supplied by local companies in Eskişehir. Alfalfa was hand-picked from the field, dried and ground. Inulin was also purchased from a local commercial company. Three different medium formulations containing these products were created and optimized according to the standard amount used in the preparation of MRS (Man, Rogosa and Sharpe, Biolife) medium, the total solid content was 52 g/L. Sesame hulls and MRS were used in medium P1; molasses, whey, alfalfa, sesame hulls and inulin were used in medium P2; and molasses, whey, sesame hulls, and inulin were used in medium P3. Since the formulations are within the scope of Postbiotech Company’s (Turkey) “know-how,” the exact usage amounts are not specified here. MRS was used only in the first formulation (P1) at a rate of 20 g/L. The MRS was not used in the postbiotics designated as P2 and P3. Additionally, the media were sterilized during the preparation of formulations P1, P2, and P3 by adding an inorganic solution containing dipotassium ammonium phosphate (-2 g/L), magnesium sulfate (0.2 g/L), sodium acetate, manganese sulfate (0.05 g/L), Tween 80 (1 ml/L) was added to the medium and the media were sterilized. Lacticaseibacillus rhamnosus strain was inoculated on MRS agar to obtain primary culture. . For each formulation, 1% (v/v) inoculum from the primary culture (L. rhamnosus) (1.108 CFU/mL) was transferred to the medium (P1, P2 and P3) and incubated at 37oC for 48 hours. pH changes and bacterial growth were monitored at regular intervals during fermentation. The taxonomic identity of Lacticaseibacillus rhamnosus was confirmed by performing a BLASTn search on the 16S rRNA (∼1258 bp) gene sequences against the NCBI nucleotide database, and the species identity was determined to be 99% similar (Accession number PV055674).
As a result of incubation, a series of filtration processes were carried out to obtain postbiotics from the fermentation broth. The culture broths collected from the fermentor were first filtered through a coarse filter to remove solid residues. After the culture broth was filtered through 0.5 µm and 0.22 µm pore diameter filters, it was checked that there was no microorganism growth in the postbiotic products by culturing and after performing the analyses described below, they were used in chicken studies.
Lipid determination
For total lipid determination, the Barnes and Blackstock (1973) method was employed. An aliquot of the sample was mixed with concentrated H₂SO₄ and incubated at 80°C for 10 minutes. After cooling, phosphovanillin was added to the samples, and absorbance was measured using an ELISA reader at 540 nm. The total lipid content (% mg) was calculated using the formula provided below, with results expressed in mg/ml.
% mg total lipid = (Sample OD / Standard OD) x 800
Protein determination
The Biuret method was used to determine the total protein content in the samples. After treatment of the samples with the biuret reagent, they were measured in ELISA at a wavelength of 540 nm (Itzhaki & Gill, 1964). For calibration with bovine serum albumin (BSA), stock standard protein solutions were prepared at various concentrations and the amount of protein was calculated according to the standard graph.
Reducing sugar assay - DNS method
The reducing sugar analysis of postbiotics was carried out according to the 3,5-dinitrosalicylic acid (DNS) method proposed by Miller (1959). This method is based on a redox between DNS and glucose or other reducing sugars. The amount of reduced sugar was calculated as glucose equivalent.
DPPH free radical scavenging capacity and total amount of phenolic content
DPPH radical scavenging test offers the first approach to evaluating antioxidant activity (Shahidi & Zhong, 2015). The study was carried out according to the method of Arora and Chandra (2010). Folin Ciocalteu determination was performed by colorimetric measurement based on the principle of electron transfer from phenolic/antioxidant compounds to phosphomolybdic and phosphotungstic acid complexes in an alkaline medium and the total phenolic content was calculated as gallic acid equivalent (Singleton, Orthofer, & Lamuela-Raventós, 1999).
L-lactic acid and acetic acid assay
Determining the amount of L-lactic acid requires two enzyme reactions. The amount of NADH formed in the reaction is stoichiometric with the amount of L-lactic acid. NADH is measured by the increase in absorbance at 340 nm. The method was carried out according to the method suggested by Bergmeyer (2012). The measurement of acetic acid is based on the formation of NADH, which is measured by the increase in absorbance at 340 nm. Since the reaction in the method is an equilibrium reaction, the amount of acetic acid present was calculated by the equation (Bergmeyer, 2012).
