Optimization of Tiliacora triandra Leaf Extraction and Probiotic Fermentation for Developing a Functional Freeze-Dried Feed Supplements

Abstract

Tiliacora triandra (Yanang) leaf contains polyphenols, flavonoids, and mucilage polysaccharides with antioxidant and prebiotic functions, making it a promising substrate for probiotic fermentation. This study aimed to optimize Yanang extraction and sterilization to preserve bioactive mucilage and support probiotic survivability during freeze-drying–based encapsulation, and evaluate antimicrobial activity against poultry pathogens. Yanang extract was prepared under different leaf processing conditions and used as a substrate for Pediococcus acidilactici V202, Lactiplantibacillus plantarum TISTR 926, Streptococcus thermophilus TISTR 894, Bacillus subtilis RP4-18, and Bacillus licheniformis 46-2. Fermentation at 37 °C for 24 h revealed that lactic acid bacteria (P. acidilactici V202, L. plantarum TISTR 926, S. thermophilus TISTR 894) reduced pH (<4.10, p < 0.001) while maintaining high viable counts (>8.67 log CFU/mL, p < 0.01), whereas Bacillus strains (B. subtilis RP4-18, B. licheniformis 46-2) retained a higher pH (>5.00) and lower viability (<8.50 log CFU/mL). Total soluble solids decreased across treatments, with the lowest observed for B. subtilis RP4-18 (1.97 °Brix, p = 0.007). Freeze-dried probiotics encapsulated in enzyme-extracted rice bran carriers had comparable physicochemical properties (p > 0.05), while compared with Bacillus strains (p < 0.01), lactic acid bacteria had superior tolerance to simulated gastrointestinal and thermal stress. Supernatant from Yanang extract inhibited B. cereus WU22001, S. aureus ATCC25923, Escherichia coli ATCC25922, and Salmonella typhimurium WU241001 (MIC/MBC 25–50% v/v). These results indicate that Yanang extract supports effective probiotic fermentation, and rice bran encapsulation enhances survivability and antimicrobial functionality for potential functional feed applications.

1. Introduction

The global escalation of antimicrobial resistance (AMR) in both human and veterinary medicine, combined with increasingly stringent food safety standards, has catalyzed a paradigm shift in the animal production industry [1]. Once routine in poultry and livestock feeds, regulation-driven restrictions and bans on antibiotic growth promoters (AGPs) have intensified the search for novel, sustainable feed additives capable of safeguarding animal health and optimizing productivity without reliance on antibiotics [2]. These factors all impact broiler production systems, which are a key livestock sector in Thailand, the world’s fourth-largest exporter of broiler meat [3]. In this context, due to their roles in modulating the gut microbiota, enhancing immune competence, and exerting direct or indirect antimicrobial effects, natural feed additives including probiotics, prebiotics, and phytobiotics have emerged as leading candidates [4]. Among the strategies gaining momentum, the fermentation of plant-derived substrates is particularly attractive. Such substrates offer the simultaneous advantages of acting as prebiotics supporting the growth of beneficial gut microorganisms and as reservoirs of phytobiotic secondary metabolites, many of which possess inherent antimicrobial and antioxidant properties [4,5]. Through the enzymatic conversion of complex compounds into more active and absorbable forms, the fermentation process facilitated by selected probiotic strains can increase the bioavailability and bioactivity of phytochemicals [6]. Notably, microbial enzymes such as β-glucosidase are capable of transforming phenolic glycosides found in plant matrices into aglycones with enhanced antioxidant and antimicrobial properties [7]. Despite the obvious benefits of natural feed additives, achieving the full potential of such synergistic systems requires careful optimization of extraction and processing parameters to maintain both the viability of probiotics and the integrity of sensitive phytochemicals.

Tiliacora triandra (locally known as “Yanang”) is a perennial herb native to Thailand and Southeast Asia. Traditionally, it has been used in both folk medicine and regional cuisine, and serves as an important component of traditional herbal remedies due to its well-recognized medicinal properties [8]. The leaves are renowned for their myriad health benefits, including cooling, anti-inflammatory, and antioxidant properties, which are primarily ascribed to their rich content of bioactive phytochemicals such as polyphenols, alkaloids, flavonoids, chlorophyll, and, distinctively, abundant mucilaginous polysaccharides [9,10]. Not only do these compounds confer functional health benefits, but they also present unique technological advantages. Yanang mucilage, a plant-derived gum, is mainly composed of xylose along with notable amounts of other neutral sugars. FT-IR analysis suggests that its structure resembles a mixed (1,3)- and (1,4)-D-xylan polysaccharide [11]. This polysaccharide is gaining attention as a potential prebiotic substrate and as a natural encapsulating or cryoprotective matrix, providing functional advantages over conventional plant-derived materials [12]. Extraction and processing methods profoundly impact the yield and functional quality of phytochemicals and mucilage obtained from Yanang leaves. Several parameters including solvent type, extraction temperature (ranging from 25 to 85 °C), duration (60–180 min), and leaf-to-solvent ratio have been identified as critical for optimizing polysaccharide and polyphenol yields [12,13]. In particular, aqueous extraction often maximizes total phenolic content retention, whereas organic solvents such as ethanol facilitate broader bioactive compound solubilization and fit well with subsequent processing steps [14]. Modern extraction techniques (ultrasonic-assisted extraction, maceration) are increasingly being adopted to further enhance yield, functionality, and stability of both mucilage and phytochemicals.

Rice bran is a by-product obtained from rice milling, with rice being a major agricultural crop in Thailand. In 2019, global production of milled rice was approximately 60 million tons, and rice bran accounted for about 5 to 8% of this total. Approximately 90% of rice bran is used as feed for livestock and poultry, while the remainder is processed for rice bran oil extraction [15]. This by-product represents an abundant, low-cost substrate suitable for microbial applications. It is rich in carbohydrates, proteins, dietary fiber, and antioxidants, and contains bioactive compounds such as γ-oryzanol, which exhibits antioxidant, anti-inflammatory, hypolipidemic, antidiabetic, anticancer, and immunomodulatory activities [16]. These properties make rice bran a promising alternative to expensive commercial probiotic culture media, while also serving as a natural encapsulating material that enhances probiotic survival during processing, including freeze-drying or spray-drying [17,18]. Recent studies demonstrated that nutrient broth supplemented with 10% (w/v) rice bran significantly promoted the growth of P. acidilactici V202 within 12 h without affecting pH (p < 0.05), highlighting its potential to accelerate probiotic proliferation, reduce incubation time, lower production costs, and increase bacterial yield for feed applications [19]. Thus, rice bran serves as a sustainable substrate for probiotic fermentation, promoting bacterial growth, enabling cost-efficient feed production, and valorizing milling by-products within a zero-waste, circular, and green bioeconomic framework.

