Effect of encapsulated Spirogyra sp. extract on small intestinal microbial balance, protein digestibility and performance of broilers

Mulyono Mulyono, Muhammad Yusronridho Riyanto, Vitus Dwi Yunianto and Lilik Krismiyanto

Department of Animal Science, Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang 50275, Central Java, Indonesia
qmulyo@gmail.com

Abstract

This study evaluated the effects of encapsulated Spirogyra sp. extract (EES) on small intestinal microbial balance, protein digestibility and broiler performance. The experiment involved 200 Ross strain broilers, aged 8 days, with an average body weight of 203.7 ± 12.66 grams. A completely randomized design was used with five dietary treatments and four replicates per treatment (each 10 chickens). The treatments included: T0, control diet; T1, diet with 0.9% Spirogyra sp. extract; T2, diet with 0.3% encapsulated Spirogyra sp. extract; T3, diet with 0.6% encapsulated Spirogyra sp. extract; and T4, diet with 0.9% encapsulated Spirogyra sp. extract. Measured parameters included total populations of lactic acid bacteria (LAB) and Escherichia coli, small intestine pH, crude protein digestibility and broiler production performance.

Data were analyzed using analysis of variance (ANOVA) at a 5% significance level, followed by Duncan's multiple-range test when significant differences were detected.

The treatments significantly (p<0.05) increased LAB populations, reduced Coliform populations, lowered small intestinal pH. Additionally, protein consumption, protein digestibility, feed intake and body weight gain increased, while the feed conversion ratio (FCR) decreased. These findings indicate that supplementing broiler diets with 0.6% encapsulated Spirogyra sp. extract enhances health, protein utilization and overall performance.

Keywords: broiler, functional feed, health, Lactobacillus, performance, H/L ratio, Spirogyra sp

Introduction

The intensification of the global broiler industry is necessitated by the escalating consumer demand for poultry meat, placing immense pressure on producers to maximize production efficiency. The economic viability of modern broiler operations is intrinsically linked to key performance indicators, particularly feed efficiency, which is fundamentally governed by the synergistic relationship between gastrointestinal integrity and immune competence (Grigore et al 2025; Xie et al 2024). However, the genetic selection for rapid growth has rendered commercial broiler strains highly susceptible to metabolic disorders, environmental stressors and infectious diseases, posing substantial hurdles to achieving optimal productivity, especially in tropical climates (Oke et al 2024; Bahari et al 2025; Khazali et al 2025).

In response to these challenges and the global legislative push to phase out antibiotic growth promoters (AGPs), research has intensified on natural, sustainable alternatives (Wang et al 2018; Abd El-Ghany and Soyadı, 2020; Rafeeq et al 2022). Among these, phytobiotics, bioactive compounds derived from plants (Rachwał and Gustaw, 2025; Rahman et al, 2022; Lillehoj et al 2018) and algae (Bouafir et al 2025) have emerged as a viable strategy. These compounds exert pleiotropic beneficial effects, including antimicrobial, antioxidant and immunomodulatory activities, which collectively enhance animal health and performance (Farag et al 2025; Urban et al 2025). A primary mechanism of action for phytobiotics is the modulation of the gut microbiota, fostering a eubiotic state characterized by a proliferation of beneficial microorganisms like LAB (Plaza-Diaz et al 2019; Obianwuna et al 2024; Yogalakshmi and Karthi, 2025). A healthy gut ecosystem is paramount, as it reinforces the intestinal barrier, inhibits pathogen colonization through competitive exclusion and improves nutrient digestion and absorption, thereby positively impacting broiler growth and feed utilization (Teodoro et al 2015; Wang et al 2018; Naeem and Bourassa, 2025).

A promising, yet underexploited, source of phytobiotics is the freshwater green macroalga Spirogyra sp., which is abundant in various regions, including Southeast Asia. This alga is a rich reservoir of bioactive phytochemicals, including flavonoids, saponins, tannins and phenols, which have demonstrated potent biological activities (Windyaswari et al 2019; Wizi et al 2022; Yongkhamcha and Buddhakala, 2023). However, a significant limitation hindering the widespread adoption of such phytobiotics is the inherent instability and low bioavailability of their active compounds. These molecules are susceptible to degradation in the harsh acidic and enzymatic environment of the upper gastrointestinal tract, which severely curtails their bio-efficacy at the target sites in the small intestine (Sugiharto et al 2023; Chen et al 2025). This presents a critical knowledge gap and a technological barrier to unlocking the full potential of Spirogyra sp. as a feed additive.