D-3 hydroxybutyric acid assay
The amount of INT-formazan formed in the reaction is stoichiometric with the amount of D-3-hydroxybutyric acid INT-formazan is measured by the increase in absorbance at 492 nm, then the concentration of D-3-hydroxybutyric acid can be calculated (Bergmeyer, 2012).
Experimental design, diets, and management of birds
The study was conducted in an experimental broiler farm of the Agricultural Faculty of Eskisehir Osmangazi University in Eskisehir, Türkiye (39°45′42″ N and 30°28′40″ E, an altitude of 813 m above sea level). For the bird study, ethics committee approval was obtained from the Animal Experimentation Local Ethics Committee (HADYEK) at Eskişehir Osmangazi University (Approval number: 1020/2024). One-day-old mixed-sex Ross 308 broiler chicks (n = 288; 144 ♀ and 144 ♂) were obtained from a commercial hatchery (Hastavuk, Balıkesir, Türkiye) and were randomly allocated into four groups (one control group and three postbiotic groups: P1, P2, and P3) according to a completely randomized design. Each group consisted of six replicate pens, with 12 chicks (6 ♀ and 6 ♂) per pen, resulting in a total of 24 replicates. The number of animals and the replicate structure used in the experiment were determined based on the sample size equation model commonly used in broiler chicken feeding studies, taking into account the expected effect size, level of variation, and similar literature studies (Steel &Torrie, 1980). The basal diets of groups were supplemented or not with 0.2% (v/w) different liquid postbiotics as follows: without postbiotic (Control), P1, P2 and P3 g kg−1. The basal diets were formulated to meet the feeding standards of mixed-sex Ross 308 broilers (Aviagen, 2019) based on a corn-soybean meal (Table 1). The chickens were fed the diets in a mash form with ad libitum access to feed and water. All birds’ general health was assessed twice daily. All standard commercial practices (Aviagen, 2019) were followed throughout this 42-day study.
The chicks were housed in an experimental poultry house that featured environmentally controlled sawdust litter and fluorescent lighting. Each pen in the poultry house measured 2.5 × 2 m, and each pen was equipped with a nipple drinker, a set of droppers, and a hanging broiler feeder (tube type). The ambient temperature was set at 32 ± 1°C for the first three days, gradually decreased to 20°C, and maintained at the end of the third week to ensure the well-being of the animals. The ambient relative humidity was maintained within the 60-70% range. During the experiment, ambient temperature and relative humidity values were controlled in accordance with the recommended rearing guide for Ross 308 broiler chickens (Aviagen, 2019). It was determined that the applied temperature and humidity combinations were within the reported thermoneutral range for poultry; therefore, the temperature-humidity index (THI) did not exceed levels that would adversely affect animal welfare throughout the experiment. The lighting cycle was set to 24 h per day during the first week, 18 h per day for the following four weeks, and 23 h per day during the final week. Lighting was provided using 125-W incandescent lamps, in accordance with the Ross 308 management guidelines (Aviagen, 2019).
Performance, sample collection, and calculations
Body weight (BW) and feed intake (FI) were measured in replicate pens on days 21 and 42. Subsequently, the daily BW gain (DWG, g d˗1), daily FI (DFI, g d˗1), and feed conversion ratio (FCR, g feed: g gain) were calculated. The FCR was calculated as FI divided by DWG (g feed/g gain). Notably, the mortality rate was 0%, and no abnormal signs were observed during the experiment. To determine the carcass yield and internal organs weights and lengths, two birds (one ♀ and one ♂) per replicate (12 birds per treatment) were selected, weighed and slaughtered on day 42. The carcass yield was determined from eviscerated weight and pre-slaughter BW. The weights of the internal organs and the length of the whole gastrointestinal tract (GUT) and its segments were collected and performed as described by Kop-Bozbay and Ocak (2019), who used the same procedures. All measurements taken were expressed as a percentage of BW just before the slaughter of each bird (g per 100 g BW).