Recent advances in formulation technologies, particularly freeze-drying (lyophilization), have markedly improved the stability and shelf-life of feeds containing sensitive bioactive compounds [20]. This technique enables moisture removal at low temperatures, thereby preserving the structural integrity, viability, and bioactivity of encapsulated probiotic cells as well as plant-derived compounds [21]. Previous studies have demonstrated that natural mucilage serves not only as a biocompatible encapsulant, protecting probiotics from thermal, oxidative, and dehydration stresses, but also as an effective prebiotic and cryoprotectant thereby providing dual functional roles during processing and gastrointestinal transit [22]. Building on this evidence, Yanang mucilage represents a promising candidate for the development of plant-based and sustainable feed additives, meeting consumer and industry demands for natural, multifunctional ingredients. While extraction methodologies and the technological applications of Yanang mucilage have gained research interest, specific processes for optimization particularly to maintain both high-phytochemical content and mucilage functionality under conditions suitable for probiotic fermentation and feed processing remain underexplored. Furthermore, few studies have systematically examined how optimized plant-based substrates support the growth, survival, and functional bioactivity of robust probiotics. Thus, it is imperative to assess the interactions among plant-derived phytobiotics, prebiotics, and probiotic strains and their impact on the efficacy of functional plant-based feed additives in livestock nutrition.

The present study highlights a systematic strategy to address challenges and promote Thai innovation globally through a freeze-dried phytosynbiotic feed additive, utilizing Yanang leaf extract as both a bioactive substrate and a natural encapsulating matrix for probiotic fermentation. The specific objectives were to: (i) optimize extraction and sterilization conditions of Yanang leaf extract to preserve its bioactive and mucilaginous properties while enhancing the growth and viability of P. acidilactici V202; (ii) evaluate the effects of probiotic fermentation on pH, total soluble solids, viable probiotic counts, product yield and quality; and (iii) assess the antimicrobial efficacy of the resulting freeze-dried phytosynbiotic products against relevant poultry foodborne pathogens. The findings are expected to provide a preliminary framework for utilizing mucilaginous plant extracts as a fermentation substrate and for protecting probiotics through freeze-drying, supporting the development of functional plant-based feed additives for antibiotic-free poultry production.

2. Materials and Methods

This study was conducted at the Animal Nutrition Laboratory, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Phitsanulok, Thailand. All experimental procedures were approved by the Naresuan University Animal Care and Use Committee (Approval ID: 68 01 008).

2.1. Experiment 1: Extraction Optimal Conditions for Yanang Leaf Extract for Use as a Natural Medium for P. acidilactici V202 Fermentation

2.1.1. Experimental Design and Fermentation Procedure

Fresh Yanang leaves were obtained from a local supplier (Phitsanulok, Thailand). The leaves with only uniformly mature and undamaged leaves were thoroughly washed under running tap water to remove debris, and excess moisture was gently blotted off. Depending on the experimental design, leaves were either processed immediately (fresh) or stored at −20 °C prior to use as a natural medium for further fermentation. Yanang leaf extracts were treated using a factorial design to evaluate six key treatment factors:

(1)

Leaf freezing (unfrozen or frozen at −20 °C);

(2)

Leaf blanching (unblanched or blanched by immersion in boiling water for 2 min);

(3)

Leaf preparation process (unfrozen and unblanched leaves, frozen and unblenched leaves, frozen first, then blanched leaves, blanched leaves and unfrozen, and blanched first, then frozen leaves);

(4)

Leaf-to-water ratio (1:3, 1:4, or 1:5 w/v);

(5)

Water temperature during extraction (4 °C, 25 °C, or 100 °C);

(6)

Heat sterilization (no sterilization [control], pasteurization at 75 °C for 5 min, steam sterilization at 100 °C for 10 min, or autoclaving at 121 °C for 15 min).

This factorial approach enabled systematic evaluation of the effects of treatment conditions on Yanang extract quality and its capacity to serve as a natural medium for probiotic culture.

2.1.2. Fermentation and Determination of pH, Total Soluble Solids, and Viable Counts

P. acidilactici V202, a probiotic lactic acid bacteria (LAB) strain capable of producing beneficial organic acids and exhibiting antimicrobial activity against pathogenic bacteria, was employed for fermentation. This laboratory strain was originally isolated from the goat vagina [23]. An active culture was cultivated overnight in de Man Rogose Sharpe (MRS) broth at 37 °C. Each 100 mL aliquot of Yanang leaf extract was inoculated with 1% (v/v) (~7 Log CFU/mL) of the active culture. The fermentation was performed at 37 °C for 24 h under static conditions [24]. The pH was determined using a digital pH meter (HI2002, Hanna Instruments, Padova, Italy). A refractometer (Hanna Instruments, Padova, Italy) was used to measure total soluble solids [25]. Total viable count (TVC) was determined by 10-fold serial dilution and spread plating on plate count agar (PCA), incubated at 37 °C for 24 h, and expressed as Log CFU/mL according to [26]. Whereas, the population of P. acidilactici V202 was serially diluted and enumerated on MRS agar under anaerobic conditions at 37 °C for 24 h, and expressed as Log CFU/mL according to Mitsuwan et al. [27].

2.1.3. Statistical Analysis

The effects of the six key treatment factors on the response variables were analyzed using a Generalized Linear Model (GLM). The statistical model included all treatment factors as independent variables: Y_ijklmno = μ + leaf freezing_i + leaf blanching_j + leaf preparation process_k + yanang leaf:water ratio_l + water temperature for extraction_m + heat sterilization_n + ε_ijklmno where Y_ijklmno is the observed response, μ is the overall mean, each main term represents the effect of treatment factors at its level, and ε_ijklmno is the random error. After model fitting, least squares means (LSMEANS) were computed for each factor level to facilitate adjusted group mean comparisons. When a significant main effect was detected (p < 0.05), the means among levels of that factor were further compared via Duncan new multiple range tests (DMRTs) to identify significant pairwise differences. All statistical analyses, including model fitting, hypothesis testing, LS means calculation, and post hoc comparisons, were conducted using The SAS University Edition for Windows, Release 9.4 [28]. Statistical significance was declared at p < 0.05.