To circumvent these limitations, microencapsulation offers a strategic technological solution. By enveloping the bioactive extract within a protective polymer matrix (e.g., maltodextrin), this technology can shield the compounds from premature degradation and facilitate their controlled and targeted release within the small intestine (Vinceković et al 2021; Gokul et al 2024; Chen et al 2025). This study was therefore designed to investigate the efficacy of an encapsulated Spirogyra sp. extract (EES) on broiler health and performance. We hypothesized that the enhanced stability and intestinal delivery of bioactive compounds via encapsulation would lead to significant improvements in: (i) gut microbial balance and intestinal pH, (ii) protein digestibility and utilization and (iii) overall growth performance (ADG and FCR), compared to both a non-supplemented control and a diet containing a non-encapsulated extract.

Materials and methods

Algal collection and extraction

Fresh Spirogyra sp. green algae (approx 10 kg) was sourced from a local supplier in Kendal, Central Java, Indonesia. The biomass was thoroughly washed and dried in a forced-air oven at 50^oC to a constant weight, after which it was pulverized into a fine powder using a mechanical grinder. The algal powder was then subjected to solvent extraction by macerating it in 96% ethanol at a 1:10 (w/v) ratio. To enhance the extraction efficiency, the suspension was subjected to ultrasonication (50 Hz) at 37^oC for 60 minutes to facilitate cellular disruption and maximize the release of bioactive compounds. The resulting hydroethanolic solution was filtered and the solvent was removed under reduced pressure using a rotary evaporator to yield a viscous, crude Spirogyra sp. extract (ES).

Figure 1. Fresh Spirogyra sp. (A), Spirogyrasp. powder (B) and encapsulated Spirogyra sp. extract (C)

Microencapsulation of the extract

A portion of the crude extract (ES) was prepared for encapsulation. The ES was first solubilized in distilled water (1:3 ratio, v/v). This aqueous solution was then homogenized with a maltodextrin solution (1:5 ratio, v/v) which served as the wall material. The final emulsion was freeze-dried (lyophilized) to produce a stable, powdered encapsulated Spirogyra sp. extract (EES).

Animals, housing and diets

This study used two hundred day-old Ross 308 broiler chicks, unsexed, with an initial body weight of 203.7 ± 12.66 g. The chicks were randomly allocated to one of five feed treatment groups in a completely randomized design. Each treatment group consisted of four replicate cages, with 10 chickens per cage. The broilers were kept in open house cages. Chickens were housed in wire-floored colony cages (100 × 100 × 80 cm) with a height of 80 cm from the floor. Feed and water were provided ad libitum during the 35-day experimental period.

A basal diet was formulated to meet or exceed the nutritional requirements stipulated by the NRC (1994) for the starter (d 8–21) and finisher (d 22–35) phases (Table 1). The five dietary treatments were as follows:

T0 (Control): Basal diet.

T1 (ES 0.9%): Basal diet + 0.9% non-encapsulated Spirogyra sp. extract.

T2 (EES 0.3%): Basal diet + 0.3% encapsulated Spirogyra sp. extract.

T3 (EES 0.6%): Basal diet + 0.6% encapsulated Spirogyra sp. extract.

T4 (EES 0.9%): Basal diet + 0.9% encapsulated Spirogyra sp. extract.

The respective additives (ES and EES) were incorporated into the basal diet.

Performance production and nutrient utilization

Parameters measured included growth rate during the period (days 8-35), feed consumption, mortality and the temperature and relative humidity of the poultry house. Broiler chicks were weighed at 7 days of age and weekly until the end of the 35-day study. Feed consumption was measured daily by subtracting the remaining feed from the total feed provided. Weight gain was measured as the difference between final and initial body weight and feed efficiency was calculated from this value.

Protein intake and crude protein digestibility

Protein intake (g) was calculated by multiplying the average daily feed intake (g) by the protein content of the diet (%). Protein digestibility was assessed using a modified total collection method (Mulyono et al 2021). Twenty broiler chickens (one per replicate) were transferred to individual metabolism cages for a four-day collection period. Crude protein digestibility (CPD) was determined using the total collection method over four days (days 29-32). One chicken per replicate (n=4 per treatment) was housed in individual metabolic cages. The feed was marked with 1% ferric oxide (Fe₂O₃) at the beginning and end of the collection period. Total excreta were collected daily, sprayed with 0.2 N HCl to prevent nitrogen loss and stored. At the end of the collection period, excreta were collected per bird, weighed and dried. The CP content of the diet and dry excreta was determined using the AOAC (1990) official method. Apparent crude protein digestibility (CP) was calculated as: CPD (%) = (CP Intake - CP Excreted)/ CP Intake × 100%

Sample collection and physiological analyses

On day 35, one bird per replicate (n=4 per treatment) was randomly selected for sampling. Blood samples were collected from the brachial vein, after which the birds were humanely euthanized by cervical dislocation.