To determine the serum concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), 4 ml of blood was collected from all slaughtered animals into heparinized tubes. The analyses were made by MG Veterinary Engineering Informatics Industry and Trade Ltd., Konya,Turkey.
Metagenomics analyses
The metagenomic sequencing method was used to investigate functional changes in the gut microbiota. Metagenome analysis was performed by BMLabosis BM Lab. Sist. Ltd. Şti. (Ankara, Turkey) as a service procurement. A total of 10 fecal samples were on day 42 to extract the genomic DNA (5 samples from Control and 5 samples from the P2 group). For microbial diversity analysis in chicken fecal samples, the V3-V4 regions of the 16S rRNA gene were amplified using PCR. The resulting amplicons were sequenced using the Illumina MiSeq platform with paired-end sequencing. Raw sequencing data were processed using QIIME2 bioinformatics software, including quality control, filtering, merging, and taxonomic classification. Alpha dilution analysis was performed for each sample using Chao1, Shannon’s, and Observed OTU indices. Bacterial diversity among the samples was revealed using Principal Coordinate Analysis and dendrogram generation. Finally, the microbial composition was determined and performed to evaluate differences between groups (Bolyen et al., 2018)
Statistical analysis
For all data, the pen was employed as the experimental unit. Kolmogorov-Smirnov and Levene’s test were performed for normality and homogeneity of variances. Data were subjected to one-way ANOVA using SPSS software (Version 21.0, Chicago, IL, USA). The differences among the means were accepted as significant at p≤ 0.05 by using Duncan’s multiple range test. For metagenome analysis, differences in microbial composition between groups were assessed using QIIME2 bioinformatics software. The Chao1, ACE, Shannon, and Simpson index were used to assess the alpha diversity of microbiota. The Adonis statistical analysis method was chosen to test the significance of differences in community structure of grouped samples. The Kruskal-Wallis test was used to analyze differences in the relative abundance of major phyla and genera.
Statistical analysis of Table 2 was performed using GraphPad Prism version 8.0.1 (GraphPad Software Inc., CA, USA) Two-way ANOVA. The results were expressed as mean ± standard deviation (SD). Statistical significance was defined as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Results and Discussion
The analysis results of the postbiotics components, lactic acid, acetic acid, DPPH, total phenolic, glucose, total protein and lipid, D3-Hydroxybutyric acid, are given in Table 2. While lactic acid ratios were determined to be close to each other in the three postbiotics, the acetic acid ratio was determined to be the highest in P1. DPPH % and phenolic amount were also found to be higher in P1 than in P2 and P3. In contrast to these findings, total lipid, protein and butyric acid content were analyzed to be the highest in P2 and P3, respectively.
The fermentation performance of Lacticaseibacillus rhamnosus was markedly influenced by the composition of the culture medium, as reflected in the final pH values and metabolite profiles obtained after 48 h of fermentation (Table 2). The pH values ranged between 4.50 and 5.13, indicating successful acidification across all experimental groups.
Lipid determination
Total lipid analysis was performed to evaluate the nutritional and functional potential of the postbiotic formulations, as lipids are essential bioactive components that contribute to energy supply and play a role in cellular structure and immune modulation (German, 2011).
Total lipid amounts of P1, P2, and P3 formulations are shown in Table 2. Total lipid content followed a trend similar to that of protein, with P2 (10.66 ± 1.240 mg/mL) and P3 (9.54 ± 0.351 mg/mL) displaying higher values than P1 (3.41 ± 0.760 mg/mL). Although LAB are not major lipid producers, increased lipid content in fermented products may originate from two sources: substrate-derived lipids and microbial biomass accumulation. Alfalfa and molasses contain minor but significant amounts of fatty acids and lipid precursors, which may be released during fermentation. Additionally, enhanced microbial growth in nutrient-rich media leads to increased synthesis of membrane phospholipids, contributing to the total lipid fraction detected in the postbiotic matrix (López-Gómez et al., 2020).
