Phycoerythrin nanoemulsion-enriched probiotic sausage: enhancing Bacillus coagulans viability via Aliinostoc sp. 2 exopolysaccharides

Abstract

Sausage is a widely consumed meat product that provides a favorable environment for the growth of foodborne pathogens due to its nutrient composition. This study aimed to produce a probiotic sausage enriched with 1% (w/w, based on total batter weight) phycoerythrin nanoemulsion and evaluate its effect on the viability of Bacillus coagulans microencapsulated with exopolysaccharides from Aliinostoc sp. 2. The cyanobacterial strain was cultured, and its exopolysaccharides and phycoerythrin pigment were extracted and purified to prepare the nanoemulsion. B. coagulans was microencapsulated, and the minimum inhibitory concentration (MIC) of the nanoemulsion was determined. Sausages were subjected to four treatments: control, 1% phycoerythrin nanoemulsion, B. coagulans (10⁷ cfu/g), and 1% phycoerythrin nanoemulsion + B. coagulans (10⁷ cfu/g). Physicochemical (pH, water activity, moisture, protein), microbial, color, lipid oxidation (TBA, peroxide value), antioxidant (DPPH, ABTS), and sensory analyses were performed on raw and cooked samples. Moisture content in cooked sausages was highest in the 1% nanoemulsion + B. coagulans treatment and lowest in the control, while protein content remained similar across treatments. Probiotic survival at the end of storage increased by approximately 18–22% in sausages with nanoemulsion + B. coagulans compared to B. coagulans alone. Total microbial counts (non-lactic bacteria), mold/yeast, psychrotrophic aerobic bacteria, and Staphylococcus aureus were significantly reduced in the combined treatment, and all samples remained free of coliforms, E. coli, and Salmonella. Color stability (L*, a*, ΔE) was better maintained in the nanoemulsion + B. coagulans treatment, while TBA and peroxide values indicated slower lipid oxidation. Antioxidant activity measured by DPPH and ABTS assays was highest in this treatment. Sensory scores for aroma, flavor, color, texture, and overall acceptability declined over 30 days in all samples but remained significantly higher in sausages with B. coagulans and nanoemulsion. Incorporation of 1% phycoerythrin nanoemulsion based on total batter weight, together with microencapsulated B. coagulans, enhanced probiotic viability, antioxidant capacity, color stability, and sensory quality, without making absolute claims regarding safety. These findings suggest a promising approach for developing functional meat products with improved shelf-life, nutritional quality, and consumer acceptability.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-48610-3.

Introduction

Functional and fortified food products have gained considerable attention in recent years, and probiotics are a central component of this trend. Probiotics are live microorganisms that, when consumed in adequate amounts, provide health benefits to the host. However, incorporating probiotics into complex food matrices such as sausages requires careful consideration of technological properties, stability, and resistance to processing conditions¹. Factors such as antimicrobial compounds, environmental acidity, water activity, oxidation–reduction potential, additives, oxygen concentration, heat treatments, and starter cultures can directly affect probiotic viability^2,3. This is particularly relevant for products like sausages that undergo thermal processing.

One of the main challenges in developing heat-treated probiotic meat products is the significant reduction in microbial viability caused by high temperatures. Common probiotic strains, including Lactobacillus and Bifidobacterium, are highly sensitive to heat and experience considerable population declines during processing, cooling, and storage^4,5. In contrast, Bacillus coagulans is a spore-forming, thermotolerant probiotic, making it a promising candidate for heat-processed foods. Its spores exhibit high resistance to heat, low pH, and environmental stress, and can germinate and multiply in the human gut, distinguishing it from conventional probiotics⁶.

The stability of B. coagulans in complex food systems such as sausages is influenced by thermal conditions, physicochemical characteristics, and the type of carrier used. Therefore, strategies are required to protect this thermally resistant probiotic from processing-induced stress. Microencapsulation using strong cyanobacterial polysaccharides has been identified as an effective approach to maintain both probiotic viability and functional bioactivity⁷.

Microencapsulation protects bioactive compounds against adverse environmental conditions such as oxygen, light, temperature, and moisture, while enabling controlled release at the target site. Food-grade encapsulating materials include polysaccharides (starch, alginate, cellulose, chitosan, gums), proteins (caseinates, gelatin, zein, whey protein), and lipids (fatty acids, waxes, oils)^8,9. Microbial polysaccharides, particularly cyanobacterial exopolysaccharides (EPS), have attracted growing interest due to their structural stability, technological advantages, and biofunctional properties. In 2023, Aliinostoc sp. 2, isolated from rice paddies in Golestan Province, Iran, was identified as a potential source of phycoerythrin and EPS¹⁰.

Cyanobacterial EPS exhibit complex structures, including 40–50 monosaccharides, diverse functional side groups (acyl, sulfate, amino acids), and potential layered or helical spatial conformations. These polymers have technological applications in food, as well as biological functions including antioxidant, antibacterial, anti-inflammatory, anticoagulant, and UV-protective properties¹¹. Cyanobacteria also produce oxygen and other bioactive substances, and their polysaccharides can form stable biofilms, contributing to environmental resilience¹². Among cyanobacterial metabolites, phycocyanin and phycoerythrin pigments are valued for their coloration and functional characteristics. Phycoerythrin is particularly noteworthy for its pigmentation, antioxidant activity, and antibacterial properties, making it suitable for food and pharmaceutical applications. However, these pigments are sensitive to light and heat, limiting their direct use in thermally processed foods. Approaches such as nanoemulsion formation and microencapsulation can enhance pigment stability, functional longevity, and usability in food matrices^13,14.

The consumer demand for probiotic-enriched foods has increased substantially in recent years, with such products comprising 60–70% of the functional food market. The World Health Organization emphasizes that probiotics can improve immune system function, gut health, and overall well-being when properly consumed. Algae-derived products, such as Spirulina, Chlorella, and Dunaliella, may stimulate probiotic growth and provide favorable habitats for their survival. Oligosaccharides from these microalgae also have prebiotic properties that enhance beneficial gut bacteria viability¹⁵.

Despite extensive research on Spirulina-derived phycocyanin, there is limited literature on the use of phycoerythrin obtained from Aliinostoc sp. 2, especially in combination with probiotics in heat-processed meat products. This gap in knowledge represents a valuable opportunity for research and potential innovation in functional meat products¹⁶. Therefore, the present study aimed to evaluate the effects of phycoerythrin nanoemulsion derived from Aliinostoc sp. 2 on the viability of Bacillus coagulans microencapsulated with EPS from the same strain in heat-processed sausages. This method offers a natural alternative to synthetic additives and seeks to enhance probiotic survival, antioxidant properties, and overall product quality. It is hypothesized that the combination of phycoerythrin nanoemulsion and B. coagulans will improve the technological and functional characteristics of the final product while providing additional nutritional and health benefits^17,18.

Materials and methods

Materials

• Microorganisms and Strains

• Aliinostoc sp. 2, Cyanobacteria Culture Collection (CCC), Islamic Azad University, Science and Research Branch, Alborz, Iran

• Bacillus coagulans ATCC 7050, American Type Culture Collection, Manassas, Virginia, USA

• Culture Media and Chemicals

• BG110 medium, Sigma-Aldrich, St. Louis, Missouri, USA

• Phosphate buffer (food grade, pH 7.2), Merck, Darmstadt, Germany

• Sodium phosphate, Merck, Darmstadt, Germany

• Castor oil, Merck, Darmstadt, Germany

• Tween 80, Sigma-Aldrich, St. Louis, Missouri, USA

• PEG-200, Merck, Darmstadt, Germany

• Ethanol 96%, Merck, Darmstadt, Germany

• Tryptic Soy Agar (TSA), HiMedia Laboratories, Mumbai, India

• Peptone water, Merck, Darmstadt, Germany

• Baird-Parker agar, HiMedia Laboratories, Mumbai, India

• VRBG agar, Merck, Darmstadt, Germany

• YGC agar (Yeast Glucose Chloramphenicol), Merck, Darmstadt, Germany

• Lauryl Sulfate broth, Merck, Darmstadt, Germany

• EC broth, Merck, Darmstadt, Germany

• Methanol (analytical grade), Merck, Darmstadt, Germany

• DPPH (2,2-diphenyl-1-picrylhydrazyl), Sigma-Aldrich, St. Louis, Missouri, USA

• ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), Sigma-Aldrich, St. Louis, Missouri, USA

