STUDIES ON THE IMPACT OF LACTOBACILLUS PLANTARUM AND LACTOBACILLUS CASEI CULTURED IN DIFFERENT NUTRIENT MEDIA ON THE GROWTH PERFORMANCE AND ON THE HELMINTHIASIS

TRADITION AND MODERNITY IN VETERINARY MEDICINE, 2025, vol. 10, No 2(19): 65–73 STUDIES ON THE IMPACT OF LACTOBACILLUS PLANTARUM AND LACTOBACILLUS CASEI CULTURED IN DIFFERENT NUTRIENT MEDIA ON THE GROWTH PERFORMANCE AND ON THE HELMINTHIASIS Fatihah Istafaro Maulidiya1, Herinda Pertiwi1*, Galuh Hesti Dwi Indrawati1, Nabila Hanina Hibatul Haqqi1, Nur Jannah Eka Agustina1, Loh Teck Chwen2, Mohamad Yusril Nur Mahendra1, Miyayu Soneta Sofyan1 1Veterinary Paramedic Program, Department of Health, Faculty of Vocational Studies Universitas Airlangga - Kampus B UNAIR - Jalan Dharmawangsa Dalam 28-30, Surabaya 60286 Indonesia 2Departement of Animal Science, Faculty of Agriculture Universiti Putra Malaysia, Darul Ehsan, Serdang Selangor 43400 Malaysia E-mail: [email protected] ORCID: 0009-0001-7729-8576 F.M.; 0000-0003-1546-2497 H.P.; 0009-0003-6846-9198 G.I.; 0009-0008-6422-9011 N.H.; 0009-0003-3827-1033 N.A.; 0000-0002-0752-2923 L.C.; 00090003-9246-1815 M.M.; 0000-0001-5561-8084 M.S. (Submitted: 5 August 2025; Accepted: 7 November 2025; Published: 27 November 2025) ABSTRACT The overuse of antibiotics in broiler production raises public health concerns. This study evaluated the potential of Lactobacillus plantarum and Lactobacillus casei cultured in sweet potato and tempeh infusions as probiotic alternatives. A total of 150 broiler chicks were divided into three groups: control (P1), sweet potatobased probiotic (P2), and tempeh-based probiotic (P3). Probiotics were administered via drinking water for 35 days. Growth performance, including average daily gain (ADG) and final body weight (FBW) and helminth egg counts were assessed. Results showed that both probiotic treatments improved growth, with P3 showing significantly higher weight gain. Probiotic supplementation also reduced fecal egg counts of Heterakis gallinarum, Ascaridia galli, Cysticola columbae, and Schistosoma mansoni. The improvements are attributed to gut microbiota modulation and immunostimulatory effects of Lactobacillus spp. This study supports the use of sweet potato and tempeh infusions as cost-effective media for probiotics, offering a promising alternative to antibiotics in poultry farming. Key words: Broiler, Lactobacillus spp., Poultry, Sweet potato, Tempeh. Introduction Over the past two decades, the use of antibiotics as growth promoters in livestock feed has been prohibited across European Union countries due to public health concerns associated with antimicrobial resistance (Pinho et al., 2025). The European Commission officially enforced this ban through Regulation (EC) No. 1831/2003, which came into effect on January 1, 2006 (OECD, 2022). Since then, the sale of veterinary antimicrobials in 25 European countries has declined by approximately 53% between 2011 and 2022 (EMA, 2023), reflecting a significant transition toward more responsible antimicrobial stewardship. Globally, it is estimated that around 70–75% of all antimicrobials produced are used in food-producing animals, underscoring the substantial contribution of the livestock sector to overall antimicrobial consumption (FAO, 2017; OECD, 2022). These restrictions have consequently stimulated research into safer and more sustainable alternatives to maintain animal health and productivity. 66 F. Maulidiya, H. Pertiwi, G. Indrawati, N. Haqqi, N. Agustina, L. Chwen, M. Mahendra, M. Sofyan The use of antibiotics in animal feed, particularly as growth promoters, may lead to the accumulation of residues in animal-derived products and accelerate the development of antibiotic-resistant bacteria, posing a major challenge to the poultry industry (Abou-Jaoudeh et al., 2024). In parallel, the ongoing search for safe and effective anthelmintic treatments remains an urgent concern (Boros et al., 2022). Among potential alternatives, probiotics have gained increasing attention due to their demonstrated anti-parasitic, antimicrobial, and immunomodulatory properties (Saracino et al., 2021). Within this context, Lactobacillus has emerged as the most widely used probiotic genus in poultry production, known to enhance growth performance, feed conversion