Protein determination
Total protein analysis was conducted to assess the nutritional quality of the postbiotic formulations, as protein-rich components are influence growth performance, carcass yield, and body composition in broiler chickens. P1 total protein content was 19.93±1.593 mg/ml, P2 total protein content was 64.42±3.981 mg/ml, and P3 total protein content was 8.30±1.076 mg/ml. Of the three postbiotics, the highest protein content was obtained in P2. The fact that P2 contains a rich source of protein/peptide such as whey powder and alfalfa may allow L. rhamnosus to produce more peptides/amino acids through its robust proteolytic systems (Yousefi, Shokrollahi, & Gernaey, 2025).
LABs, including L. rhamnosus, possess sophisticated proteolytic systems consisting of cell envelope proteinases, peptide transporters, and intracellular peptidases that hydrolyze complex proteins into biologically active peptides and free amino acids (Savijoki, Ingmer, & Varmanen, 2006).
Despite containing whey powder, the relatively low protein content in P3 suggests that the presence of sesame hulls may inhibit proteolytic efficiency by binding proteins within a fibrous matrix or through interaction with phenolic compounds. Similar inhibitory effects of dietary fiber on protein bioavailability have also been reported in plant-based fermentation systems (Gil-Sánchez et al., 2019). Feeding high protein diets has resulted in increased carcass yield and decreased fatness. In contrast, reported that when fed low-protein diets, de novo fat synthesis was accelerated, resulting in higher levels of saturated fatty acids and monounsaturated fatty acids in the liver and higher levels of triglycerides and cholesterol in the blood plasma (Aletor et al., 2003). According to our study, an increase in yield was observed in chickens fed with high protein content postbiotic supplemented feed.
Reducing sugar assay - DNS method
Reducing sugar analysis was performed to evaluate substrate utilization efficiency during fermentation, as reducing sugars serve as the primary carbon source for lactic acid bacteria and are directly associated with organic acid production. The amount of reducing sugar was determined as 1.98±0.100 for P1, 8.99±1.324 for P2 and 6.64±0.401 mg/mL for P3, respectively (Table 2). The elevated residual sugar levels in P2 and P3 indicate that the initial carbohydrate load exceeded the metabolic consumption capacity of L. rhamnosus within the 48-h fermentation period. Molasses-based media are known to contain high concentrations of sucrose and reducing sugars, which may not be fully metabolized during batch fermentation, particularly when nitrogen is not growth-limiting (John et al., 2021). According to Martínez-Trujillo et al. (2020), in a study carried out at a bioreactor scale (1L), the joint action of oxidases and hydrolases released 18 g/L reducing sugars after 24 h (60% of which corresponds to glucose). In another study, Lactobacillus delbrueckii and Lactobacillus rhamnosus were produced using MRS and BHM medium. According to the measured reduced sugar amount, the amount of sugar in the medium decreased as the amount of lactic acid increased, inversely proportional to the lactic acid production (Hadiantoro et al., 2021). Therefore, in this study, the low amount of sugar significantly contributes to the high amount of lactic acid.
DPPH free radical scavenging capacity assay
DPPH radical scavenging activity was evaluated to assess the antioxidant potential of the postbiotic formulations, as antioxidant capacity is an important functional property that contributes to the protective effects of postbiotics against oxidative stress. The DPPH radical scavenging activity of the postbiotic formulations was determined as 44.77±3.639% for P1, 12.26±1.532% for P2, and 22.78±2.488% for P3 (Table 2). These values demonstrate moderate antioxidant potential, particularly in P1. In comparison, literature reports higher activities for certain strains. For example, Lactiplantibacillus plantarum C88 and O7S1 strains showed 53.05% and 88.78% activity, respectively (Li et al., 2012; Aliouche, Sifour, & Ouled-Haddar, 2024). Other studies revealed that postbiotics from L. casei had the highest antioxidant capacity among several LAB strains (Noori et al., 2023), while L. reuteri-derived postbiotics exhibited 52.3 mg/mL scavenging activity (Jalali et al., 2024). Davarzani et al. (2024) reported 48.81% antioxidant activity in postbiotic yoghurt by day 14. The antioxidant capacity detected in P1 can be supported by the fact that sesame hulls are rich in lignans, flavonoids, and other phenolic compounds with strong antioxidant properties. It has also been reported that during fermentation, microbial enzymes such as β-glucosidase and esterase can increase antioxidant capacity by releasing bound phenolics (Filannino, Di Cagno, & Gobbetti, 2018).