2.2. Experiment 2: Probiotic Fermentation Using Yanang Leaf Extract as a Natural Medium and Production of Freeze-Dried Probiotics

2.2.1. Experimental Design

A completely randomized design (CRD) was employed, consisting of five probiotic strain treatments, each with five replicates (n = 5). Probiotic strains capable of producing beneficial organic acids, metabolites, and exhibiting antimicrobial activity against pathogenic bacteria were used in this study. P. acidilactici V202, as mentioned above [23], S. thermophilus TISTR 894, and L. plantarum TISTR 926 were sourced from the Thailand Institute of Scientific and Technological Research. Meanwhile, B. subtilis RP4-18 and B. licheniformis 46-2, laboratory strains, were obtained from King Mongkut’s University of Technology Thonburi.

2.2.2. Yanang Leaf Extract Preparation Under Optimal Conditions

Two pre-treatment methods were applied: freezing at −20 °C until use, and blanching in hot water (100 °C) for 2 min to inactivate endogenous enzymes. The pre-treated leaves were then chopped, and homogenized with distilled water at a 1:3 (w/v) ratio using a blender. The homogenized samples were then filtered through sterile muslin cloth to obtain a crude extract. The crude extract was sterilized by pasteurization at 75 °C for 5 min and cooled down before being used as a natural medium for probiotic fermentation. The overall workflow for Yanang extract preparation under various pre-treatment and sterilization conditions is shown in Figure 1.

2.2.3. Determination of Yield, Product Quality, and Stability

The fermentation substrate consisted of sterile Yanang leaf extract, which was inoculated with a mixed culture of five activated probiotic strains at 1 × 10⁸ CFU/mL. Fermentation was conducted in sterile Duran flasks under static conditions at 37 °C for 24 h. Following fermentation, the broth was mixed with sterilized extracted rice bran (1.0 mm) at an 85:15 (w/w) ratio to serve as a carrier, and homogenized at 1200–2000 rpm for 15–20 min. The mixtures were then loaded into pretreated wall material pores by adsorption under mild vacuum to enhance penetration efficiency. The loaded materials (300 mL per tray) were frozen at −18 to −20 °C for 24 h. Primary drying was performed by lyophilization at −40 °C and 0.5 mbar for 24 h. Secondary drying was conducted at 20 °C and 0.5 mbar for 24 h. Then, the samples were stored in vacuum bags, exhibiting residual moisture content below 1% and water activity (Aw) less than 0.2. Freeze-drying efficiency was assessed by calculating the product yield (%), a widely used parameter in food and pharmaceutical research. It was determined using the formula freeze-dried product yield (%), which was calculated as (Weight of freeze-dried product/Initial total solids) × 100, that represents the proportion of the original solid material retained after water removal and reflects the effectiveness of the freeze-drying process [29]. The viability of probiotics in the freeze-dried products was evaluated to determine the survival rate of probiotics after processing. Freeze-dried samples were first rehydrated in sterile saline solution (0.85% NaCl, w/v) under aseptic conditions to restore the bacterial cells to a viable state. The rehydrated suspensions were then subjected to a series of tenfold serial dilutions to ensure an appropriate range of colony counts. TVC and LAB probiotic (LAP) were determined as described above. Bacillus-based probiotics (BBP) were plated and enumerated on nutrient agar under anaerobic conditions at 37 °C for 24 h according to Dumitru et al. [30]. Freeze-drying efficiency (%) was determined following Mitsuwan et al. [27] using the equation: freeze-drying efficiency (%) = (N/N₀) × 100, where N represents the number of viable cells released from the microcapsules after freeze-drying, and N₀ is the number of free cells initially incorporated into the microcapsules. This calculation provides a quantitative measure of the survival rate of probiotics during the freeze-drying process.

The quality of the dried encapsulated powders was assessed through instrumental analyses. All measurements were conducted in triplicate, and reported as mean values. Bulk density and tapped density of the powder were determined following the United States Pharmacopeia (USP) guidelines. A known amount of powder was gently poured into a 100 mL graduated cylinder to measure its initial volume for bulk density calculation, after which the cylinder was mechanically tapped until a constant volume was achieved to determine tapped density. Bulk and tapped densities were calculated as the ratio of the powder mass to the respective volumes, and the compressibility index, representing powder flowability, was calculated as [(tapped density − bulk density)/tapped density] × 100 [31]. Color parameters (L*, a*, b*) were measured using a colorimeter (HunterLab, MiniScan EZ 45/0 (LAV)) calibrated according to the CIELAB system, where L* represents lightness (0–100), a* represents redness–greenness, and b* represents yellowness–blueness. Chroma (C*) and hue angle (h°) were calculated from a* and b* values according to the CIELAB system [32]. The Aw of the freeze-dried probiotic was measured with a water activity meter (AQUALAB 4TE water activity meter) at room temperature (25 ± 3 °C) [33].

Tolerance of probiotics to various stress conditions was evaluated using their encapsulated forms. Acid tolerance was assessed by immersing 5.0 ± 0.5 g of microcapsules in 20 mL of 0.2 M phosphate buffer (pH 2.0), followed by incubation in simulated gastric fluid (0.3% pepsin, pH 2.5) at 40–42 °C for 2 h. Pancreatin tolerance was evaluated by suspending the samples in simulated intestinal fluid (1% pancreatin in deionized water, pH 7.0) and incubating at 40–42 °C for 3 h according to Mitsuwan et al. [27]. All treatments were performed in a thermostatically controlled water bath with gentle agitation at the specified times and temperatures. Thermal tolerance was assessed by exposing the samples to 100 °C for 3 min to simulate feed pelleting conditions prior to microbial enumeration. TVC, LAP, and BBP were determined in triplicate as described above. Probiotic tolerance (%) was calculated as (N/N₀) × 100, where N is the number of viable cells after treatment and N₀ is the initial cell number in the microcapsules [34]. This calculation provides a quantitative measure of probiotic survival under gastrointestinal conditions and thermal stress during the manufacturing process.