efficiency, nutrient absorption, gut microbiota balance, intestinal health, and immune function in broilers (Pertiwi & Mahendra, 2021). However, the cultivation of these bacteria requires suitable media that can support their growth and ensure the successful isolation of potential probiotic strains (Bonnet et al., 2020). An ideal culture medium should be cost-effective, practical, and capable of maintaining the viability and metabolic activity of Lactobacillus spp. (Aumiller et al., 2023). Formulating appropriate cultivation media remains challenging due to the fastidious nutritional requirements and strain variability of Lactobacillus spp. (Harina et al., 2023). To promote optimal bacterial growth, culture media are typically enriched with amino acids, peptides, nucleic acid derivatives, fatty acid esters, minerals, vitamins, buffering agents, and carbohydrates (Rawat et al., 2023). Common nitrogen sources such as soy peptones, tryptone, beef extract, or yeast extract are essential but often expensive, representing a major cost component in culture media production (Tao et al., 2023; Yuniarti et al., 2023). This economic constraint highlights the need for alternative, low-cost substrates capable of supporting bacterial proliferation without compromising growth efficiency. Agricultural by-products and plant-derived materials have recently gained considerable attention as sustainable alternatives to conventional culture media components. Among these, sweet potatoes (Ipomoea batatas) represent a particularly promising substrate due to their high content of carbohydrates, amino acids, vitamins, and minerals, which can effectively support probiotic growth (Bernabeu et al., 2024). Moreover, oligosaccharides derived from sweet potatoes function as prebiotics that stimulate the proliferation of Lactobacillus and enhance their survivability in the gastrointestinal environment (Nguyen et al., 2021; Giang et al., 2021). In addition to sweet potatoes, tempeh—a traditional fermented soy-based product—also offers a nutrient-rich medium for probiotic cultivation. It provides abundant protein and bioactive compounds, including genistein, which contribute to the overall nutritional and functional quality of the culture substrate (Nurkolis et al., 2024). Notably, co-fermentation of Lactobacillus plantarum with Rhizopus oligosporus during tempeh production has been reported to improve lipid metabolism and glucose regulation. Furthermore, L. plantarum produces β-glucosidase, an enzyme that hydrolyzes glucoside isoflavones into more bioavailable aglycones (Huang et al., 2018). Collectively, the integration of natural substrates such as sweet potatoes and tempeh could enhance both the growth efficiency and functional potential of Lactobacillus spp. in probiotic applications for poultry production systems. The functional benefits of these probiotics extend beyond bacterial cultivation. Lactobacillus species play a central role in maintaining intestinal microbiota homeostasis, which is critical for nutrient utilization, feed conversion efficiency, and immune defense against enteric pathogens (Naeem & Bourassa, 2025). Their metabolic activity facilitates nutrient absorption, thereby improving energy availability for growth and productivity (Abdel-Moneim et al., 2020). Additionally, Studies on the impact of Lactobacillus plantarum and Lactobacillus casei … 67 Lactobacillus spp. inhibit pathogen adhesion and colonization in the intestinal mucosa through competitive exclusion mechanisms, contributing to the prevention of enteric infections in broilers (Yaqoob et al., 2022). These probiotics also produce bacteriocins and digestive enzymes that stabilize gut function, enhance nutrient digestibility, and modulate immune responses (Dempsey & Corr, 2022; Huang et al., 2022; Huang et al., 2023). Considering the nutritional potential and prebiotic properties of sweet potatoand tempehbased substrates, the present study aims to evaluate the effects of probiotic supplementation cultured in these alternative media on growth performance and helminth egg counts in broiler chickens. Materials and Methods The study was conducted in compliance with animal welfare and ethical regulations as stipulated in Indonesian Law No. 18/2009 on Animal Husbandry and