• Potassium persulfate, Merck, Darmstadt, Germany

• Meat and Food Ingredients

• Frozen beef (semimembranosus and biceps femoris muscles), local commercial slaughterhouse, Tehran, Iran

• Frozen bovine fat, local commercial supplier, Tehran, Iran

• Ice (food grade), Local supplier, Tehran, Iran

• Salt (NaCl), Local supplier, Tehran, Iran

• Sodium nitrite, Local supplier, Tehran, Iran

• Sucuk combi seasoning, Wiberg, Austria

• Starter culture (Biobak L), Chr. Hansen, Hørsholm, Denmark

• Equipment

• Growth chamber, Binder, Tuttlingen, Germany

• Magnetic stirrer, Heidolph Instruments, Schwabach, Germany

• Ultrasonicator, Hielscher, Teltow, Germany

• Zetasizer Nano ZS (for DLS and zeta potential), Malvern Instruments, Worcestershire, UK

• Freeze dryer, Labconco, Kansas City, Missouri, USA

• Scanning Electron Microscope (MIRA3-LMU), Tescan, Brno, Czech Republic

• pH meter, Metrohm, Herisau, Switzerland

• Water activity meter, AquaLab, Pullman, Washington, USA

• Colorimeter, Minolta, Osaka, Japan

• Spectrophotometer (UV–Vis), Shimadzu, Kyoto, Japan

Culture of Aliinostoc sp. 2

The cyanobacterial strain Aliinostoc sp. 2 was obtained from the Cyanobacteria Culture Collection (CCC) of the Islamic Azad University, Science and Research Branch (Alborz Herbarium). The strain was cultured, and the purity and axenic status of the cultures were confirmed. Pure cultures were grown on solid BG11₀ medium in a growth chamber at 28 ± 2°C under continuous fluorescent illumination with a light intensity of 300 μmol photons m⁻² s⁻¹ for a period of 30 days¹⁹.

Isolation of exopolysaccharides from Aliinostoc sp. 2

Thirty-day-old cultures were used for exopolysaccharide (EPS) extraction. After separating the biomass using filter paper, the cells were washed with distilled water. Subsequently, ten times the biomass weight of phosphate buffer was added. The mixture was centrifuged at 4000 rpm, and four volumes of 96% ethanol were gradually added to the resulting supernatant. A cream-colored precipitate -representing the polysaccharides- formed over time, and the mixture was refrigerated for 24 h. The precipitate was then collected by centrifugation at 4000 rpm for 10 min, and the obtained exopolysaccharide was isolated according to the method of²⁰. The extracted EPS was subsequently used for microencapsulation of the probiotic bacteria.

Isolation and extraction of phycoerythrin

To extract phycoerythrin, 500 mL of the 14-day-old culture was centrifuged at 4000 rpm, and the pellet was washed with food-grade phosphate buffer (pH 7.2) and then lyophilized. Two grams of freeze-dried biomass were suspended in 500 mL of sodium phosphate buffer (0.1 M, pH 7.2). The phycoerythrin was isolated by freezing and thawing in the dark, by doing the freeze–thaw steps²¹ using − 20 °C as the freezing temperature and the room temperature as the thawing temperature. Subsequently, it was centrifuged at 10,000 × g for 30 min at 0 ± 5°, and the suspension containing phycoerythrin was obtained and lyophilized²². The freeze-dried pigment was stored at − 20 °C until further use.

A UV–Visible spectrophotometer was used to do spectrophotometric analysis in the range of 250–700 nm. The purity and concentration of the pigments were calculated according to the equations described by²³. Maximum absorbance values at 620 nm (PE), 565 nm (PC), and 650 nm (APC) were used for calculations.

Preparation of phycoerythrin nanoemulsion

The nanoemulsion was prepared using castor oil as the oil phase, Tween 80 as surfactant, and PEG-200 as co-surfactant. Phycoerythrin (PE) was incorporated at 0.1%, 0.5%, and 1% (w/v), corresponding to actual pigment concentrations of 0.015, 0.075, and 0.15 mg/mL, respectively. The emulsifier-to-oil ratio was set at 1.5:1 (w/w) and the co-surfactant-to-oil ratio at 0.5:1 (w/w), consistent with previously published nanoemulsion formulations for hydrophilic pigments²⁴.

Initially, the required amount of PE was dissolved in a glass beaker, and the oil, surfactant, and co-surfactant were added. The mixture was gently stirred using a magnetic stirrer and then ultrasonicated to obtain a homogeneous nanoemulsion.

The average particle size and polydispersity index (PDI) of the nanoemulsion were measured using dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, UK). Zeta potential was also determined to assess colloidal stability. The nanoemulsion was found to be stable during storage at 4 °C and under the sausage fermentation and cooking conditions applied in this study, ensuring consistent functional performance in the food matrix²⁴.

Encapsulation of the probiotic bacterium

The probiotic strain Bacillus coagulans (ATCC 7050) was initially adjusted to a 0.5 McFarland standard and cultivated on TSA agar for 24 h. A single colony was subsequently transferred into sterile Ringer’s solution to obtain a fresh bacterial suspension.

Encapsulation was performed using 2 g of freeze-dried polysaccharide dissolved in 98 mL of distilled water. The bacterial suspension was incorporated into the polysaccharide solution under sterile conditions. The resulting encapsulated preparation was transferred into sterile plates and stored at − 20 °C to facilitate the encapsulation process prior to its incorporation into the sausage formulation.

Scanning Electron Microscopy (SEM) analysis

The morphological characteristics and particle size of microcapsules containing 0.5% encapsulated probiotic bacteria were evaluated using scanning electron microscopy (SEM).

The encapsulated samples were initially frozen at − 80 °C for 1 h to stabilize their structure. Subsequently, the frozen samples were transferred to a freeze dryer and lyophilized for 3–4 h to completely remove moisture. This step was performed to prevent structural deformation during SEM analysis under vacuum conditions. After freeze-drying, a small amount of the powdered sample was mounted onto aluminum stubs using double-sided carbon adhesive tape. To improve electrical conductivity and prevent surface charging during imaging, the samples were sputter-coated with a thin layer of gold under vacuum conditions. SEM micrographs were obtained using a MIRA3-LMU scanning electron microscope (USA) operated at an accelerating voltage of 15 kV. Images were captured at different magnifications, including 20,000×. Particle diameters were determined directly from SEM images using the scale bar provided. Measurements were performed on multiple particles located in different regions of the micrographs to obtain a representative size distribution²⁵.

Evaluation of encapsulation efficiency

Encapsulation efficiency (EE%) was determined to quantitatively evaluate the performance of the microencapsulation system. The initial viable cell count of Bacillus coagulans prior to encapsulation was determined using the standard plate count method on TSA agar and expressed as log CFU/mL. Following encapsulation, the microcapsules were centrifuged at 4000 rpm for 10 min to separate free (non-encapsulated) cells from the encapsulated fraction. The supernatant containing free cells was collected and serially diluted for viable cell enumeration. To determine the number of encapsulated cells, the pellet was resuspended in sterile Ringer’s solution and gently homogenized to release entrapped bacteria, followed by serial dilution and plating. Encapsulated Bacillus coagulans microcapsules were stored at − 20 °C prior to incorporation into the sausage formulation.

Encapsulation efficiency was calculated according to the following equation:

All experiments were conducted in triplicate²⁶.

Determination of Minimum Inhibitory Concentration (MIC)

The antimicrobial activity of the phycoerythrin nanoemulsion (PE-NE) was evaluated using the broth microdilution method according to CLSI guidelines (National Committee for Clinical Laboratory Standards, 1997). Serial dilutions of the phycoerythrin nanoemulsion were prepared in appropriate growth medium, and the minimum inhibitory concentration (MIC) was defined as the lowest concentration that inhibited visible growth of Bacillus coagulans after incubation. It should be emphasized that this assay was conducted under in vitro conditions to characterize the intrinsic antimicrobial activity of the pigment. The concentration applied in the sausage formulation referred to the percentage of nanoemulsion incorporated into the meat matrix and did not correspond to the purified pigment concentration used in the MIC assay.

