Abstract
Introduction:
Plant-based beverages are in high demand as dairy alternatives due to dietary restrictions, sustainability concerns, and growing interest in functional foods. However, many non-dairy beverages exhibit poor nutritional profiles and lack essential long-chain omega-3 polyunsaturated fatty acids (LC w-3 PUFAs). Ensuring probiotic viability during processing and storage also poses a significant technological challenge. This study aimed to develop and characterize a functional beverage enriched with omega-3 fatty acids and the probiotic Lactiplantibacillus plantarum NR_115605.1 from Macadamia integrifolia nuts, utilizing alginate-based encapsulation systems.
Methods:
The beverages were prepared by soaking macadamia nuts for three to four hours at different nut-to-water ratios (1:2 and 1:4), followed by thermal treatment, wet milling, filtration, ingredient addition, pasteurization, and homogenization. Omega-3 fatty acids derived from fish by-products were incorporated in both encapsulated and emulsified forms, while probiotic cells were encapsulated in alginate matrices. The physicochemical, nutritional, and microbiological properties of the beverages were evaluated and compared with commercial cow’s milk and almond-based beverages. Probiotic viability was monitored under simulated gastrointestinal conditions and during 15 days of refrigerated storage.
Results:
The highest processing yield was obtained using a 1:4 substrate-to-water ratio. From a nutritional standpoint, the developed macadamia beverages exhibited a comparable protein content to cow’s milk and significantly higher than almond-based alternatives. A single 240 mL serving provided at least 85% of the minimum recommended daily intake of EPA and DHA. Furthermore, L. plantarum demonstrated robust tolerance to simulated gastric and bile environments, maintaining probiotic viability above the generally accepted threshold for probiotic functionality throughout 15 days of refrigerated storage.
Discussion/Conclusion:
The identified critical quality attributes support the technological feasibility of the developed formulation. Overall, this study presents a promising strategy for the development of a non-dairy functional beverage combining high protein content, targeted omega-3 delivery, and probiotic stability through alginate-based encapsulation systems, providing a foundation for future product development and pilot-scale evaluation.
1 Introduction
Dietary patterns have undergone significant changes in recent years, driven by increased consumer awareness of health, sustainability, and ethical considerations. In this context, plant-based diets, including vegan, vegetarian, and flexitarian approaches, have gained considerable global popularity, stimulating the development of innovative plant-based foods with added functional value (; ).
Functional foods enriched with LC ω-3 PUFAs and probiotic microorganisms are of particular technological and nutritional interest. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) play essential roles in cardiovascular health, neurological development, immune modulation, and metabolic regulation (). Recently, enriching plant milks with omega-3 fatty acids has gained traction as a practical way to boost their nutritional value and satisfy the growing demand for functional dairy alternatives (). However, their incorporation into food matrices remains challenging due to their high susceptibility to oxidative degradation, which leads to off-flavors and nutritional losses. Consequently, encapsulation strategies have been extensively investigated to improve oxidative stability, sensory acceptance, and bioavailability of omega-3 fatty acids in food systems (; ).
Probiotics are defined as live microorganisms that confer health benefits when consumed in adequate amounts, provided that they survive food processing, storage, and gastrointestinal conditions, including low pH and bile salts (). Because their clinical efficacy depends entirely on reaching the gut intact, exploring their specific mechanisms of action and expanding their therapeutic applications in human nutrition remains a major focus of functional food design (). Encapsulation using biopolymers such as alginate has proven effective in enhancing probiotic survival, making this approach particularly suitable for functional food applications (; ; ).
While dairy products have traditionally served as the primary vehicles for probiotic delivery, the rapid expansion of the plant-based beverage sector has opened new opportunities for non-dairy probiotic formulations suitable for consumers with lactose intolerance, milk protein allergies, or plant-based dietary preferences (). However, turning plant matrices into acceptable dairy alternatives is often hindered by poor nutrient profiles, unstable flavor behaviors, and processing bottlenecks that restrict consumer adoption (). Among plant-derived raw materials, Macadamia integrifolia nuts represent a promising yet underexplored matrix due to their favorable lipid profile rich in monounsaturated fatty acids, as well as their appreciable protein and dietary fiber content (; ). Nevertheless, macadamia nuts naturally contain negligible amounts of EPA and DHA, limiting their intrinsic omega-3 nutritional value.
To date, no studies have reported the development of macadamia-based beverages simultaneously fortified with omega-3 fatty acids and probiotic microorganisms. Therefore, this study aimed to develop a functional macadamia nut beverage enriched with omega-3 fatty acids derived from fish by-products and the probiotic Lactiplantibacillus plantarum, employing alginate-based encapsulation systems. The nutritional, physicochemical, and microbiological properties of the developed beverages were evaluated and compared with conventional cow’s milk and a commercial almond-based beverage, and probiotic viability was assessed during refrigerated storage.
2 Materials and methods
2.1 Materials
Three batches of dried Macadamia integrifolia (cv. HAES 344) nuts were obtained from the commercial supplier Vivo y Sano in Ciudad del Este, Paraguay, for use in beverage production. Lactobacillus plantarum NR_115605.1 was provided by the Faculty of Sciences and Natural Resources (FACEN–Universidad Nacional de Asunción, Paraguay).