2.2.4. Determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)

The freeze-dried encapsulated powders were added to Mueller-Hinton broth (MHB), and incubated under aerobic conditions at 37 °C for 24 h. The samples were centrifuged at 5000× g for 10 min, followed by filtration using 0.45 µm filters to obtain cell-free supernatant (CFS) [35]. Antimicrobial activity of CFS was investigated using a broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Serial two-fold dilutions of each CFS were prepared in MHB. The bacterial pathogens (B. cereus WU22001, S. aureus ATCC25923, E. coli ATCC 25922, and S. typhimurium WU241001) were tested at a cell density of 10⁵ CFU/mL. The plates were incubated under aerobic conditions at 37 °C for 18 h [36]. Vancomycin and gentamicin (Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls. The MIC was defined as the lowest product concentration that prevented visible growth, while the MBC was the lowest concentration yielding no colony formation upon subculture onto agar [37].

2.2.5. Statistical Analysis

Data were analyzed by one-way analysis of variance (ANOVA) using the General Linear Model (GLM). Where significant differences were detected, treatment means were compared by Tukey’s honest significant difference (HSD) test. The statistical model used was: Yij = μ + τi + εij, where Yij is the response of the ith probiotic strain in the jth replicate, μ is the overall mean, τi is the effect of the probiotic strain, and εij is the random error. Significance was considered at p < 0.05. All statistical analyses were performed using Jamavi version 2.3 [38]. Tukey’s Honestly Significant Difference (HSD) test was applied for multiple comparisons when significant differences were detected (p < 0.05).

3. Result and Discussion

3.1. Experiment 1: Extraction Optimal Conditions for Yanang Leaf Extract for Use as a Natural Medium for P. acidilactici V202 Fermentation

Preliminary results identified the optimal conditions for using Yanang leaf extract as a natural substrate for P. acidilactici V202 fermentation, highlighting that processing parameters significantly influenced the physicochemical and microbial properties (

Table 1. The influence of different extraction conditions of Yanang leaf extract on viable count, pH, and total soluble solids of P. acidilactici V202 fermentation.

Extraction Conditions	pH	TSS (°Brix)	Viable Count (Log CFU/mL)
			PCA (P)	MRS Agar (M)	P–M
Freezing process
Unfrozen	4.52 A	1.98	8.15 B	7.89 B	0.31
Frozen at −20 °C	4.35 B	2.01	8.23 A	8.05 A	0.19
SEM	0.086	0.021	0.012	0.046	0.091
p-value	<0.001	0.363	0.001	0.001	0.216
Blanching process
Unblanched	4.65 a	2.10	7.89 B	7.63 B	0.26
Blanched for 2 min with hot water	4.54 b	2.11	8.12 A	8.10 A	0.02
SEM	0.092	0.021	0.090	0.010	0.001
p-value	0.014	0.496	0.001	0.001	0.001
Preparation process
Unfrozen and unblanched leaves	4.75 a	1.99	8.20 b	8.13 b	0.06
Frozen and unblenched leaves	4.59 b	2.02	8.20 b	8.13 b	0.07
Frozen then blanched leaves	4.70 b	2.00	8.24 a	8.23 a	0.02
Blanched and unfrozen leaves	4.65 b	1.99	8.12 c	8.10 c	0.03
Blanched then frozen leaves	4.80 a	2.02	8.14 c	8.09 c	0.05
SEM	0.069	0.018	0.011	0.012	0.004
p-value	0.039	0.567	0.025	0.019	0.057
Water temperature
Cool temperature at 4 °C	4.65 a	1.99	8.12	8.00 a	0.18 b
Room temperature at 25 °C	4.59 a	2.02	8.16	8.06 a	0.11 b
Hot temperature at 100 °C	4.90 b	2.00	8.10	7.89 b	0.22 a
SEM	0.062	0.039	0.05	0.05	0.028
p-value	0.025	0.612	0.43	0.001	0.024
Yanang leaves: water ratio (w/v)
1:3	4.65 b	2.10	8.14	8.10 a	0.18 b
1:4	4.90 a	2.02	8.01	7.91 b	0.11 b
1:5	4.90 a	2.01	8.00	7.82 b	0.22 a
SEM	0.062	0.021	0.079	0.028	0.014
p-value	0.025	0.427	0.434	0.019	0.024
Sterilization process
Non-sterilization	4.61 B	1.97	8.36 B	8.29 B	0.08
Pasteurization at 75 °C, 5 min	4.27 C	2.03	8.79 A	8.66 A	0.13
Steam sterilization at 100 °C, 10 min	4.86 A	1.97	8.49 B	8.58 A	−0.10
Autoclavation at 121 °C, 15 min	5.07 A	2.00	8.27 B	8.22 B	0.05
SEM	0.093	0.001	0.063	0.061	0.046
p-value	<0.001	0.363	0.007	0.007	0.374

SEM = standard error of the means. ^a,b,c Different superscripts in the same column indicate significant differences (p < 0.05). ^A,B,C Different superscripts in the same column indicate significant differences (p < 0.01).

and Figure 2). Freezing Yanang leaves prior to juice extraction significantly reduced the extract pH compared to unfrozen leaves (p < 0.001). The growth of P. acidilactici V202 on PCA and MRS agar was significantly higher in extracts from frozen leaves (8.23 and 8.05 Log CFU/mL, respectively; p = 0.001), likely due to increased availability of nutrients from disrupted leaf tissues. Blanching for 2 min in hot water decreased pH and probiotic counts on A-B agar, while increasing probiotic counts on PCA and selective agar had no effect on total soluble solids. When combined with freezing, blanching further elevated pH and resulted in the highest probiotic counts on PCA and selective agar, whereas blanching alone produced the lowest values; total soluble solids remained unchanged, and probiotic counts on A-B agar did not decrease. Extraction temperature influenced the leaf characteristics, with hot water yielding the highest pH and probiotic counts on A-B agar but reducing probiotic counts on selective agar relative to cool and room temperature extractions. Total soluble solids and probiotic counts on PCA were unaffected. Leaf-to-water ratio also modulated the results, as a 1:3 ratio produced the highest probiotic counts on selective agar but the lowest pH, whereas higher ratios (1:4 and 1:5) increased pH and particularly enhanced probiotic counts on A-B agar, without affecting total soluble solids or probiotic counts on PCA.