Preparation of sausage and application of treatments

The minimum inhibitory concentration (MIC) of phycoerythrin was previously determined under in vitro conditions against Bacillus coagulans to evaluate the intrinsic antimicrobial activity of the pigment. It should be noted that the concentration applied in the sausage formulation referred to the percentage of phycoerythrin nanoemulsion (PE-NE), not the purified pigment itself²⁷. All MIC determinations were performed in triplicate to ensure reproducibility and reliability of the results. The prepared nanoemulsion contained 0.15 mg/mL of phycoerythrin; therefore, incorporation of 1% (w/v) PE-NE into the sausage matrix resulted in a substantially lower final pigment concentration than the reported MIC value. The selected level was chosen to ensure functional performance in the food matrix while maintaining probiotic viability.

Sausage formulation was produced using 73.5% frozen beef (semimembranosus and biceps femoris muscles from adult cattle, purchased from a local commercial slaughterhouse in Tehran, Iran) and 15.5% frozen bovine fat, which were minced sequentially through grinder plates of 20 mm and 5 mm. These minced components were then mixed with ice (15.5%), The final salt content of the sausage formulation was 1.8% (w/w), sodium nitrite (0.012%), Sucuk combi seasoning supplied by Wiberg (3.7%), and a commercial microbial starter (Biobak L, 0.055%) was used to initiate fermentation, while encapsulated Bacillus coagulans at 10⁷ CFU/g served as a functional probiotic strain²⁸.

After homogenization, the meat emulsion was divided into four treatment groups: Control (without phycoerythrin nanoemulsion and without probiotic) 1: Sausages containing encapsulated Bacillus coagulans at 10⁷ CFU/g, 2: Sausages containing 1% (w/v) phycoerythrin nanoemulsion, 3: Sausages containing both 1% (w/v) phycoerythrin nanoemulsion and 10⁷ CFU/g encapsulated Bacillus coagulans.

The emulsified meat was transferred to a filler and stuffed into 40 mm fibrous casings. Fermentation was carried out at 24–25 °C and 70–80% relative humidity for 24 h, during which the pH decreased from 5.8 at stuffing to 4.9 after 24 h fermentation²⁹. The sausages were then cooked at 68 °C until the core temperature reached 62 °C. Although this core temperature is moderate, product safety was ensured by prior fermentation (pH reduction) and low water activity (0.93–0.95), and confirmed by microbiological analyses showing the absence of Salmonella, E. coli, and coagulase-positive Staphylococcus aureus. The sausages were then cooked at 68 °C until the core temperature reached 62 °C.

After cooking, samples were held at 22–23 °C with 60–70% relative humidity for 24 h to complete maturation (water activity 0.93–0.95). Finally, products were vacuum-packaged and stored at 4 °C for 30 days.

Sampling was performed after stuffing (day 0), 12 h and 24 h post-stuffing, and at the end of maturation. All treatments were analyzed in triplicate at each time point³⁰ (Figs. 1 and 2).

Fig. 1.

Preparation of sausage mixtures for four treatments. (a) Individual spice components used for formulation, (b) Mixing of spices and seasoning into the meat emulsion, (c) Incorporation of salt into the minced meat, (d) Addition of salt for homogenization, (e) Addition of phycoerythrin nanoemulsion to the meat batter, (f) Portioning the meat emulsion into equal pieces for uniform, (g) Shaped sausages ready for casing and fermentation.

Fig. 2.

Depicts the vacuum packaging and cold storage process. (a,b) Stuffed sausages in colored fibrous casings for different treatments. (c) Filling of sausages into the vacuum packaging machine. (d) Vacuum-packed sausages prior to cold storage. (e) Storage of vacuum-packed sausages at 4 °C for maturation and shelf-life study.

The entire sausage production process was performed in one independent batch under controlled conditions, with analytical triplicates at each time point to ensure reliability.

Determination of pH

The pH of the samples was measured according to the National Standard 1028 using a calibrated pH meter³¹. For this purpose, 10 g of each sample was homogenized with 90 mL of distilled water. The pH meter was calibrated with standard buffers at pH 4 and 7 before measurements. Readings were taken at four time points: immediately after stuffing (time zero), 12 h, 24 h post-stuffing, and at the end of the maturation period (24 h after cooking).

Water activity measurement

A water activity meter was used to measure water activity (a_w) according to the Iranian National Standard No. 9657. The measurements were made at the same time points: right after stuffing, 12 h after stuffing, 24 h after stuffing, and at the culmination of the maturation period (24 h after cooking).

Determination of moisture content

The moisture content of the sausage samples was determined according to the Iranian National Standard No. 745. Sample weights were recorded before and after cooking. The samples were then dried in an incubator at 75 °C for 7–8 h until a constant weight was reached. Although AOAC guidelines typically recommend drying temperatures of 100–105 °C for meat products, the lower temperature of 75 °C was intentionally selected to prevent thermal degradation of sensitive components, including phycoerythrin nanoemulsion and encapsulated Bacillus coagulans probiotics, which are critical for the functional properties of the enriched sausages. Drying to constant weight ensured accurate and reproducible determination of moisture content. Measurements were performed at four time points: immediately after stuffing, 12 and 24 h post-stuffing, and at the end of the maturation period (24 h after cooking).

Determination of protein content

Protein content was analyzed using the Kjeldahl method in accordance with Iranian National Standard No. 924. One gram of each sample was mixed with 0.5 g copper sulfate and 4.5 g potassium sulfate as catalysts and digested with 15 mL of 98% sulfuric acid in a Kjeldahl digestion tube. Digestion was carried out at 300 °C for 2.5–3 h until a clear solution was obtained. After cooling, 75 mL of distilled water was added, and the solution was titrated with 0.1 N hydrochloric acid in the presence of a boric acid indicator. The acid volume at the first color change to pink was recorded. Protein analyses were performed at the same four time points: immediately after stuffing, 12 h, 24 h post-stuffing, and 24 h after cooking³².

Protein content (%) was then obtained by multiplying the total nitrogen by a conversion factor of 6.25, which is the standard for meat products (AOAC), to account for the average nitrogen content of proteins.

Microbiological analyses

All microbiological analyses were performed at multiple time intervals: immediately after stuffing (0 h), 12 h and 24 h post-stuffing, at the end of the maturation period (24 h after cooking), and on days 5, 15, and 30 of refrigerated storage. All analyses were conducted in triplicate.

Sample preparation

For each analysis, 10 g of the sample was aseptically weighed in a sterile plastic container and diluted with nine volumes of sterile buffered peptone water. The mixture was homogenized using a stomacher according to Iranian National Standard No. 9899. Subsequently, 1 mL of the initial suspension was aseptically transferred into 9 mL of sterile diluent and mixed using a vortex for 5–10 s to achieve a 10^–2 dilution. Serial dilutions were prepared as needed following the Iranian National Standard No. 1–8923. The detection limit for all microbial enumerations was 1 log CFU/g.

Enumeration of probiotic bacteria (Bacillus coagulans)

Probiotic bacteria were enumerated on TSA agar modified with 0.15% bile salts, sodium acetate, and ammonium citrate to selectively inhibit non-target microorganisms. The medium was adjusted to pH 6.1. This selective medium has been validated for B. coagulans enumeration in previous studies³³. Anaerobic plates were incubated at 37 °C for 48–72 h. Viability after cooking reflects the heat-resistant spores of B. coagulans, ensuring survival through the cooking process.

Total viable counts (non-lactic bacteria)

0.1 mL of the appropriate dilution was plated on PCA medium³⁴, incubated at 37 °C for 48 h, and colonies were expressed as log CFU/g following Iranian National Standard No. 2395, 2018.

Identification and enumeration of pathogenic bacteria

Total coliforms 10 g of sample was inoculated into 90 mL of peptone water, incubated at 37 °C for 18 h, and streaked onto VRBG agar³⁵. Colonies were verified on nutrient agar and reported as log CFU/g (Iranian National Standard No. 2946, 2017).

Coagulase-positive Staphylococcus aureus Enumerated on Baird-Parker agar; colonies confirmed using brain–heart infusion broth with rabbit plasma. A positive coagulase test was indicated by firm clot formation (Iranian National Standard No. 2395, 2018).

Escherichia coli Enumerated using double-strength Lauryl Sulfate broth and EC broth at 44 °C, confirmed with indole test (Iranian National Standard No. 2946, 2007).

Yeast and mold 0.1 mL plated on YGC agar, incubated at 25 °C for 5 days, reported as log CFU/g (Iranian National Standard No. 3-10899, 2018).

Aerobic Mesophilic Bacteria (APC) 0.1 mL plated on nutrient agar, incubated at 22 °C for 72 h, counted as CFU/g.