Intestinal microbiota and ph: Intestinal digesta were aseptically collected from the ileum. For microbial analysis, serial dilutions were plated on de Man Rogosa Sharpe (MRS) agar for Lactic acid bacteria (LAB) and Eosin Methylene Blue (EMB) agar for Coliforms. Plates were incubated under appropriate conditions and colony-forming units (CFU) were counted and expressed as log CFU/g of digesta. The pH of the digesta was measured immediately using a calibrated digital pH meter.

Statistical analysis

Data were analyzed using one-way anova at a 5% significance level via the SPSS software (Version 21). When significant differences were detected, the analysis was followed by Duncan's Multiple Range Test for mean separation.

Results and discussion

Intestinal microbiota and pH modulation

The dietary inclusion of Spirogyra sp. extract, particularly in its encapsulated form (EES), altered the intestinal microbial population and luminal pH of the broiler chickens (p<0.001). The key findings are detailed in Table 2. Supplementation with EES increased the LAB population with increasing doses and concomitantly decreased the Coliform population. The most significant effect was observed in the group receiving 0.9% EES (T4), which showed the highest LAB count (8.08 log cfu/g) and the lowest Coliform count (2.97 log cfu/g).

In line with the shift in microbial populations, small intestinal pH decreased significantly in all supplemented groups compared to the control group. Lower pH values ​​were recorded in the T3 (6.29) and T4 (6.21) treatment groups, indicating a more acidic intestinal environment.

The results clearly demonstrate that the encapsulated Spirogyra sp. extract acts as a potent modulator of the gut ecosystem, fostering a health-promoting environment and inhibiting pathogenic bacteria. This shift is a cornerstone for improving nutrient digestion and overall broiler performance. The observed increase in LAB populations alongside a decrease in Coliforms points to a significant reshaping of the intestinal microbiota. This is likely driven by the rich profile of bioactive compounds, such as flavonoids, in Spirogyra sp., which are known for their selective antimicrobial properties (Thumvijit et al 2013; Bhakta et al 2022; Sruthy et al 2025). These compounds appear to confer a competitive advantage to beneficial commensal bacteria, such as LAB, while suppressing the growth of potential pathogens, such as E. coli (Coliforms). LAB are a primary source of probiotics and are critical for maintaining gut health through various mechanisms, including immunomodulation and the production of antimicrobial metabolites (Mesbahzadeh et al 2018; Ashaolu, 2020). By reducing the pathogenic load, the host is less challenged and the activity of beneficial flora is no longer inhibited by pathogenic competitors (Champa et al 2016; Ashayerizadeh et al 2018).

The proliferation of LAB directly contributes to the acidification of the intestinal lumen. These bacteria ferment carbohydrates into organic acids, primarily lactic acid and short-chain fatty acids (SCFAs) (Feng et al 2019). This resulting decrease in pH creates a self-perpetuating beneficial cycle: the acidic environment is optimal for the growth of LAB but hostile to acid-sensitive pathogens such as Coliforms (Chukwudi et al 2025). Notably, the intestinal pH in the most effective treatment groups (T3 and T4) remained within the optimal physiological range of 5.5-6.6, which is crucial for the function of endogenous digestive enzymes and overall gut homeostasis (Ao et al 2008). The administration of 0.9% encapsulated Spirogyra sp. extract effectively established a healthier intestinal milieu characterized by a favorable microbial balance and an optimal pH, laying a robust foundation for improved nutrient digestibility, enhanced immunity and superior growth performance.

Protein consumption and digestibility

The dietary inclusion of encapsulated Spirogyra sp. extract (EES) had a significant effect (p<0.05) on both protein consumption and the apparent digestibility of protein (Table 3).

A dose-dependent response was observed in protein intake, with the 0.9% EES treatment (T4) resulting in a significantly higher intake (21.32 g) than the control group (20.65 g).