Frozen Yanang leaves prior to juice extraction markedly improved their suitability as a substrate for P. acidilactici V202 fermentation, primarily through the formation of ice crystals that disrupted cellular structures and enhanced the release of soluble sugars, amino acids, and micronutrients [39], which in turn promoted higher probiotic growth compared with fresh extracts. This effect aligns with observations using other plant-based substrates, where freezing enhances the extraction of bioactive and fermentable compounds, and the slight reduction in pH in frozen extracts may further create a favorable environment for LAB [40]. In addition, brief blanching for 1–2 min in hot water provided an effective means to inactivate oxidative enzymes such as polyphenol oxidase and peroxidase, reduce enzymatic degradation, remove surface contaminants, and improve extract stability without significant nutrient loss [41]. By combining freezing with short blanching, the extract benefits from both maximal nutrient release and enhanced stability, producing a nutrient-rich, manageable substrate that supports microbial proliferation and metabolic activity [42]. Moreover, preparing the extract using room temperature (25 °C) water after blanching further preserves heat-sensitive compounds and essential nutrients, contributing to a balanced chemical environment conducive to probiotic growth. Importantly, adjusting the leaf-to-water ratio to 1:3 (w/v) yields a concentrated extract with optimal bioactive compound content, including polyphenols and xylans, which enhances bacterial growth and metabolic activity while maintaining a workable viscosity for efficient fermentation [43].

Collectively, these findings demonstrate that integrating freezing, brief blanching, room temperature extraction, and an optimized leaf-to-water ratio constitutes a minimal processing strategy that effectively balances nutrient availability, enzyme inactivation, extract stability, and fermentability, thereby supporting high-yield and robust P. acidilactici V202 fermentation. This approach not only underscores the importance of pre-treatment conditions in plant-based probiotic substrates but also provides a practical framework for optimizing fermentation processes in functional feed additive applications.

The effects of heat sterilization on Yanang leaf extract are summarized in Table 1. Sterilization significantly affected pH (p < 0.001), with autoclaving producing the highest and pasteurization the lowest, while total soluble solids were unaffected (p = 0.363). Probiotic counts differed among treatments (p = 0.007), with pasteurization supporting the highest viability on PCA (8.79 Log CFU/mL) and selective agar (8.66 Log CFU/mL), and steam sterilization maintaining comparable counts (8.58 Log CFU/mL). Non-sterilized and autoclaved extracts had lower viability, and probiotic balance (A–B) was not significantly affected (p = 0.374). Overall, pasteurization favored probiotic growth, whereas stronger heat treatments altered pH and microbial outcomes. Further, the physical appearance and color of Yanang leaf extract were markedly influenced by pre-fermentation sterilization, showing clear temperature-dependent alterations (Figure 2). The non-sterilized extract (A) retained a bright green, transparent appearance, suggesting intact chlorophyll and minimal thermal degradation. Pasteurization at 75 °C for 5 min (B) caused only slight darkening while maintaining clarity, indicating that moderate heat minimally affects pigment stability. Steaming at 100 °C for 10 min (C) resulted in a darker green with minor turbidity, likely due to partial chlorophyll degradation and protein coagulation. Autoclaving at 121 °C for 15 min (D) produced a pronounced brownish-green color and increased opacity, reflecting extensive pigment breakdown and potential Maillard reactions.

These results demonstrated that the sterilization method markedly influenced the fermentation performance of P. acidilactici V202 and the bioactive properties of Yanang leaf extract. Pasteurization at 75 °C favored the highest probiotic viability, likely because moderate heat preserves nutrients and reduces microbial competition, supporting bacterial growth. In contrast, autoclaving at 121 °C reduced probiotic counts, probably due to degradation of fermentable substrates and formation of inhibitory compounds, but it enhanced antimicrobial activity, potentially through heat-induced release or transformation of phenolic and alkaloid compounds that synergize with organic acids. Steam sterilization at 100 °C provided a balance, maintaining relatively high viability while also improving antibacterial effects. In addition, these results underscore the thermal sensitivity of chlorophyll and other heat-labile phytochemicals in Yanang leaf extract. Mild heat treatments, such as pasteurization, effectively preserve both the visual and functional properties of the extract, making it suitable as a substrate for P. acidilactici V202 fermentation. In contrast, intensive sterilization, particularly autoclaving, accelerates pigment degradation and reduces phytochemical stability, potentially limiting the extract’s capacity to support probiotic growth [44]. These observations align with previous studies demonstrating that higher temperatures and prolonged heating result in greater losses of chlorophyll, phenolic compounds, and antioxidant activity in Yanang extract [45]. Non-sterilized and mildly pasteurized samples exhibited superior color retention and stability, whereas autoclaving markedly diminished both quality and bioactivity [46]. Optimizing pre-fermentation sterilization is crucial for preserving the color, bioactive compounds, and functional properties of Yanang leaf extract, thereby ensuring its effectiveness as a substrate for probiotic fermentation and its potential use in functional foods. Thus, this study demonstrated that aqueous extraction of Yanang leaves using a green solvent approach produces a natural culture medium suitable for LAB. Leaf pre-treatment, extraction temperature, leaf-to-water ratio, and sterilization significantly affect phytochemical content and mucilage functionality. Collectively, these factors determine the extract’s overall quality and underscore its potential as a sustainable, plant-based substrate for probiotic fermentation, with broad applications in food, feed, and biotechnology industries.

3.2. Experiment 2: Probiotic Fermentation Using Yanang Leaf Extract as a Natural Medium and Production of Freeze-Dried Probiotics

Fermentation of Yanang leaf extract with different probiotic strains led to distinct changes in pH, brix, and viable cell counts (

Table 2. pH, total soluble solids, and viable probiotic counts of Yanang leaf extract fermented with different probiotic strains.

Probiotic Strains	pH	TSS (°Brix)	Viable Count (Log CFU/mL)
			PCA (P)	Selective Agar (S)	P–S
P. acidilactici V202	4.02 B	2.07 AB	8.79 A	8.66 A	0.13
L. plantarum TISTR 926	4.06 B	2.10 A	8.67 A	8.59 A	0.08
S. thermophilus TISTR 894	4.01 B	2.00 BC	8.69 A	8.56 A	0.13
B. subtilis RP4-18	5.19 A	1.97 C	8.49 B	8.34 B	0.15
B. licheniformis 46-2	5.11 A	2.00 BC	8.46 B	8.26 B	0.19
SEM	0.147	0.015	0.040	0.043	0.020
p-value	<0.001	0.007	0.009	<0.001	0.511

ND = Not detect. SEM = standard error of the means. TSS = total soluble solids. ^A,B,C Different superscripts in the same column indicate significant differences (p < 0.01).