Salmonella Detection was performed according to Iranian National Standard No. 1810 (2017) using a presence/absence approach in 25 g of sample, including pre-enrichment in buffered peptone water³⁶, selective enrichment in MSRV medium, and confirmation on nutrient agar. Colonies were not reported as CFU/g due to regulatory requirements for food safety.

Color stability analysis of inoculated samples

The inoculated sausages were evaluated at 0 h (immediately after filling the casing), 12 h, 24 h post-filling, the end of the process (24 h after cooking), on days 5, 15 and 30 of refrigeration storage using a colorimeter. Overall color changes were calculated and reported according to the following formula³⁷.

Determination of Thiobarbituric Acid (TBA) value

Lipid oxidation was assessed using the TBA method according to Strange et al. (1977). Samples were reacted with trichloroacetic acid (TCA) and perchloric acid, centrifuged, and the supernatant was combined with TBA solution, then incubated in a hot water bath for 1 h. After cooling, the absorbance was measured at 530–532 nm, and the TBA index was calculated as TEP. Measurements were conducted at 0 h, 12 h, 24 h, at the end of processing, and on days 5, 15, and 30 during refrigerated storage.

Peroxide Value (PV) determination

The main product of lipid oxidation was peroxide, which was measured by the use of hexane to extract the fat. Five times their weight was combined with hexane, which was left at room temperature, 24 h to permit the extraction of oil. It was then filtered, and hexane was evaporated at 50 °C to get the leftover oil that was weighed. In the case of PV measurement, 30 mL of acetic acid-chloroform mixture and 0.5 mL of saturated potassium iodide were put in each 5 g of oil and left to react in darkness after 1 min. Distilled water was then followed by adding an equal amount of water, some drops of starch solution were added, and the mixture was titrated with sodium thiosulfate until the solution became clear. The amount of sodium thiosulfate used was the PV, in milliequivalents per 1000g fat. Measures were conducted at 0 h, 12 h, 24h, end of processing, and days 5, 15, and 30 of refrigerated storage.

Determination of antioxidant activity of inoculated sausages

DPPH method

The antioxidant activity of the sausage samples was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical assay. For extraction, 5 g of each sausage sample was homogenized with 10 mL of methanol:water (80:20, v/v) and shaken at room temperature for 30 min. The mixture was centrifuged at 5000 rpm for 10 min, and the supernatant was collected as the sausage extract. One milliliter of extract was mixed with 1 mL of 0.002% DPPH solution in methanol and incubated in the dark at room temperature for 30 min³⁸. The absorbance was measured at 517 nm using a spectrophotometer. Ascorbic acid was used to prepare a calibration curve (0–50 µg/mL), and the coefficient of determination (R²) was calculated. Antioxidant activity was expressed as IC₅₀ in µg/mL. All measurements were performed in triplicate at 0 h (immediately after filling), 12 h, 24 h, 24 h after cooking, and on days 5, 15, and 30 of refrigerated storage.

ABTS radical cation scavenging assay

ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation scavenging activity was determined as described previously, with modification for sausage extracts³⁹. ABTS stock solution (7 mM) was mixed with 2.45 mM potassium persulfate and incubated in the dark at room temperature for 12 h to generate ABTS⁺ radicals. Sausage extracts were prepared as above. Then, 0.1 mL of extract was added to 0.9 mL of ABTS⁺ solution and vortexed for 45 s. Absorbance was read at 734 nm after 1 min. A calibration curve using ascorbic acid (0–50 µg/mL) was constructed, and R² was reported. Results were expressed as IC₅₀ in µg/mL. Assays were performed in triplicate at all sampling times (0 h, 12 h, 24 h, 24 h post-cooking, and days 5, 15, 30 of storage)⁴⁰.

The percentage radical scavenging activity for both assays was calculated as:

Calibration curves with R² ≥ 0.995 were obtained for both DPPH and ABTS, ensuring accurate determination of IC₅₀ values.

Sensory evaluation

The sensory qualities of the sausage samples were evaluated according to the domestic requirements of Gooshtiran Company. The evaluation was conducted by a trained panel of 30 assessors (15 females and 15 males) aged 28–40 years⁴¹.

All panelists provided written informed consent, and the study protocol was approved by the Ethical Committee of Islamic Azad University-Science and Research Branch, Approval No. IR.IAU.SRB.REC.1404.110.

A 5-point hedonic scale was used, where 1 = very poor, 2 = poor, 3 = acceptable, 4 = good, and 5 = very good. Sensory testing was conducted in a controlled room at 25 °C, with sufficient lighting and absence of external odors.

Measurements were performed at 0 h (after filling casing), 12 h, 24 h, at the completion of cooking (24 h post-cooking), and on days 5, 15, and 30 of refrigerated storage. All evaluations were performed in triplicate for each treatment to ensure reproducibility and reliability of the sensory scores.

Statistical analysis

Experimental data were analyzed using factorial three-way ANOVA to assess the main effects of treatment, processing stage, and storage time, as well as their interactions. All analyses were performed using SPSS software, version 26, at a significance level of p ≤ 0.05. When the overall effect was significant, Duncan’s multiple range test was applied to identify statistically significant differences among mean values.

All treatments were prepared with three independent biological replicates (separate sausage batches), and measurements were performed in triplicate as analytical replicates at each sampling point (0 h raw, 12 h raw, 24 h raw, 24 h cooked, and days 5, 15, and 30 of refrigerated storage).

Prior to ANOVA, data were checked for normality using the Shapiro–Wilk test and for homogeneity of variances using Levene’s test, confirming that assumptions for ANOVA were satisfied.

Results

MIC and spectroscopic analysis of phycoerythrin nanoemulsion

Spectroscopic analysis of the phycoerythrin pigment revealed a maximum absorbance at 568 nm with an absorbance value of 1.63. The concentration and purity of phycoerythrin were determined using appropriate formulas, yielding 0.15 ± 0.001 mg/mL and 0.55 ± 0.02, respectively. The purity value indicates that the nanoemulsion is slightly below the standard threshold for food-grade pigment (0.7)⁴², and minor impurities did not adversely affect the antioxidant, antimicrobial, or color properties.

The yield of phycoerythrin relative to freeze-dried biomass was 3.8%, and the yield of exopolysaccharides (EPS) relative to biomass was 7.5%.

The antimicrobial activity of the phycoerythrin nanoemulsion (PE-NE) against Bacillus coagulans was evaluated using the broth microdilution method according to CLSI guidelines. The average minimum inhibitory concentration (MIC) was calculated as 0.78 ± 0.001 mg/mL (Supp Fig. 1).

It should be noted that the MIC was determined under in vitro conditions using the purified pigment. The concentration applied in the sausage formulation referred to the incorporation of 1% (w/v) of the nanoemulsion, which contains 0.15 mg/mL of phycoerythrin. Therefore, the actual amount of pigment present in the sausage matrix was substantially lower than the MIC determined in vitro, ensuring that the probiotic Bacillus coagulans remained viable while still allowing evaluation of the pigment’s functional properties.

Characterization of phycoerythrin nanoemulsion

Phycoerythrin nanoemulsions were successfully prepared at concentrations of 0.1%, 0.5%, and 1% (w/v), corresponding to actual pigment concentrations of 0.015, 0.075, and 0.15 mg/mL, respectively. The physicochemical properties of the nanoemulsions were evaluated, including particle size, polydispersity index (PDI), and zeta potential, to assess stability (Table 1, Figs. 3 and 4).

Table 1.

Physicochemical characterization of phycoerythrin nanoemulsion at different concentrations.

Sample (w/v)	Z-Average (nm)	PDI	Zeta potential (mV)
0.1%	159.9	0.444	25.4
0.5%	189.9	0.375	22.4
1%	206.2	0.430	18.7

Fig. 3.

Particle size distribution of phycoerythrin nanoemulsions at different concentrations determined by DLS: 0.1% (A), 0.5% (B), and 1% (C) w/v. Particle size increased with higher pigment concentration, while distributions remained relatively narrow.

Fig. 4.

Zeta potential distribution of phycoerythrin nanoemulsions at different concentrations: 0.1% (A), 0.5% (B), and 1% (C) w/v.

The results (Table 1, Fig. 3) indicated that the particle size increased with increasing phycoerythrin concentration. Specifically, the 0.1% formulation exhibited the smallest particle size (159.9 nm), while the 1% formulation showed the largest (206.2 nm). PDI values ranged from 0.375 to 0.444, suggesting moderate homogeneity and confirming the formation of stable nanoemulsions.