Health and its amendment Law No. 41/2014. All experimental procedures followed national guidelines ensuring proper housing conditions, continuous access to feed and water, and appropriate environmental management to minimize stress and ensure animal welfare throughout the experimental period. A completely randomized design (CRD) was employed in this study. A total of 150 day-old chicks (DOC) were randomly allocated into three treatment groups, each consisting of 50 birds: Control, P1: chickens received plain drinking water; P2: chickens received drinking water containing probiotics prepared in sweet potato infusion; P3: chickens received drinking water containing probiotics prepared in fermented soybean cake (tempeh) infusion. All treatments were administered continuously for 35 days. During this period, the birds had no other water sources besides those containing the designated probiotic or control solutions. Prior to administration, the probiotic preparation—containing Lactobacillus casei (1.62 × 10⁶ CFU/mL) and Lactobacillus plantarum (0.25 × 10⁶ CFU/mL) – was incubated in each infusion medium at room temperature (24–30 °C) for six days. The resulting probiotic solution was then provided to the broiler chickens via drinking water at a ratio of 15% probiotic infusion to 85% water. The chicks used in this experiment were healthy, non-infected commercial broilers. No artificial infection was introduced; the presence of helminths was naturally occurring and monitored as part of the study. Body weight was recorded weekly to evaluate growth performance. In the 5week, fecal samples were collected from each group to examine the presence of helminth eggs under a microscope at 100–400× magnification. All collected data were analyzed using one-way ANOVA with SPSS version 16. Differences between groups were considered statistically significant at P < 0.05. Results Supplementation with Lactobacillus plantarum and Lactobacillus casei in both sweet potato and fermented tempeh infusion media significantly increased the ADG and FBW of broilers compared to the control group (P<0.05). During the fourth week, the ADG of chickens in P3 (tempeh infusion) was 74.03 ± 17.27 g, which was significantly higher than both the control group P1 (39.69 ± 30.75 g) and the sweet potato infusion group P2 (39.51 ± 5.13 g). At the end of the 5-week experiment, the final body weight was highest in P3 (1409.0 ± 161.78 g), followed by P2 (1104.0 ± 83.23 g), and lowest in the control group P1 (1085.8 ± 77.15 g) (Table 1). 68 F. Maulidiya, H. Pertiwi, G. Indrawati, N. Haqqi, N. Agustina, L. Chwen, M. Mahendra, M. Sofyan Table 1: Average daily gain and final body weight of broiler chicken drank with Lactobacillus plantarum and Lactobacillus casei in sweet potato and tempeh infusion water Parameters P1 P2 P3 Week 1 36.85±5.80 32.36±6.13 32.14±5.18 Week 2 23.02±12.95 28.71±12.05 27.00±8.78 Week 3 33.67±20.50 31.00±13.58 34.93±12.94 Week 4 39.69a±30.75 39.51b±5.13 74.03ab±17.27 Final Body Weight 1085.8a ±77.15 1104.0b±83.23 1409.0ab±161.78 Mean values bearing different superscript with in a row differ significantly (P<0.05) Probiotic supplementation also affected the number of helminth eggs identified in the feces of broilers at the end of the 5-week experiment (Table 2). A significant decrease (P<0.05) was observed only in the number of Heterakis gallinarum eggs, which decreased from 620.0 ± 472.00 in P1 to 101.0 ± 1.00 in P2 and 101.0 ± 1.00 in P3. In contrast, the number of Ascaridia galli eggs increased from 101.50 ± 1.29 in P1 to 540.00 ± 173.20 in P2, while P3 showed 180.00 ± 138.56 eggs, and the differences were not consistent. Similarly, Cysticola columbae and Schistosoma mansoni eggs showed either minor increases or remained similar to the control (Table 2). Table 2: Helminth eggs identification and total egg/gram feces of broiler chickens at the end of the 5-week experiment, drank with Lactobacillus plantarum and Lactobacillus casei in sweet potato and tempeh-infused water Helmint Eggs P1 P2 P3 Heterakis galinarum 620.0a ± 472.00 101.0b ± 1.00 101ab ±1.00 Ascaridia galli 101.50 a ±1.29 540.00 b ±173.20 180.00 ab ±138.56 Cysticola columbae 160.25±119.83 180.33±138.27 101.00±1.00 Schistosoma mansoni 101.00 a ±0.81 180.00 b ±134.56 101.00 ab ±1.00 