). Inoculation of Yanang leaf extract with P. acidilactici V202, L. plantarum TISTR 926, and S. thermophilus TISTR 894 significantly lowered the pH to below 4.1, whereas B. subtilis RP4-18, and B. licheniformis 46-2 maintained higher pH values (>5.0; p < 0.01). Total soluble solids decreased across all treatments, with the lowest value observed for B. subtilis RP4-18 and slightly higher levels for L. plantarum TISTR 926 and B. licheniformis 46-2 (~2.00–2.10; p < 0.01). Probiotic viability measured on PCA was highest for LAB (>8.67 Log CFU/mL) and exceeded that of Bacillus strains (<8.50 Log CFU/mL; p < 0.01). Enumeration on selective agar revealed a similar trend, with P. acidilactici V202 reaching 8.66 Log CFU/mL and B. licheniformis 46-2 the lowest at 8.26 Log CFU/mL (p < 0.01). As a result, LAB strains can thrive better than Bacillus strains in Yanang leaf extract. Generally, LAB are more tolerant of acidic compounds and the high tannin content, and better adapt to the readily available fermentable sugars (carbohydrates) that Yanang leaves contain. In contrast, Bacillus strains are often more sensitive to these conditions. Differences between PCA and selective agar counts (P–S) were minimal (<0.20 Log CFU/mL) and not statistically significant (p > 0.05), indicating high recovery efficiency across treatments (Table 2). Colony formation of probiotic bacteria cultured in Yanang leaf extract using a drop plate method are shown in Figure 3. Fungal growth was observed in the non-inoculated control. Among the tested strains, P. acidilactici V202 formed dense and uniform colonies, indicating efficient substrate utilization and robust growth. Although with slightly fewer uniform colonies, L. plantarum TISTR 926 also grew effectively, while S. thermophilus TISTR 894 had moderate growth with smaller colonies, reflecting strain-specific differences in metabolic activity on plant-based substrates. These results clearly demonstrate that LAB can inhibit the growth of other microbes and thrive well on Yanang leaf extract.

These observations are in line with previous reports indicating that plant-derived substrates rich in polyphenols, polysaccharides [9] and other bioactive compounds can selectively stimulate certain probiotic strains while others are less compatible due to variations in enzymatic capacity and tolerance to phytochemicals [47]. The superior growth of P. acidilactici V202 may be attributed to its ability to metabolize complex carbohydrates and withstand the inhibitory effects of plant antioxidants [48]. Studies on fermentation using herbal and plant extracts, including Yanang, suggest that such substrates can enhance probiotic viability and stability, likely through their inherent antioxidant and prebiotic properties [49]. The interaction between substrate and microbial strain is therefore critical, as selecting compatible probiotics and substrates can optimize fermentation performance, viable cell counts, and functional properties of the final product. In addition, lactic acid production during fermentation contributes to a significant decrease in pH, creating conditions unfavorable for many undesirable microorganisms [50]. This acidification promotes selective proliferation of beneficial LAB, enhancing both fermentation efficiency and microbial safety. The inhibitory effects of low pH and undissociated lactic acid disrupt nutrient uptake and enzymatic functions in competing microbes [51], whereas LAB such as P. acidilactici V202 possess adaptive mechanisms that enable survival and growth under acidic conditions. This selective advantage explains the absence of microbial growth in non-inoculated controls and reinforces the potential of LAB-dominated fermentation to suppress pathogenic and spoilage microorganisms. Further, Yanang leaf extract may further enhance this selectivity through its phytochemicals [10], which can act as stressors favoring tolerant strains while inhibiting less-adapted microflora. This combined chemical and microbiological mechanism highlights the dual benefits of LAB fermentation in plant-based substrates. It supports the robust growth of beneficial probiotics and suppresses harmful microorganisms [52], ultimately improving both the functional and safety attributes of Yanang-based fermented feed products. Consequently, the findings of this study indicate that Yanang extract holds promise as a natural culture medium for LAB fermentation, providing a sustainable plant-based alternative to conventional substrates. Its functional properties may enhance probiotic viability, while further investigations are needed to optimize its applications and evaluate broader implications for food, feed, and health-related industries.

Another important aim of this study was to develop a probiotic fermentation product using Yanang leaf extract as a natural substrate, formulated into a freeze-dried feed supplement with rice bran as a carrier. The investigation focused on probiotic viability, product yield, and overall quality. The effects of Yanang leaf extract on the quality attributes of freeze-dried feed supplements are summarized in

Table 3. Probiotic viability, product yield, and overall quality of freeze-dried probiotics fermented in Yanang leaf extract and encapsulated in an extracted rice bran carrier.

Freeze-Dried Probiotic Products	Freeze-Drying Efficiency		Aw	Bulk Density (g/100 mL)	Tapped Density (g/100 mL)	Compressibility of Powder Ratio (%)
	Product Yield (%)	Viability (%)
P. acidilactici V202	20.22	96.71	0.20	24.33	28.33	14.12
L. plantarum TISTR 926	19.93	95.94	0.20	24.67	29.00	14.87
S. thermophilus TISTR 894	20.18	96.70	0.20	24.67	29.33	15.90
B. subtilis RP4-18	20.24	94.18	0.19	25.00	29.33	14.76
B. licheniformis 46-2	20.18	94.16	0.19	24.00	28.33	15.27
SEM	0.130	0.223	0.001	0.165	0.215	0.488
p-value	0.96	0.054	0.511	0.415	0.388	0.878

. Product yield ranged between 19.93% and 20.24%, with no significant differences among treatments (p > 0.05). In terms of probiotic survival, viability remained above 94% for all tested strains, indicating the protective effect of Yanang extract during freeze-drying. The highest viability was recorded for P. acidilactici V202 (96.71%) and S. thermophilus TISTR 894 (96.70%), whereas B. subtilis RP4-18 (94.18%) and B. licheniformis 46-2 (94.16%) exhibited comparatively lower values. Although statistical comparison revealed no significant differences (p = 0.054), the data suggested a trend towards strain-dependent variability in post-drying survival. In addition, Aw values ranged from 0.19 to 0.20, with no significant differences observed among the treatments (p > 0.05), indicating favorable stability for storage. Bulk density values varied between 24.00 and 25.00 g/100 mL, whereas tapped density ranged from 28.33 to 29.33 g/100 mL, with no significant variation across strains (p > 0.05). The compressibility ratio of the powders ranged from 14.12% to 15.90%, suggesting good flowability, with no significant differences among the treatments (p > 0.05).