Zeta potential measurements revealed a decreasing trend with increasing phycoerythrin concentration. The highest zeta potential was observed at 0.1% (25.4 mV), and the lowest at 1% (18.7 mV), indicating sufficient colloidal stability in all formulations (Table 1, Fig. 4). No visible aggregation or phase separation was observed during storage at 4 °C or after heating at 68 °C for 60 min, demonstrating stability under typical sausage processing conditions.

The stable physicochemical characteristics of the nanoemulsions, as confirmed by DLS and zeta potential analysis, ensure consistent performance in the sausage matrix, contributing to enhanced color retention, antioxidant activity, and antimicrobial effects.

Morphological evaluation of microcapsules

The SEM micrographs (Fig. 5) revealed that the formed microcapsules were predominantly spherical to sub-spherical in shape. The particles exhibited relatively smooth surfaces with slight surface indentations. These surface depressions are likely attributed to the freeze-drying process, during which sublimation of ice crystals may have caused partial shrinkage of the capsule wall structure.

Fig. 5.

Scanning electron micrograph (SEM) of microcapsules containing 0.5% encapsulated probiotic bacteria after freeze-drying. The microcapsules exhibit predominantly spherical to sub-spherical morphology with relatively smooth surfaces and slight surface indentations attributed to the lyophilization process. Particle diameters ranged from 4.21 to 15.25 µm. The image was captured at 20,000 × magnification with a scale bar of 1 µm and an accelerating voltage of 15 kV.

The measured particle size ranged from 4.21 to 15.25 µm. Most of the particles were distributed within the range of approximately 5–10 µm, indicating a relatively uniform particle size distribution. This size range is considered appropriate for food applications, particularly for incorporation into chewing gum formulations, as particles smaller than 20 µm generally do not negatively affect mouthfeel.

The predominantly spherical morphology contributes to improved mechanical stability and enhanced protection of probiotic cells against environmental stresses. Moreover, the absence of significant cracks or structural collapse suggests that the encapsulation and freeze-drying processes effectively preserved the integrity of the capsule wall. Limited particle agglomeration was observed, indicating acceptable process conditions and good dispersion characteristics.

Encapsulation efficiency of probiotic cells

The encapsulation efficiency (EE) of Bacillus coagulans using cyanobacterial exopolysaccharides was determined in triplicate experiments. The highest observed encapsulation efficiency was 73.4 ± 2.1%, calculated based on the viable cell counts before and after encapsulation. This result indicates that the exopolysaccharide matrix effectively entrapped the probiotic cells with minimal loss of viability.

pH evaluation

Statistical analysis, including the three-way interaction (treatment × processing stage × storage time), indicated that the pH of raw sausages generally decreased over the storage period. Among the treatments, sausages containing 1% (w/v) phycoerythrin nanoemulsion combined with Bacillus coagulans (10⁷ cfu/g) exhibited the lowest pH, with statistically significant differences compared to other treatments (p ≤ 0.05).

In cooked sausages, distinct trends were observed. Control sausages and those containing 1% (w/v) phycoerythrin nanoemulsion alone displayed a slight but statistically significant increase in pH over time (p ≤ 0.05). In contrast, sausages treated with Bacillus coagulans (10⁷ cfu/g) or the combination of 1% (w/v) phycoerythrin nanoemulsion + Bacillus coagulans (10⁷ cfu/g) showed a decreasing pH trend throughout storage (p ≤ 0.05). The combination treatment consistently exhibited the lowest pH among all cooked samples.

Notably, the inclusion of the Interaction column in Supp Table 1 highlights the significant combined effect of treatment, processing stage, and storage time (p ≤ 0.05), confirming that the observed pH changes are influenced by both individual factors and their interactions.

Water activity determination

Statistical analysis, including the interaction between treatment and storage time, indicated that at 24 h post-cooking, the water activity of the control and the Bacillus coagulans (10⁷ cfu/g) treatments did not differ significantly (p > 0.05). At Day 30, most treatments also showed no significant differences, except for the combination treatment of 1% (w/v) phycoerythrin nanoemulsion + Bacillus coagulans (10⁷ cfu/g), which exhibited a significantly higher water activity compared to the control (p ≤ 0.05).

The Interaction column in Supp Table 2 highlights that the effect of treatment on water activity depends on storage time, with some treatments showing significant differences in water activity over time (p ≤ 0.05). These results suggest that both treatment and storage time contribute to the observed variations in water activity of cooked sausage samples.

Measurement of moisture content

Statistical analysis, including the three-way interaction (treatment × processing stage × storage time), indicated that the moisture content of raw sausages remained relatively stable over the storage period, with no significant differences among treatments at most time points (p > 0.05).

In contrast, cooked sausages exhibited a marked decrease in moisture content during storage. The lowest moisture content was observed in the control sausage at Day 30, whereas the highest moisture content was recorded in the treatment containing 1% (w/v) phycoerythrin nanoemulsion combined with Bacillus coagulans (10⁷ cfu/g), showing a statistically significant difference compared to the other treatments (p ≤ 0.05) (Supp Table 3).

The Interaction column in Supp Table 3 highlights that the effect of treatment on moisture content depended on both processing stage and storage time, indicating that moisture changes were influenced by the combined effects of these factors (p ≤ 0.05).

Protein content measurement of samples

One-way ANOVA indicated no significant differences in protein content among sausage treatments 24 h post-cooking (p > 0.05), suggesting that incorporation of phycoerythrin nanoemulsion and/or Bacillus coagulans did not affect protein content under the evaluated conditions.

Total probiotic bacteria count and total microorganism count (non-lactic bacteria)

The factorial ANOVA revealed a significant three-way interaction among treatment, processing stage (raw/cooked), and storage time for total probiotic bacteria count (p < 0.05), indicating that changes in probiotic viability depended simultaneously on formulation, processing condition, and storage duration.

In raw sausages containing Bacillus coagulans (10⁷ cfu/g) and 1% (w/v) phycoerythrin nanoemulsion + Bacillus coagulans (10⁷ cfu/g), probiotic counts increased significantly during storage (p < 0.05). No significant difference between these two treatments was observed 24 h after preparation of raw sausages. In cooked samples, although thermal processing initially reduced probiotic counts, a subsequent increase was observed during storage. By day 30, sausages containing B. coagulans alone exhibited significantly higher probiotic counts compared to earlier storage intervals (p < 0.05). The significant interaction effect confirms that the behavior of probiotic bacteria differed depending on whether the product was raw or cooked and on the presence of the nanoemulsion.

For total microorganism count (non-lactic bacteria), factorial ANOVA also showed a significant interaction among treatment, processing stage, and storage time (p < 0.05). In both raw and cooked sausages, total bacterial counts increased over time; however, the rate of increase varied among treatments. The slowest increase was observed in sausages containing 1% (w/v) phycoerythrin nanoemulsion + B. coagulans (10⁷ cfu/g), indicating a potential synergistic antimicrobial effect of the combined treatment. These results suggest that the combination may help maintain microbial stability during storage under the tested conditions (Table 2).

Table 2.

Total probiotic bacteria count and total microorganism count (non-lactic bacteria) (log cfu/g) in probiotic sausages during processing and storage.