Total amount of phenolic content
Total phenolic content was determined to evaluate the contribution of phenolic compounds to the antioxidant capacity of the postbiotic formulations, as these compounds are known to act as effective free radical scavengers. Regarding phenolic content (Table 2), P1 exhibited the highest value at 21.88±1.041 µg/mL gallic acid equivalent (GAE), followed by P2 (9.20±1.428 µg/mL) and P3 (0.19±0.009 µg/mL). These are lower than values reported in other studies, such as 541.60 mg/kg from L. acidophilus (Nasri et al., 2024) and 250.8 mg/mL in mixed cultures (Jalali et al., 2024). High values up to 2336.11 mg/L GAE were also reported for Pediococcus acidilactici postbiotics (İncili et al., 2021; 2022). The results here suggest that although phenolic content is comparatively low, it contributes to the antioxidant potential observed, particularly in P1.
L-lactic acid (L-lactate) and acetic acid assay
L-lactic acid was the dominant organic acid detected in all fermented samples, confirming the primarily homolactic metabolism of L. rhamnosus. Lactic acid contributes to antimicrobial activity and gut health. The highest lactic acid concentration was observed in P2 (5.63 ± 0.787 g/L), followed by P3 (4.42 ± 0.267 g/L) and P1 (4.19 ± 0.702 g/L). The elevated lactic acid production in P2 reflects the efficient utilization of soluble sugars derived from molasses (mainly sucrose and glucose) and partially hydrolyzed fructans from inulin.
Molasses is widely recognized as an economical and effective carbon source for LAB fermentation due to its high sugar content and presence of growth-promoting micronutrients (Chen et al., 2019; John et al., 2021). Inulin, although not fully fermentable by all LAB strains, can be partially hydrolyzed into fructose units, providing an additional carbon supply and enhancing metabolic activity (Gibson et al., 2017). Studies focusing on L. rhamnosus have demonstrated that fermentable oligosaccharides significantly increase lactic acid yield by sustaining glycolytic activity during prolonged fermentation (Zhang et al., 2023). The highest lactic acid activity was observed in P2 with 5.63±0.787 g/L (Table 2). Hussain et al. (2021) reported lactic acid levels of 1.621±0.06 g/L and 1.653±0.05 g/L for L. delbrueckii PL11 and PL13 strains, respectively. Similarly, L. plantarum showed 5.11% lactic acid content in a study by Ozma et al. (2024). Yoon et al. (2006) reported initial lactic acid levels around 0.11-0.12% for L. casei, L. plantarum, and L. delbrueckii. Nasri et al. (2024) documented 192.95±25.95 g/kg and 287.16±21.08 g/kg lactic acid in MRS-Pb and W-Pb postbiotics, respectively. Moreover, Chang et al. (2021) reported levels ranging from 30.2 to 42.6 g/L among six L. plantarum strains. Kıvanc, Yilmaz and Çakir (2011) found values between 0.16 and 7.79 mg/mL depending on the isolate. The lactic acid levels in this study are within the effective range and are particularly high in P1, likely due to partial use of MRS broth, a known enhancer of lactic acid production.
Regarding acetic acid, the highest level was measured in P1 with 22.81±2.371 g/L, while P2 and P3 showed similar, lower levels (Table 2). P1 consisted of MRS medium supplemented with sesame hulls, a formulation characterized by moderate carbohydrate availability and a relatively high buffering capacity. Under such conditions, LAB metabolism may shift toward mixed-acid fermentation routes, including the acetyl-phosphate pathway, to maintain redox balance (Papagianni, 2012). Increased acetate formation under carbohydrate-limited or stress conditions has been widely documented in LAB fermentations (Pessione & Cirillo, 2016). Hussain et al. (2021) recorded acetic acid values of approximately 0.98 g/L for L. delbrueckii strains. Chang et al. (2021) noted values from 15.4 to 35.1 mM across various L. plantarum strains, and Nasri et al. (2024) found 18.47±2.05 g/kg and 5.53±0.58 g/kg for MRS-Pb and W-Pb, respectively. Acetic acid was also the dominant organic acid in L. rhamnosus and L. reuteri postbiotics (Jalali et al., 2024). Since the study design aimed to prioritize lactic acid production, relatively lower acetic acid levels-especially in P2 and P3-were expected and consistent with the intended fermentation profile.