The stability of the freeze-dried feed supplement containing probiotics encapsulated with Yanang leaf extract in a rice bran carrier was assessed under simulated gastrointestinal and heat stress conditions (Figure 4). Probiotic viability was moderately reduced after pepsin treatment at pH 2.5, indicating a degree of acid tolerance across strains. In contrast, exposure to pancreatin at pH 7 maintained high survival rates, suggesting that the encapsulation system effectively protected cells during simulated intestinal digestion. Heat treatment at 100 °C for 3 min resulted in the greatest reduction in viable counts; however, substantial populations of P. acidilactici V202, L. plantarum TISTR 926, and S. thermophilus TISTR 894 remained detectable, whereas B. subtilis RP4-18, and B. Licheniformis 46-2 exhibited comparatively lower tolerance. These results demonstrate that the developed microencapsulation system conferred both gastrointestinal stability and considerable thermal resistance, supporting its potential application in probiotic feed formulations.

Stereomicroscopy underscored that the freeze-dried feed supplements encapsulated with Yanang leaf extract in a rice bran carrier exhibited a uniform powdery structure with good dispersion (Figure 5). Colorimetric analysis revealed minimal variation among the different probiotic strains, with all values remaining within a narrow range, indicating consistent visual attributes. Both LAB and spore-forming probiotics displayed comparable lightness (L*), redness (a*), and yellowness (b*), resulting in a similar light-yellow appearance primarily derived from the rice bran–yanang mixture. These results suggest that probiotic type had minimal influence on product color, highlighting the stability of the encapsulation system and its potential acceptability for use in poultry diets.

As reflected by reductions in pH and total soluble solids indicative of lactic acid production and substrate utilization, this study demonstrates that Yanang leaf extract serves as an effective natural substrate for probiotic fermentation, supporting the growth and metabolic activity of various probiotic strains, which is in agreement with previous reports on plant-based fermentation systems [52]. Further, probiotic performance was strain-dependent, with LAB such as P. acidilactici V202 and S. thermophilus TISTR 894 demonstrating superior proliferation compared to Bacillus species. This advantage may be attributed to the xylan-rich polysaccharides and mucilaginous constituents of Yanang, which not only provide fermentable carbohydrates but also establish a protective matrix favorable for LAB growth [53]. Its polysaccharide network forms a protective barrier around bacterial cells, reducing ice crystal-induced membrane damage, and improving cell viability post-lyophilization [11,12]. Similarly, mucilage from Aloe vera has been used successfully as a wall material in spray drying to encapsulate probiotics, showing good protection and preserving probiotic viability [54]. Bustamante et al. [55] demonstrated that blending flaxseed mucilage with flaxseed-soluble protein for spray drying encapsulation significantly enhanced the viability of L. acidophilus La-05 cells, providing effective protection during the drying process. Furthermore, encapsulation of Bifidobacterium infantis and L. plantarum using blends of mucilage and soluble protein extracted from chia seeds and flaxseed resulted in sustained high viability after 45 days of refrigerated storage [56]. Similar protective effects have been reported in other mucilage-rich plant extracts, which promote survival of probiotics under gastrointestinal and thermal stress conditions [47,49]. In addition, Fermentation with Yanang leaf not only supported probiotic growth but also influenced colony morphology and viable cell counts, likely due to the presence of bioactive phytochemicals and antioxidants, underscoring its dual role as both a nutrient source and a functional medium. The incorporation of rice bran as a carrier further enhanced probiotic stability and activity by providing additional polysaccharides and reducing sugars essential for bacterial proliferation, acting as a prebiotic and stabilizing matrix, while simultaneously improving the nutritional profile through increased amino acids and vitamins and decreased anti-nutritional factors [57,58]. The combined strategy of freeze-drying and encapsulation proved highly effective in maintaining probiotic viability, with the xylan-rich Yanang mucilage forming a biocompatible matrix [53] that reduces ice-crystal damage and stabilizes cell membranes, and rice bran components supporting survival during both freezing and drying [16]. Consequently, the resulting powders exhibited low Aw value (<0.20) and uniform densities, rendering them suitable for long-term storage and large-scale handling.

The high survival rates observed following freeze-drying, particularly when rapid freezing and suitable encapsulation were applied, highlight the effectiveness of protective strategies in minimizing structural damage to probiotic cells [59]. Although variations were evident between LAB and Bacillus species, survival exceeded 94% immediately post-process, suggesting that strain-specific physiological properties influence tolerance to lyophilization. This observation aligns with previous studies indicating that LAB typically demonstrate enhanced tolerance to dehydration when protected by appropriate cryoprotective and encapsulating agents [60]. Lyophilization, achieved by sublimation of water under low temperature and vacuum, not only preserves cell integrity but also provides long-term stability by reducing susceptibility to oxidative stress and thermal degradation during storage and transportation [61]. These findings emphasize the importance of optimizing both pre-freezing conditions and encapsulation matrices to ensure maximal recovery and functional performance of probiotics in practical applications. Under simulated gastrointestinal conditions, the Yanang–rice bran matrix provided robust protection, particularly against pancreatin, confirming its effectiveness in maintaining probiotic functionality during intestinal transit, and although some reduction in viability occurred under acidic and thermal stress, sufficient populations remained viable, illustrating the synergistic effect of plant-derived mucilage and rice bran in promoting probiotic survival and offering a sustainable, cost-effective alternative to synthetic encapsulants [58]. Thus, this study demonstrates that freeze-drying is an effective microencapsulation strategy for preserving LAB, and that the combined use of Yanang leaf extract and extracted rice bran as a natural matrix offers a promising approach to enhance probiotic viability. Its functional properties may enhance probiotic viability under gastrointestinal stresses and elevated temperatures and support broader applications in animal nutrition and feed industry development.