Prepared sausage samples	0 h Raw	12 h Raw	24 h Raw	24 h Cooked	Day 5	Day 15	Day 30	Treatment × Stage × Time
Total probiotic bacteria count
Control	0	0	0	0	0	0	0	–
1% (w/v) Phycoerythrin Nanoemulsion	0	0	0	0	0	0	0	–
Bacillus coagulans (107 cfu/g)	5.15 ± 0.02 (aA)	6.25 ± 0.01 (aB)	7.06 ± 0.02 (aD)	6.25 ± 0.01 (aB)	6.86 ± 0.02 (aC)	7.28 ± 0.00 (aE)	6.90 ± 0.02 (aC)	(p < 0.05)*
1% (w/v) Phycoerythrin Nanoemulsion + Bacillus coagulans (107 cfu/g)	5.08 ± 0.01 (bA)	6.22 ± 0.01 (bB)	7.00 ± 0.02 (aC)	6.05 ± 0.01 (bD)	6.56 ± 0.02 (bE)	7.23 ± 0.01 (bF)	6.73 ± 0.05 (bG)	(p < 0.05)*
Total microorganism count (non-lactic bacteria)
Control	4.19 ± 0.01 (aA)	5.25 ± 0.01 (aB)	5.71 ± 0.04 (aC)	3.27 ± 0.00 (aD)	3.80 ± 0.03 (cE)	5.11 ± 0.01 (aF)	6.19 ± 0.01 (aG)	(p < 0.05)*
1% (w/v) Phycoerythrin Nanoemulsion	4.21 ± 0.00 (aA)	4.83 ± 0.02 (bB)	5.35 ± 0.00 (bC)	2.63 ± 0.02 (bD)	3.25 ± 0.00 (aE)	4.30 ± 0.01 (bF)	4.89 ± 0.01 (bG)	(p < 0.05)*
Bacillus coagulans (107 cfu/g)	4.18 ± 0.01 (aA)	5.17 ± 0.01 (aB)	5.57 ± 0.06 (aC)	3.23 ± 0.01 (aD)	3.50 ± 0.02 (bE)	4.58 ± 0.04 (cF)	5.62 ± 0.03 (cG)	(p < 0.05)*
1% (w/v) Phycoerythrin Nanoemulsion + Bacillus coagulans (107 cfu/g)	4.19 ± 0.01 (aC)	4.67 ± 0.02 (cD)	5.04 ± 0.02 (cE)	2.48 ± 0.05 (cA)	3.19 ± 0.01 (aB)	4.23 ± 0.01 (dC)	4.66 ± 0.02 (dD)	(p < 0.05)*

Different lowercase letters within the same column indicate significant differences among treatments (p < 0.05).

Different uppercase letters within the same row indicate significant differences over time (p < 0.05).

*Interaction effect (treatment × processing stage × storage time) was evaluated using factorial ANOVA and was significant at p < 0.05.

According to Iranian National Standard No. 2303, the permissible limit for total bacterial count is 5 × 10³ cfu/g. Based on this standard, raw sausages containing 1% (w/v) phycoerythrin nanoemulsion, B. coagulans (10⁷ cfu/g), and their combination remained within the acceptable microbial limit up to 12 h post-processing, whereas control raw sausages exceeded this limit after 12 h. For cooked sausages, samples containing 1% (w/v) phycoerythrin nanoemulsion + B. coagulans (10⁷ cfu/g), as well as nanoemulsion alone, remained within the permissible microbial limit up to day 30. Cooked sausages containing B. coagulans alone were within the limit up to day 15, whereas control sausages exceeded the allowable limit after day 5. These observations indicate that the combined treatment may contribute to enhanced microbial stability under the tested conditions.

Identification and enumeration of total coliforms, Escherichia coli, and Salmonella

No total coliforms, E. coli, or Salmonella were detected in any sample throughout the 30-day storage period under laboratory-tested conditions.

Identification and enumeration of coagulase-positive Staphylococcus aureus

According to the Iranian National Standard No. 2303, the permissible limit for total bacteria should be less than 1 cfu/g. Based on this standard, raw sausages prepared on the same day and consumed within 12 h, as well as cooked sausages free of coagulase-positive Staphylococcus aureus, were considered suitable for consumption. Sausages prepared more than 12 h before were not accepted.

One-way ANOVA performed 24 h after filling the sausage casings showed that S. aureus counts in sausages containing 1% w/v phycoerythrin nanoemulsion + Bacillus coagulans 10⁷ cfu/g were significantly lower than those in the control group (Fig. 6).

Fig. 6.

Coagulase-positive Staphylococcus aureus counts (Log cfu/g) in Four Treatments of Raw and Cooked Sausages. Lowercase letters indicate significant differences among treatments.

Identification and enumeration of molds, yeasts, and psychrotrophic aerobic bacteria

One-way ANOVA analysis of mold and yeast counts revealed that their numbers increased over time in both raw and cooked sausages. However, this increase was significantly slower in sausages containing 1% w/v phycoerythrin nanoemulsion + Bacillus coagulans 10⁷ cfu/g. According to Iranian National Standard No. 2303, the permissible limit for total bacteria is 2 cfu/g. Therefore, only raw and cooked sausages prepared with 1% w/v phycoerythrin nanoemulsion + Bacillus coagulans 10⁷ cfu/g were acceptable up to 12 h and 30 days post-preparation, respectively. In contrast, raw sausages containing only 1% w/v phycoerythrin nanoemulsion were suitable for consumption for up to 24 h after preparation.

Importantly, analysis of the interaction between treatment, processing stage, and storage time (Treatment × Stage × Time) indicated a statistically significant effect (p < 0.05) on mold and yeast growth, confirming that the combined effect of phycoerythrin nanoemulsion and Bacillus coagulans slowed microbial proliferation more effectively than either treatment alone (Table 3).

Table 3.

Counts of total probiotic bacteria and total microorganisms (non-lactic bacteria) in prepared raw and cooked sausages (log cfu/g) across different treatments and storage times.

Prepared sausage samples	0 h Raw	12 h Raw	24 h Raw	24 h Cooked	Day 5	Day 15	Day 30	Treatment × Stage × Time
Total probiotic bacteria count
Control	0	0	0	0	0	0	0	–
1% (w/v) Phycoerythrin Nanoemulsion	0	0	0	0	0	0	0	–
Bacillus coagulans (107 cfu/g)	5.15 ± 0.02 (aA)	6.25 ± 0.01 (aB)	7.06 ± 0.02 (aD)	6.25 ± 0.01 (aB)	6.86 ± 0.02 (aC)	7.28 ± 0.00 (aE)	6.90 ± 0.02 (aC)	(p < 0.05)*
1% (w/v) Phycoerythrin Nanoemulsion + Bacillus coagulans (107 cfu/g)	5.08 ± 0.01 (bA)	6.22 ± 0.01 (bB)	7.00 ± 0.02 (aC)	6.05 ± 0.01 (bD)	6.56 ± 0.02 (bE)	7.23 ± 0.01 (bF)	6.73 ± 0.05 (bG)	(p < 0.05)*
Total microorganism count (non-lactic bacteria)
Control	4.19 ± 0.01 (aA)	5.25 ± 0.01 (aB)	5.71 ± 0.04 (aC)	3.27 ± 0.00 (aD)	3.80 ± 0.03 (cE)	5.11 ± 0.01 (aF)	6.19 ± 0.01 (aG)	(p < 0.05)*
1% (w/v) Phycoerythrin Nanoemulsion	4.21 ± 0.00 (aA)	4.83 ± 0.02 (bB)	5.35 ± 0.00 (bC)	2.63 ± 0.02 (bD)	3.25 ± 0.00 (aE)	4.30 ± 0.01 (bF)	4.89 ± 0.01 (bG)	(p < 0.05)*
Bacillus coagulans (107 cfu/g)	4.18 ± 0.01 (aA)	5.17 ± 0.01 (aB)	5.57 ± 0.06 (aC)	3.23 ± 0.01 (aD)	3.50 ± 0.02 (bE)	4.58 ± 0.04 (cF)	5.62 ± 0.03 (cG)	(p < 0.05)*
1% (w/v) Phycoerythrin Nanoemulsion + Bacillus coagulans (107 cfu/g)	4.19 ± 0.01 (aC)	4.67 ± 0.02 (cD)	5.04 ± 0.02 (cE)	2.48 ± 0.05 (cA)	3.19 ± 0.01 (aB)	4.23 ± 0.01 (dC)	4.66 ± 0.02 (dD)	(p < 0.05)*

Values are presented as mean ± SE (n = 3). Lowercase letters indicate significant differences among treatments at each time point, while uppercase letters indicate significant differences over storage time within each treatment. The column “Treatment × Stage × Time” shows the interaction effect among treatment, processing stage, and storage time; (p < 0.05)* indicates a statistically significant interaction, whereas “–” indicates no observable interaction.

Similarly, one-way ANOVA of psychrotrophic aerobic bacteria counts demonstrated an overall increase over time in both raw and cooked sausages. However, sausages containing 1% w/v phycoerythrin nanoemulsion + Bacillus coagulans 10⁷ cfu/g showed a significantly slower increase. The interaction among treatment, stage, and storage time was also significant (p < 0.05), indicating that the combination treatment effectively controlled the growth of psychrotrophic aerobic bacteria over the storage period (Table 3).

These results suggest that the incorporation of phycoerythrin pigment in combination with Bacillus coagulans reduced bacterial counts and effectively extended the shelf life of prepared sausages.