D-3-hydroxybutyric acid (D-3 hydroxybutyrate) assay
D-3-hydroxybutyric acid analysis was performed to evaluate the presence of short-chain fatty acids in the postbiotic formulations, as these metabolites are associated with intestinal health, energy metabolism, and epithelial integrity. In the D-3-Hydroxybutyric Acid method, according to Table 2, P1, P2 and P3 was found to be 2.16±1.726, 35.76±5.438, 21.7±1.646 g/100g, respectively. In a study using L. plantarum species, the butyric acid content (GC-MS) of the postbiotic was calculated as 2.41% (Ozma et al., 2024). In our results, the simultaneous presence of excess carbohydrates and amino acids in P2 likely promoted overflow metabolism and activation of alternative metabolic routes leading to D-3-hydroxybutyric acid formation. Such metabolites are increasingly recognized as functional postbiotic components with potential health benefits, including anti-inflammatory and gut-protective effects (Canfora, Jocken, & Blaak, 2015). In the determination of organic acids in L. rhamnosus and L. reuteri postbiotic extracts, butyric acid was detected the least among organic acids (Jalali et al., 2024). In another study conducted with L. acidophilus, L. helveticus, L. plantarum, L. rhamnosus and B. bifidum species, butyric acid was not detected in any species (Ibrahim et al., 2024). In the literature, it is clearly seen that butyric acid determination is done less or its amounts are detected less in the studies conducted on postbiotic extracts. Therefore, it is clearly seen that the butyric acid amounts obtained in this study were successful.
Zootechnical Performance
Performance data are presented in Table 3. Compared with the control group, the supplementation of broiler diets with postbiotics obtained in the study resulted in increased BW on days 21 and 42 (P<0.05). Furthermore, on days 0-21, DWG was higher in all postbiotic groups than in the control animals (P<0.05), while DFI and FCR were not affected (P>0.05). In the overall phase, all postbiotic group animals exhibited higher DWG and lower FCR than the control birds (P<0.05). A higher carcass yield was observed in P2 and P3 birds compared to the control group (P<0.05).
As demonstrated in Table 4, except for relative duodenum weight, other internal organ weights and lengths were unaffected by postbiotic addition to the diet (P>0.05). However, a statistically significant difference was observed in the relative duodenum weight of P2 group birds compared to both control and P1 birds (P<0.05). Dietary postbiotic supplementation did not influence broilers’ serum AST and ALT concentrations (P > 0.05; Table 4).
In recent years, postbiotics have gained attention for their potential to improve poultry performance and serve as alternatives to antibiotics (Danladi et al., 2022; Fang et al., 2024; Monika et al., 2024; Atan Çırpıcı & Kırkpınar, 2025). This study demonstrated the benefits of adding P1, P2, and P3 postbiotics to broiler diets as innovative feed additives, resulting in decreased feed conversion ratio (FCR) and increased live weight and carcass yield. These results are consistent with Monika et al. (2024), who found that 0.2% postbiotic supplementation from Lactobacillus acidophilus led to the highest body weight and optimal FCR. Similarly, Fang et al. (2024) reported positive effects from a Bacillus subtilis-derived postbiotic. However, Danladi et al. (2022) noted that while postbiotic and paraprobiotic supplementation significantly increased feed intake, other growth parameters, including final body weight and mortality, were not significantly affected throughout the treatment. In this study, no statistically significant differences were found between the P1, P2, and P3 postbiotic groups in terms of live weight, daily live weight gain, feed consumption, and feed conversion ratio (P>0.05). This finding suggests that different postbiotic formulations can exhibit similar levels of biological activity and that the applied 0.2% dose may have elicited a common physiological response across postbiotics. The fact that the main mechanisms of action of postbiotics operate through common pathways, such as balancing the gut microbiota, producing short-chain fatty acids, and supporting epithelial integrity (Monika et al., 2024), may explain the lack of significant performance differences between the groups. Furthermore, it is possible that the differences in chemical composition between the formulations did not translate into a metabolic superiority sufficient to differentiate growth performance. However, the higher total protein and lipid content determined in the P2 postbiotic formulation (Table 2) theoretically presents a biochemical profile that could support growth performance. Proteins play a significant role in muscle tissue development and cellular regeneration, whereas lipids are crucial for meeting metabolic energy requirements, particularly in broiler chickens during periods of rapid growth. In this context, the numerical improvements observed in live weight, feed conversion ratio, and carcass yield in the P2 group (Table 3) may be related to this chemical composition. However, the lack of a statistically significant performance difference between the groups suggests that these biochemical advantages may be limited or offset by the combined mechanisms of action of other postbiotics.