The supernatant of probiotic fermentation products using Yanang leaf extract as a natural substrate and freeze-dried with rice bran as a carrier was evaluated for antimicrobial activity, specifically determining the MIC and MBC (

Table 4. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against pathogens of freeze-dried probiotic supernatants prepared with Yanang leaf extract in an extracted rice bran carrier.

Item	MIC/MBC (%v/v)
	B. cereus WU22001	S. aureus ATCC25923	E. coli ATCC 25922	S. Typhimurium WU241001
P. acidilactici V202	50/>50	25/>50	25/>50	50/>50
L. plantarum TISTR 926	50/>50	50/>50	25/>50	50/>50
S. thermophilus TISTR 894	50/>50	50/>50	25/>50	50/>50
B. subtilis RP4-18	50/>50	50/>50	50/>50	50/>50
B. licheniformis 46-2	50/>50	50/>50	50/>50	50/>50
Vancomycin, µg/mL	1/2	0.5/1	NA	NA
Gentamicin, µg/mL	NA	NA	0.5/1	1/1

NA = Not applicable. MIC = Minimal inhibitory concentration. MBC = Minimal bactericidal concentration.

). The MIC values against B. cereus WU22001 ranged from 25% v/v (P. acidilactici V202) to 50% v/v (all other strains), while all tested probiotics exhibited MBC values greater than 50% v/v for this pathogen. For S. aureus ATCC25923, the MIC ranged from 25% v/v (P. acidilactici V202) to 50% v/v, with corresponding MBC values also exceeding 50% v/v. Against E. coli 25922, the lowest MIC (25% v/v) was observed for P. acidilactici V202, L. plantarum TISTR 926, and S. thermophilus TISTR 894 while B. subtilis RP4-18, and B. licheniformis 46-2 had MIC values of 50% v/v; MBC values for all probiotics exceeded 50% v/v. In the case of S. typhimurium WU241001, the MIC was 50% v/v for all probiotic strains except L. plantarum TISTR 926, and S. thermophilus TISTR 894 (25% v/v), with MBC values above 50% v/v. Compared with standard antibiotics, vancomycin demonstrated potent activity against Gram-positive bacteria (B. cereus WU22001, and S. aureus ATCC25923) with MIC values of 1 and 0.5 µg/mL, respectively, while gentamicin was effective against Gram-negative bacteria (E. coli ATCC 25922 and S. typhimurium WU241001) with MIC values of 0.5 and 1 µg/mL, respectively.

This study demonstrated that probiotic fermentation products derived from Yanang leaf extract, when formulated as freeze-dried supplements with rice bran as a carrier, exhibit notable antimicrobial activity against livestock-associated pathogens. The MIC and MBC values varied depending on the specific probiotic strain, highlighting their potential as natural bioactive additives for sustainable feed applications. Generally, probiotics show stronger activity against Gram-positive bacteria (S. aureus and B. cereus) than against Gram-negative bacteria (E. coli and S. typhimurium). However, the effectiveness of freeze-dried probiotic products against pathogenic bacteria results from a complex interaction among the probiotic strain, formulation, and target pathogen. As reflected by its lower MIC values compared with other strains, P. acidilactici V202 consistently displayed stronger inhibitory effects on B. cereus WU22001, S. aureus ATCC25923, and E. coli ATCC 25922. This observation aligns with earlier reports indicating that P. acidilactici V202 produces organic acids and bacteriocins such as pediocin, which suppress pathogen growth through acidification and the production of antimicrobial peptides [62]. These findings underscore its superior contribution to the overall antimicrobial efficacy of the system. By contrast, B. subtilis RP4-18, and B. licheniformis 46-2 required higher concentrations to inhibit pathogen growth, suggesting weaker antimicrobial activity, possibly due to differences in metabolic pathways or secondary metabolite production during fermentation. Although inhibitory activity was observed across all formulations, MBC values exceeded 50% v/v, suggesting that the antimicrobial effect was predominantly bacteriostatic rather than bactericidal. This outcome is consistent with known probiotic mechanisms that inhibit pathogen proliferation by lowering pH, competing for nutrients, and producing antimicrobial metabolites, rather than directly inducing cell death [63]. Compared with conventional antibiotics such as vancomycin and gentamicin, which had much lower MIC values in the µg/mL range, the probiotic formulations were less potent. However, their significance lies in their potential to reduce pathogen loads through natural, continuous supplementation without driving antimicrobial resistance, a key advantage over long-term antibiotic use in animal production systems. The use of rice bran as a carrier further strengthened the functional performance of the formulations. Enzymatically extracted rice bran (RB1), enriched in polysaccharides like xylose, has been shown to enhance the growth of beneficial LAB and directly inhibit pathogens such as B. cereus WU22001 and E. coli ATCC 25922 at levels comparable to inulin [54]. Its dual functionality as both a prebiotic substrate and a stabilizing carrier likely contributed to the enhanced stability and antimicrobial performance of the freeze-dried probiotic preparations in this study [64]. Accordingly, these results highlight the potential of combining Yanang leaf extract, probiotics, and rice bran to develop functional feed supplements with antimicrobial activity. While their efficacy is moderate compared with standard antibiotics, their natural origin, prebiotic-probiotic synergy, and protective matrix effects make them well suited for sustainable animal nutrition strategies. Future research should aim to optimize fermentation conditions, characterize the key antimicrobial metabolites involved, and validate the in vivo effects of these formulations in livestock feeding trials to confirm their practical benefits in animal health management.

4. Conclusions

This study demonstrated that Yanang leaf extract is an effective natural substrate for probiotic fermentation. In the first experiment, pre-treatment and sterilization of Yanang leaves, including freezing, blanching, and pasteurization, produced a capable extract that supported the growth of P. acidilactici V202. In the second experiment, fermentation with LAP efficiently reduced pH, maintained high viable counts, and promoted robust colony formation, whereas Bacillus strains had comparatively higher pH and slightly lower probiotic counts. Freeze-dried probiotics prepared using Yanang extract combined with extracted rice bran as a carrier exhibited tolerance to GI conditions and thermal stresses. Moreover, their supernatants demonstrated antimicrobial activity against the concerned pathogenic bacteria in poultry. These results suggest that Yanang-based freeze-dried probiotics have strong potential as functional feed supplements to enhance gut health, improve microbial balance, and reduce pathogen load in livestock.