Color stability analysis of inoculated samples

One-way ANOVA of the examined treatments indicated that the L* values decreased over time in both raw and cooked sausages. However, sausages containing 1% w/v phycoerythrin nanoemulsion + Bacillus coagulans 10⁷ cfu/g showed a significantly slower decrease, maintaining higher L* values compared to the other treatments. The interaction among treatment, processing stage, and storage time (Treatment × Stage × Time) was significant (p < 0.05), confirming that the combined treatment effectively slowed the lightness reduction (Supp Table 4). Similarly, the a* values, reflecting redness, generally increased over time in both raw and cooked sausages. In sausages with 1% phycoerythrin nanoemulsion + Bacillus coagulans, the increase in a* was slower, especially in cooked sausages, resulting in significantly lower a* values compared to other treatments. The significant interaction (p < 0.05) suggests that the effect of the combined treatment on redness depended on both processing stage and storage time. The b* values, representing yellowness, increased over the storage period, particularly in the combination treatment. This moderate increase in b* contributes to a brighter, more visually appealing color in sausages, which is generally desirable for consumer perception. ΔE increased over time in both raw and cooked sausages. According to established perceptibility thresholds, ΔE values below 1 are not noticeable, while values above 3 are clearly perceptible. The combination treatment showed the slowest increase in ΔE, remaining mostly below the perceptibility threshold during storage, indicating better color stability. Slower decreases in L* and a*, together with moderate increases in b* and reduced ΔE, indicate that the combination treatment helps maintain overall color quality and visual appeal.

Results of thiobarbituric acid (TBA) content and peroxide value (PV) determination

One-way ANOVA revealed that both TBA and PV values increased over time in raw and cooked sausages, reflecting progressive lipid oxidation. However, sausages containing 1% w/v phycoerythrin nanoemulsion + Bacillus coagulans 10⁷ cfu/g exhibited a significantly slower increase in both TBA and PV, maintaining lower values compared to the other treatments throughout the storage period. The interaction among treatment, processing stage, and storage time (Treatment × Stage × Time) was significant (p < 0.05)* for both TBA and PV, indicating that the effect of the combined treatment on lipid oxidation depended on both sausage type (raw vs. cooked) and storage time. This demonstrates that incorporation of phycoerythrin nanoemulsion with Bacillus coagulans effectively delayed lipid oxidation in both raw and cooked sausages (Table 4).

Table 4.

Thiobarbituric acid (TBA, ppm) and peroxide value (meq/kg O₂) in raw and cooked sausages with interaction column.

Parameter	Prepared sausage samples	Raw sausages			Cooked sausages				Treatment × Stage × Time
		0 h	12 h	24 h	24 h	Day 5	Day 15	Day 30
TBA value (ppm)
	Control	0.12 ± 0.00(aA)	0.13 ± 0.00(aB)	0.14 ± 0.00(aC)	0.33 ± 0.00(aD)	0.38 ± 0.00(aE)	0.63 ± 0.00(aF)	0.93 ± 0.00(aG)	(p < 0.05)*
	1% Phycoerythrin NE	0.12 ± 0.00(aA)	0.12 ± 0.00(cA)	0.14 ± 0.00(bB)	0.30 ± 0.00(bC)	0.33 ± 0.00(bD)	0.56 ± 0.00(bE)	0.79 ± 0.00(bF)	(p < 0.05)*
	Bacillus coagulans 107	0.12 ± 0.00(aA)	0.13 ± 0.00(bA)	0.14 ± 0.00(cB)	0.32 ± 0.00(cC)	0.36 ± 0.00(cD)	0.60 ± 0.00(cE)	0.87 ± 0.00(cF)	(p < 0.05)*
	1% Phycoerythrin NE + Bacillus coagulans 107	0.12 ± 0.00(aA)	0.12 ± 0.00(cAB)	0.13 ± 0.00(dB)	0.29 ± 0.00(dC)	0.32 ± 0.00(dD)	0.53 ± 0.00(dE)	0.74 ± 0.00(dF)	(p < 0.05)*
Peroxide value (meq/kg O2)
	Control	0.70 ± 0.03(aA)	0.87 ± 0.07(aA)	0.87 ± 0.02(aA)	1.35 ± 0.03(aB)	1.65 ± 0.03(aC)	2.10 ± 0.06(aD)	2.50 ± 0.06(aE)	(p < 0.05)*
	1% Phycoerythrin NE	0.65 ± 0.03(aA)	0.78 ± 0.03(abA)	0.82 ± 0.03(abA)	1.18 ± 0.04(abB)	1.32 ± 0.02(bB)	1.60 ± 0.06(bC)	1.97 ± 0.03(bD)	(p < 0.05)*
	Bacillus coagulans 107	0.63 ± 0.02(aA)	0.73 ± 0.02(abA)	0.73 ± 0.02(bcA)	1.00 ± 0.06(bB)	1.25 ± 0.03(bC)	1.48 ± 0.04(bD)	1.92 ± 0.06(bE)	(p < 0.05)*
	1% Phycoerythrin NE + Bacillus coagulans 107	0.65 ± 0.03(aAB)	0.62 ± 0.02(bA)	0.68 ± 0.02(cAB)	0.80 ± 0.03(cB)	1.02 ± 0.04(cC)	1.25 ± 0.03(cD)	1.60 ± 0.06(cE)	(p < 0.05)*

Values are presented as mean ± SE (n = 3). Columns are grouped into Raw Sausages (0 h, 12 h, 24 h) and Cooked Sausages (24 h, Day 5, Day 15, Day 30). Lowercase letters indicate significant differences among treatments at each time point, while uppercase letters indicate significant differences over storage time within each treatment. The column Treatment × Stage × Time shows the interaction effect among treatment, processing stage, and storage time; (p < 0.05)* indicates a statistically significant interaction, whereas – indicates no observable interaction.

Results of antioxidant activity in inoculated sausages (DPPH and ABTS assays)

Antioxidant activity decreased over time in control samples, while treatments containing phycoerythrin, particularly in combination with B. coagulans, maintained significantly lower IC50 values throughout storage. Sausages containing 1% w/v phycoerythrin nanoemulsion + Bacillus coagulans 10⁷ cfu/g exhibited the highest antioxidant activity, with significantly lower IC₅₀ values at 24 h in raw sausages and day 30 in cooked sausages compared to other treatments.

The interaction among treatment, processing stage, and storage time (Treatment × Stage × Time) was significant (p < 0.05)* for both assays, indicating that the effect of the combined treatment on antioxidant activity depended on both the type of sausage (raw vs. cooked) and storage time. These results confirm that incorporation of phycoerythrin nanoemulsion with Bacillus coagulans effectively enhanced antioxidant activity throughout the storage period (Table 5).

Table 5.

Antioxidant activity of inoculated raw and cooked sausages based on DPPH (IC₅₀ mg/ml) and ABTS assays.

Parameter	Prepared sausage samples	Raw sausages			Cooked sausages				Treatment × Stage × Time
		0 h	12 h	24 h	24 h	Day 5	Day 15	Day 30
DPPH IC50 (mg/ml)
	Control	67.01 ± 0.20(cA)	71.23 ± 0.39(aB)	73.31 ± 0.41(aC)	85.94 ± 0.40(aD)	88.83 ± 0.43(aE)	93.54 ± 0.48(aF)	97.88 ± 0.52(aG)	(p < 0.05)*
	1% Phycoerythrin NE	39.53 ± 0.12(aA)	40.74 ± 0.07(bAB)	41.71 ± 0.13(bB)	51.15 ± 0.16(bC)	100.99 ± 0.32(bD)	103.63 ± 0.59(bE)	106.77 ± 0.62(bF)	(p < 0.05)*
	Bacillus coagulans 107	66.04 ± 0.19(bC)	63.99 ± 0.31(cB)	61.89 ± 0.29(cA)	72.07 ± 0.16(cD)	72.90 ± 0.29(cDE)	73.92 ± 0.30(cE)	76.05 ± 0.32(cF)	(p < 0.05)*
	1% Phycoerythrin NE + Bacillus coagulans 107	39.74 ± 0.12(aC)	39.19 ± 0.07(dB)	38.13 ± 0.11(dA)	47.76 ± 0.07(dD)	48.12 ± 0.07(dD)	48.56 ± 0.07(dE)	48.86 ± 0.08(dE)	(p < 0.05)*
ABTS (IC50 mg/ml)
	Control	37.74 ± 0.05(bA)	38.68 ± 0.09(aB)	39.78 ± 0.09(aC)	60.77 ± 0.08(cD)	61.89 ± 0.14(cE)	62.38 ± 0.14(cF)	63.90 ± 0.15(cG)	(p < 0.05)*
	1% Phycoerythrin NE	29.71 ± 0.08(aA)	30.17 ± 0.06(bB)	30.42 ± 0.05(bB)	46.74 ± 0.05(aC)	47.11 ± 0.05(aD)	47.44 ± 0.05(aE)	48.22 ± 0.09(aF)	(p < 0.05)*
	Bacillus coagulans 107	37.64 ± 0.08(bB)	37.26 ± 0.05(cAB)	36.94 ± 0.14(cA)	60.08 ± 0.08(bC)	60.46 ± 0.13(bC)	61.57 ± 0.21(bD)	62.55 ± 0.08(bE)	(p < 0.05)*
	1% Phycoerythrin NE + Bacillus coagulans 107	29.86 ± 0.05(aB)	29.62 ± 0.03(dAB)	29.33 ± 0.05(dA)	46.60 ± 0.05(aC)	46.97 ± 0.05(aC)	47.69 ± 0.13(aD)	48.07 ± 0.08(aE)	(p < 0.05)*