Mean values bearing different superscript with in a row differ significantly (P<0.05) These results indicate that probiotic administration selectively suppressed Heterakis gallinarum infection at the end of the study, while showing limited or inconsistent effects on other helminth species. The data highlight that the effects of supplementation were species-specific, emphasizing the importance of monitoring individual helminth populations when evaluating probiotic efficacy. Discussion The results of this study demonstrated that supplementation with Lactobacillus plantarum and Lactobacillus casei cultured in sweet potato and tempeh infusion media significantly improved broiler growth performance. This was evident from the increase in average daily gain ADG and FBW compared to the control group. The highest improvement was observed in chickens receiving probiotics cultured in tempeh infusion, followed by those in sweet potato infusion. These findings indicate that the type of fermentation substrate plays an important role in determining the viability, stability, and metabolic activity of probiotics during fermentation and after entering the gastrointestinal tract (Rivas et al., 2021). Tempeh infusion medium is rich in peptides, free amino acids, and hydrolyzed isoflavones, which serve as nitrogen sources and bioactive compounds that support probiotic growth (Górska et al., 2025). During fermentation, Rhizopus oligosporus converts soybean proteins into short peptides and amino acids that are readily utilized by Lactobacillus spp., thereby enhancing bacterial adaptability and metabolic activity in the intestine (Huang et al., 2018). Furthermore, tempeh contains Studies on the impact of Lactobacillus plantarum and Lactobacillus casei … 69 genistein and daidzein in their aglycone forms, which possess antioxidant and immunomodulatory properties, promoting beneficial microbial colonization and strengthening intestinal mucosal immunity (Ahmad et al., 2015). The combination of these factors explains why the tempeh infusion group exhibited the best growth performance. Meanwhile, the sweet potato infusion medium also exerted a positive effect on broiler performance, although not as pronounced as the tempeh medium. Sweet potatoes contain complex carbohydrates, resistant starch, and oligosaccharides such as raffinose and stachyose, which serve as fermentable energy sources and prebiotic compounds for Lactobacillus (Zaman & Sarbini, 2016; Nguyen et al., 2021; Giang et al., 2021; Tekin & Dincer, 2023). These oligosaccharides enhance probiotic viability by serving as key metabolic substrates for the production of lactic and acetic acids— two important metabolites that lower intestinal lumen pH and inhibit enteric pathogens such as E. coli and Salmonella (Tran et al., 2018). In addition, the B-complex vitamins and minerals in sweet potatoes may support the enzymatic activity of L. plantarum and L. casei, enhancing their ability to synthesize hydrolytic enzymes such as proteases, amylases, and lipases, which contribute to improved nutrient digestibility (Islam, 2024). Thus, the sweet potato medium functions not only as a carrier but also as a functional substrate that reinforces probiotic metabolic activity and survival during gastrointestinal transit. The significant improvement in growth performance observed in the fourth week suggests a period of microbial adaptation followed by stabilization of the intestinal ecosystem. Lactobacillus spp. produce organic acids and bacteriocins that reduce intestinal pH, creating an unfavorable environment for pathogens such as Clostridium perfringens and Enterococcus faecalis, while promoting colonization of beneficial bacteria (Monika et al., 2024). This condition enhances villus height, reduces crypt depth, and increases the villus-to-crypt ratio, which translates into more efficient nutrient absorption. Additionally, L. plantarum and L. casei are known to produce bioactive metabolites such as exopolysaccharides (EPS) and short-chain fatty acids (SCFA), which serve as energy sources for enterocytes and help strengthen the intestinal mucosal barrier (Tao et al., 2024). From an immunological perspective, both Lactobacillus species can activate Toll-like receptor 2 (TLR2) and TLR4 on intestinal