Similarly, protein digestibility was significantly improved by the supplementation. The highest digestibility was observed in the 0.9% EES group (T4; 79.43%), which was statistically superior to the control (73.80%) and the non-encapsulated extract (T1) treatments. The 0.3% and 0.6% EES treatments (T2 and T3) also demonstrated significant improvements over the control group.

Significant increases in protein digestibility and consumption demonstrate the potential of encapsulated Spirogyra sp. extract as a potent modulator of gut function and nutrient utilization. The mechanisms driving these improvements are likely multifactorial, involving the synergistic action of algal bioactive compounds on the gut environment.

Spirogyra sp. is a rich source of phytochemicals, particularly flavonoids, which possess well-documented antioxidant and antimicrobial properties (Guleria et al 2024). These compounds can selectively inhibit enteric pathogens while promoting the proliferation of beneficial lactic acid bacteria. This modulation of the gut microbiota results in a more favorable gut pH, creating an optimal environment for the activity of endogenous digestive enzymes such as proteases, thereby enhancing the breakdown and subsequent absorption of dietary protein (Karabulut et al 2024).

This increased digestibility directly results in greater amino acid bioavailability, essential building blocks for muscle growth and tissue development. This efficient nutrient absorption positively contributes to the overall health and growth of broilers (Abd El-Hady et al 2022; Taylor-Bowden et al 2024; Abdel-Wareth et al 2024).

Most importantly, encapsulation of the extract represents a significant technological advantage. This method protects the bioactive compounds from degradation in the upper gastrointestinal tract, ensuring controlled release and targeted delivery to the small intestine. This improves the stability and bioefficacy of the active ingredients, ensuring a more consistent and potent effect compared to direct application of the non-encapsulated extract (da Silva et al 2024).

Production performance

The effects of supplementing broiler diets with non-encapsulated Spirogyra extract (ES) and encapsulated Spirogyra extract (EES) on main production parameters are presented in Table 4. The dietary treatments had a significant effect (p<0.05) on all measured performance indicators: daily feed consumption, Average Daily Gain (ADG) and Feed Conversion Ratio (FCR).

Specifically, the groups receiving EES at 0.6% (T3) and 0.9% (T4) demonstrated the best performance. These two groups showed significantly higher ADG and significantly lower (more efficient) FCR than the control (T0) and other treatments. Daily feed consumption was also significantly higher in the T3 and T4 groups.

The significant improvements in ADG and FCR confirm that the physiological benefits of EES supplementation directly contribute to enhanced production performance. This finding aligns with previous research indicating that algal additives can positively impact broiler growth and feed efficiency (Sugiharto et al 2022; Ivarsson et al 2025; Madacussengua et al 2025). The superior performance, particularly at the 0.6% and 0.9% EES inclusion levels, is a cumulative result of the improved gut health, immunity and nutrient digestibility discussed in previous sections.

The higher ADG is a direct outcome of enhanced nutrient utilization. With improved protein digestibility, more amino acids were available for muscle deposition and growth. Furthermore, a healthier gut environment reduces the metabolic energy cost associated with fighting subclinical infections, allowing more energy to be partitioned toward productive gain.

This results in a better FCR, which is the most important economic indicator in broiler production. Broilers in the T3 and T4 groups required less feed per unit of body weight gain, indicating a significant increase in feed efficiency. This is due to the bioactive compounds in Spirogyra sp. (e.g., antioxidants), which support digestive function and reduce oxidative stress, allowing birds to utilize feed more effectively (Dias Carrasco et al 2019; Khan et al 2020; Abideen et al 2025). The slight increase in feed consumption in these best-performing groups is likely a positive response; healthier birds with higher growth potential are encouraged to consume more feed to meet their metabolic needs. The encapsulated Spirogyra sp. extract acted as an effective growth promoter, with the 0.6% and 0.9% doses providing the optimal balance of gut modulation and immunostimulation to achieve the best overall production efficiency.

Conclusion

This study conclusively demonstrates that dietary supplementation with encapsulated Spirogyra sp. extract (EES) is a highly effective strategy for enhancing the health, nutrient utilization and overall productive performance of broiler chickens. The inclusion of 0.9% EES proved optimal, yielding the most significant and consistent benefits across all measured parameters. The findings validate that microencapsulation is a crucial technology for protecting and ensuring the targeted delivery of Spirogyra's bioactive compounds, establishing EES as a potent phytobiotic alternative to antibiotic growth promoters in modern poultry production.

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