Values are presented as mean ± SE (n = 3). Columns are grouped into Raw Sausages (0 h, 12 h, 24 h) and Cooked Sausages (24 h, Day 5, Day 15, Day 30). Lowercase letters indicate significant differences among treatments at each time point, while uppercase letters indicate significant differences over storage time within each treatment. The column Treatment × Stage × Time shows the interaction effect among treatment, processing stage, and storage time; (p < 0.05)* indicates a statistically significant interaction, whereas “–” indicates no observable interaction.

Sensory evaluation

The results of a one-way ANOVA indicated that participants’ acceptance scores for aroma, flavor, taste, color, texture, and overall acceptability significantly decreased over 30 days post-cooking in all treatments. However, on day 30, sausages containing Bacillus coagulans 10⁷ cfu/g and 1% w/v phycoerythrin nanoemulsion + Bacillus coagulans 10⁷ cfu/g exhibited significantly higher scores compared to the other treatments (Fig. 7).

Fig. 7.

Results of Sensory Evaluation in cooked sausages.

Discussion

The present study evaluated the effects of phycoerythrin nanoemulsion derived from Aliinostoc sp. 2 and microencapsulated Bacillus coagulans on physicochemical, microbial, antioxidant, and sensory characteristics of probiotic sausages. Overall, the combined treatment (1% phycoerythrin nanoemulsion + 10⁷ CFU/g B. coagulans) demonstrated improved performance across several quality parameters compared with control and single treatments⁴³.

The phycoerythrin concentration incorporated into the sausage matrix (0.15 mg/mL) was substantially lower than the MIC against B. coagulans (0.78 mg/mL), explaining the absence of inhibitory effects on probiotic viability during storage. Although pigment purity (OD562/OD280 = 0.55) was slightly below the typical food-grade threshold (0.7)⁴², incorporation in nanoemulsified form ensured functional efficacy while maintaining probiotic survival. The actual pigment concentration remained well below inhibitory levels, and no negative impact on antioxidant, antimicrobial, or color properties was observed.

Nanoemulsion characterization indicated moderate homogeneity and colloidal stability. Although particle size and PDI increased slightly at higher pigment concentrations and zeta potential decreased, no phase separation or aggregation occurred during storage (4 °C) or thermal processing (68 °C). Such physicochemical stability is consistent with literature reporting that nanoemulsions can enhance uniform pigment dispersion and functional stability in meat systems^44,45.

Encapsulation efficiency (73.4 ± 2.1%) was within the typical range reported for probiotic encapsulation using biopolymer matrices (70–90%)^44,45, indicating that cyanobacterial exopolysaccharides provided effective physical protection. SEM observations confirmed spherical microcapsules without structural collapse. The protective effect of spore-forming B. coagulans is consistent with previous reports demonstrating improved survival of encapsulated spores under processing and storage conditions⁴⁶.

A significant pH reduction was observed in treatments containing B. coagulans. Given that B. coagulans is a spore-forming lactic acid bacterium, partial germination followed by limited vegetative metabolic activity during refrigerated storage (~ 4 °C) may explain the gradual acidification^47–49. While extensive vegetative growth is unlikely at chilled temperatures, low-level metabolic activity and lactic acid production may contribute to pH decline. Thermal processing may also influence buffering properties of the meat matrix; however, post-cooking differences are more plausibly related to probiotic metabolic activity during storage.

In contrast to the expected water-binding effect of polysaccharides, slightly higher water activity was observed in the combined treatment at later storage stages. This may be associated with microbial metabolic activity generating soluble low-molecular-weight compounds that modify water distribution within the matrix^50,51. The significant treatment × storage interaction supports a systematic biological contribution rather than random variation.

Improved moisture retention in nanoemulsion-containing samples aligns with previous studies indicating that nanoemulsions enhance water-holding capacity and reduce moisture loss in meat systems by strengthening the protein–emulsion network during heating^52–57. Although direct microstructural analysis was not performed, the statistically significant differences observed are consistent with these mechanisms.

No significant differences in total protein content were detected among treatments. Nevertheless, potential non-covalent interactions between meat proteins and cyanobacterial-derived compounds, including phycobiliproteins and polysaccharides, may influence functional properties such as water retention and matrix stability without altering total protein levels^58–60.

The reduction in total non-lactic bacterial counts may reflect the combined antimicrobial contributions of phycoerythrin nanoemulsion and probiotic activity. Although Gram differentiation was not specifically performed, previous reports suggest that phycobiliproteins and probiotic-derived antimicrobial metabolites may exert stronger effects on Gram-positive bacteria than Gram-negative species^61–65. Further targeted studies would be required to confirm differential susceptibility.

Color stability was enhanced in nanoemulsion-treated samples. In addition to antioxidant protection, the intrinsic pigmentation properties of phycoerythrin and improved dispersion within the matrix likely contributed to maintained L*, a*, and ΔE values^64–68. These findings support the technological role of nanoformulated natural pigments in processed meat products.

Lipid oxidation indicators (TBA and peroxide values) increased during storage but remained below typical rancidity thresholds reported for sausages (≈0.5–1.0 mg MDA/kg)^68–74. The combined treatment exhibited slower oxidative progression, consistent with enhanced radical-scavenging activity observed in DPPH and ABTS assays. Lower IC₅₀ values in nanoemulsion-containing treatments suggest sustained antioxidant capacity, whereas slight increases during storage may reflect gradual pigment degradation. Similar protective effects of nanoformulated pigments and encapsulated probiotics have been reported previously⁷⁵.

Sensory scores declined over storage in all treatments, as expected; however, probiotic and combined treatments maintained significantly higher acceptability compared with control samples (p < 0.05). These results are consistent with instrumental findings related to oxidation control, moisture retention, and color stability, supporting the functional contribution of the additives.

Importantly, microbiological findings are interpreted within laboratory-scale conditions and relevant Iranian food safety standards, without extending claims beyond tested parameters. While the combined formulation demonstrated improved stability and probiotic viability, conclusions regarding long-term industrial performance require further validation.

Several limitations should be acknowledged. Only a single concentration of phycoerythrin nanoemulsion and probiotic was evaluated, storage duration was limited, and molecular-level mechanisms were not directly assessed. Future studies should investigate dose–response relationships, long-term industrial stability, metabolite profiling, and performance under commercial packaging conditions⁷⁶.

Conclusion

The incorporation of phycoerythrin nanoemulsion and microencapsulated Bacillus coagulans into sausages effectively enhanced probiotic survival, delayed lipid oxidation, maintained color stability, and improved sensory acceptance during 30 days of storage. Treatments containing both phycoerythrin and B. coagulans showed higher probiotic counts, lower TBARS and peroxide values, better color retention, and higher sensory scores compared to controls. These results suggest that this combination can serve as a promising strategy for producing functional, clean-label meat products.

However, the mechanisms underlying these effects, including bioactive compound release and microbial metabolite activity, require further investigation. Additionally, long-term studies and in vivo validation are necessary to confirm the efficacy and safety of such formulations under industrial processing and real consumption conditions.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, B.N.; methodology, N.E.; software, B.N.; validation, B.N.; formal analysis, B.N. investigation, resources, B.N.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files (S1).

Declarations

Competing interests

The authors declare no competing interests.

Informed consent

A clear statement on informed consent obtained from all the panelists.