There are several reasons for the observed improvement in performance. One reason may be the antibacterial properties of postbiotics, which combat pathogenic bacteria and inhibit toxin production in the GUT (Monika et al., 2024). Also, postbiotics enhance nutrient absorption in the intestine through various metabolites, including short-chain fatty acids, microbial cell fragments, extracellular polysaccharides, cell lysates, teichoic acids, and vitamins (Monika et al., 2024). The undoubted advantage of postbiotics is bypassing the problem of acquiring antibiotic resistance genes and virulence factors, which may occur in vivo when probiotics are used (Żółkiewicz et al., 2020). Our findings indicate that improvements in nutrient absorption and digestion lead to a decrease in FCR and, consequently an increase in BW due to the contents of P1, P2 and P3 postbiotics given in Table 3. Parallel to our results, in a study conducted by Monika et al. (2024), it was reported that the use of 0.2% postbiotic (T4) produced from Lactobacillus acidophilus resulted in better FCR (1.75) and higher body weight (1677.52 g) in broiler chickens. From an economic perspective, supplementing with postbiotics, an increase in BW and a decrease in FCR, can enhance profitability by reducing feed costs. Additionally, postbiotic supplementation may improve histomorphology, as evidenced by increased villus height in the duodenum and ileum (Kareem et al., 2016; Monika et al., 2024). This improvement can contribute to animal growth by enhancing nutrient absorption in the intestine (Sikandar et al., 2017). Although our study did not directly establish this relationship, the observed increase in growth performance and decrease in FCR could also be attributed to the rise in villus height and the reduction in crypt depth (Xu et al., 2003).
Contrary to the study conducted by Monika et al. (2024), our findings showed that the addition of postbiotics to the diet affected carcass yield. Specifically, carcass yield in groups P2 and P3 was 1.95 and 1.79 units higher than the control group, respectively. This increase is thought to be due to the higher live weight of broilers fed a diet supplemented with Lacticaseibacillus rhamnosus-derived postbiotics.
The relative weight and length of the gastrointestinal tract (GUT) organs and segments can indicate their level of development (Alshamy et al., 2018). In this study, dietary supplementation with postbiotics did not significantly impact the development of the small intestinal segments, except for the duodenum. This finding contrasts with the study by Khonyoung and Yamauchi (2019), which reported that supplementation with heat-killed Lactobacillus sakei HS-1 reduced the weights of the ileum, total small intestine and blind intestine in broiler chicks. It can be hypothesized that different types of postbiotics may have varying effects on gut health, potentially improving performance by increasing duodenal weight. However, by day 42 of the study, no significant differences were observed in the weights of any metabolic organs analyzed, except for the duodenum. Similarly, no significant differences were noted in the weights and lengths of the digestive organs. The significant difference observed in duodenal weight suggests that the effects of postbiotic supplementation on intestinal morphology may be segment-specific. The duodenum, the intestinal region where digestive enzymes are secreted in high concentrations and where initial contact with nutrients occurs, may exhibit a structure that is more sensitive to diet-related interventions (Xu et al., 2003; Khonyoung & Yamauchi, 2019). Therefore, structural changes observed in the duodenum following postbiotic administration may be related to adaptive responses occurring in the early stages of digestion and absorption. In contrast, the lack of significant differences in morphological parameters between the jejunum and ileum may be explained by these segments being involved in more stable phases of nutrient absorption and by their more balanced morphology (Kareem et al., 2016; Sikandar et al., 2017). The literature reports that the effects of postbiotic and probiotic applications on intestinal morphology are mostly more pronounced in the duodenum, while these effects may be limited or variable in the jejunum and ileum (Xu et al., 2003; Khonyoung & Yamauchi, 2019). Furthermore, the absence of significant morphological changes in the jejunum and ileum suggests that postbiotic application preserves morphological integrity in these intestinal segments, producing effects within physiological limits (Kareem et al., 2016).