epithelial cells, leading to upregulation of mucin (MUC2) and tight junction proteins such as claudin, occludin, and ZO-1 (Al-Sadi et al., 2021). This mechanism maintains epithelial barrier integrity against microbial and endotoxin invasion. Probiotics also enhance secretory IgA production and balance cytokine profiles by suppressing IL-6 and TNF-α while increasing IL-10 and TGF-β, resulting in an anti-inflammatory effect that supports immune homeostasis (Kumar et al., 2025). These synergistic effects collectively improve feed efficiency and gut health, reflected in enhanced growth performance. Regarding parasitic infection, the results showed that probiotic supplementation selectively reduced Heterakis gallinarum egg counts, whereas Ascaridia galli, Cysticola columbae, and Schistosoma mansoni remained unaffected. This species-specific response suggests that probiotic effects on helminth infection are dependent on parasite biology. The reduction in Heterakis gallinarum may be attributed to increased Th1-mediated immune responses via elevated IFN-γ, IL-2, and TNFα production, which activate macrophages and suppress nematode development (Salas-Lais et al., 2020; Tanaka et al., 2020). Additionally, probiotics may modulate the microbial environment of the cecum – the primary site of Heterakis gallinarum development—through competition for space and nutrients, as well as secretion of antimicrobial metabolites that disrupt the parasite’s life cycle (Khasanah et al., 2024). 70 F. Maulidiya, H. Pertiwi, G. Indrawati, N. Haqqi, N. Agustina, L. Chwen, M. Mahendra, M. Sofyan Conversely, Ascaridia galli and Schistosoma mansoni may be less affected because they occupy distinct colonization sites (the anterior small intestine and mesenteric blood vessels, respectively), resulting in lower exposure to probiotic metabolites (Permin et al., 2006). Furthermore, the five-week experimental duration may not have been sufficient to induce significant adaptive immune responses against parasites with longer life cycles. These findings are consistent with reports by Myhill et al. (2022) and Vinayamohan et al. (2024), which emphasize that probiotic efficacy against parasites depends on host species, infection site, and duration of exposure. Overall, this study demonstrates that L. plantarum and L. casei cultured in tempehand sweetpotato-based media enhance broiler growth performance through mechanisms involving improved bacterial viability, modulation of gut microbiota, enhancement of mucosal morphology, and strengthening of mucosal immunity. The selective anthelmintic effect against Heterakis gallinarum indicates that probiotic–parasite interactions are species-specific and complex. Further studies are warranted to identify the key metabolites responsible for immunomodulatory activity, determine the optimal dosage, and explore the potential of natural fermentation media such as sweet potato and tempeh as functional carriers to enhance the biological efficacy of probiotics in tropical poultry production systems. Conclusion In conclusion, supplementation with the probiotics Lactobacillus casei (1.62 × 10⁶ CFU/mL) and Lactobacillus plantarum (0.25 × 10⁶ CFU/mL) in sweet potato and tempeh-infused water significantly improved broiler growth performance. Chickens receiving probiotics in tempeh infusion (P3) achieved the highest final body weight (1409 ± 161.78 g) at the end of the 5-week study, which was significantly higher than both the control group (P1: 1085.8 ± 77.15 g) and the sweet potato infusion group (P2: 1104.0 ± 83.23 g). Probiotic supplementation selectively reduced the number of Heterakis gallinarum eggs in feces at the end of the experiment (P3: 101 ± 1.00 eggs/g; P2: 101 ± 1.00 eggs/g) compared to the control (620 ± 472 eggs/g), whereas the numbers of Ascaridia galli, Cysticola columbae, and Schistosoma mansoni eggs either increased or remained unchanged. These results indicate that probiotic administration, particularly in tempeh-based media, enhances growth performance and provides species-specific protection limited to Heterakis gallinarum infection at the end of the study, highlighting the need for monitoring individual helminth populations when evaluating probiotic efficacy. 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