Serum biochemical parameters such as AST and ALT provide important information about the general health status of animals. Increased AST levels are associated with heart (Wirz et al., 1990) and liver damage, while increased ALT levels indicate liver damage (Baradaran et al., 2019). In line with the results of Li et al. (2024), serum AST and ALT values in our study were similar to the control. These data indicate that the postbiotics used in the study did not adversely affect liver or heart functions. In particular, the fact that no mortality was recorded in this study suggests that these postbiotics positively affected growth performance in broiler chickens with high metabolic rates, resulting in improved health and welfare.
In conclusion, the evaluation of all findings shows that the use of 0.2% Lacticaseibacillus rhamnosus derived postbiotics as dietary supplements in broiler diets can improve growth performance. This shows the potential of using postbiotics P2, P3 and P1 as growth promoting feed additives as alternatives to antibiotics, respectively. Especially P2 positively affected body weight and feed conversion in broiler chickens, diversified the intestinal microbiota and created a healthier intestinal flora.
16S V3V4 Metagenom analysis
After DNA isolation of microorganisms in control and P2 group chicken feces samples, V3-V4 regions of 16S rRNA genes were amplified by PCR method. Metagenomic analysis was performed using QIIME2 for the regions sequenced with Illumina Miseq. As a result of the analyzed microbial diversity, Firmicutes was found to be the most dense Phylum in both groups. According to the metagenome analysis results, the dominant genus in the control group were Turicibacter (%21.86), Escherichia-Shigella (%19.31), Romboutsia (%15.78). The rate of Escherichia-Shigella (%19.3) genus was found to be dominant (Figure 1). In the P2 group, Acinetobacter (17.72%), Brachybacterium (11.21%), and Corynebacterium (8.67%) were determined as the dominant genus. Also, the potentially pathogenic Escherichia-Shigella rate (3.88%) significantly decreased (Figure 2). Acinetobacter became dominant in the P2 group (17.72%). Especially, the determination of Lactobacillus (7.5%) at the genus level in the P2 group is a remarkable data. The increase in the diversity of intestinal microbiota in the P2 group using postbiotics can be interpreted as an indicator of a healthier intestinal flora. Bacteria that support the immune system such as Lactobacillales, Acinetobacter and Brachybacterium increased in the P2 group. Pathogenic bacteria such as Escherichia-Shigella and Turicibacter significantly decreased. An increase in bacteria important for intestinal health such as Bacteroidota was also observed in the P2 group. All these results show that the use of postbiotics improves intestinal health in broilers and promotes the growth of beneficial bacteria (Figure 1 and 2).
Taxonomic content analysis for the 16S V3V4 region of the control samples (n=5; samples:feces).
Taxonomic content analysis for the 16S V3V4 region of P2 group samples (n=5; samples:feces).
Conclusıons
In our study, it was determined that the addition of postbiotics derived from Lactocaseibacillus rhamnosus to broiler rations improved growth performance and feed conversion ratio. Furthermore, postbiotic supplementation positively supported various carcass and gut-related parameters. Specifically, the increase in carcass yield in groups P2 and P3, the increase in the relative weight of the duodenum in group P2, and the shift in gut microbiota diversity towards a beneficial profile demonstrate the effects of postbiotics on digestive health and nutrient utilization.
Acknowledgments
This study was supported by the 1507 - TUBITAK SME R&D Start-up Support Program Ankara, Turkey with grant number 7230573.
Data Availability Statement
Data available upon request to authors.
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Editor de seção:
Renato Paiva