Refined cod liver oil containing 9.56% of EPA and 29.13% DHA was supplied by Omega Sur S.A. (Mar del Plata, Argentina). Commercial cow’s milk (Milkaut®) and almond beverage (La Serenísima®) were purchased from a local supermarket for use as reference products. All reagents used for analytical determinations were of analytical grade. The food-grade materials used for capsule and beverage preparation included calcium chloride and potassium sorbate (KUBO, Argentina); sodium alginate (Química Bolívar, Argentina); carboxymethylcellulose (CMC); stevia (KONY Stevia®, Argentina); sunflower oil (AGD®, Argentina); and chocolate flavoring (Fleibor®, Argentina), all of which were obtained from commercial suppliers in Mar del Plata, Argentina.
2.2 Raw material characterization
The moisture, ash, protein, and fat contents of raw nuts and beverage samples were analyzed according to the . The protein content was calculated using a 5.3 nitrogen-to-protein conversion factor. Total lipids were extracted and quantified using the . Total carbohydrate content was calculated by difference (100 minus the sum of protein, lipid, and ash contents) according to the AOAC method 986.25 (2005).
Fatty acid composition was determined by gas chromatography after methyl esterification of the lipid fraction. In brief, 60 mg of the extracted lipids were mixed with 2 mL of hexane and 0.3 mL of a KOH/methanol reagent to produce fatty acid methyl esters (FAMEs). After vigorous mixing, 2 mL of a NaCl solution and an additional 2 mL of hexane were added. The mixture was left to stand for 5 min, and the upper hexane layer was collected for analysis.
The FAMEs were analyzed using a Shimadzu GC-2010 gas chromatograph (Kyoto, Japan) equipped with a flame ionization detector (set at 260 °C) and an OmegaWax 320 capillary column. A split injector (50:1 ratio) at 250 °C was utilized. The oven temperature program was set as follows: initial temperature of 120 °C held for 20 min, increased to 200 °C at 1 °C/min, then increased to 220 °C at 5 °C/min, and held for a final 20 min. Nitrogen was used as the carrier gas at a constant flow rate of 1 mL/min. Fatty acids were identified by comparison with external standards (Supelco FAME Mix C4–C24) and quantified using GC Solution software (Shimadzu).
Nut-based and commercial beverages were also characterized for moisture, protein, fat, ash, and titratable acidity following standard AOAC methods (2005). Beverage pH was measured using a calibrated pH meter (Hanna Instruments).
Protein yield was calculated as a percentage of the retained protein content using the following equation:
Where Po2 is protein recovered in beverage, and Po1 is protein present in macadamia raw material.
2.3 Physicochemical characterization and oxidative quality of fish oil
The fish oil obtained from fish by-products was characterized. This was done prior to encapsulation. The characterization was according to the American Oil Chemists’ Society (AOCS) official methods. Primary oxidation was evaluated using the peroxide value (PV) (AOCS Cd 8-53, 2009), and secondary oxidation was monitored by determining the p-anisidine value (p-AV) using the AOCS Cd 18-90 (2009) method. Overall oxidative degradation was expressed as the total oxidation (TOTOX) value, calculated as follows: TOTOX = 2 × PV + p-AV. Hydrolytic rancidity was determined via the acid value (AV) (AOCS Ca 5a-40, 2009). A calibrated pycnometer was used to measure relative density at 20 °C, while the Gardner color scale (AOCS Td 1a-64, 2009) was employed to determine color.
2.4 Preparation of omega-3-rich fish oil capsules
To encapsulate the fish oil, sodium alginate was dispersed in distilled water and left overnight at room temperature to ensure complete hydration of the biopolymer. A discontinuous emulsion (10 g) was then prepared with a dispersed oil phase to a continuous phase ratio of 1:3. The emulsion was obtained by homogenizing at medium speed for two minutes using an Ultra-Turrax (Omni Mixer, Germany). The capsules were obtained by dropping the mixture into a 0.1 M CaCl2 solution. Finally, the capsules were dried in an oven at 30 °C for 24 hours, followed by seven days in a desiccator.
The average diameter and shape of the dry, encapsulated beads were determined by measuring 20–25 beads from 10 randomly selected batches using a Canon PowerShot G9 camera attached to a Zeiss Stemi 2000-C binocular loupe and Axiovision measurement software. Sphericity was quantitatively evaluated using the sphericity factor index described by .
To determine the encapsulation efficiency, capsule samples were collected, and the lipid content was analyzed using the Randall method (AOAC Official Method 2003.05 Randall/Soxtec Submersion Method, 2005). The efficiency was then calculated as a percentage of the retained content, as determined by the Randall method, and the initial lipid content used to make the capsules, using the following equation:
where EE is the encapsulation efficiency, Wo1 is the initial amount of oil weighed, and Wo2 is the total encapsulated oil.
2.5 Encapsulation of L. plantarum
The L. plantarum strain was identified using MALDI-TOF MS and gene sequencing. The culture was activated for 24 hours and propagated in Man–Rogosa–Sharpe (MRS) broth at 37 °C under anaerobic conditions. The cells were then harvested by centrifugation at 3,500 rpm for 20 minutes at 4 °C, after which they were washed three times with phosphate-buffered saline (PBS).
To encapsulate the probiotics, a pre-emulsion consisting of 1% xanthan gum and 1% sodium alginate in a 1:3 ratio was mixed with the concentrated bacterial biomass using a high-speed homogenizer in three cycles. Capsules were formed by dripping the mixture into a 0.1 mol/L CaCl2 solution under magnetic stirring at 20 rpm. The probiotic capsules were stored at 4 °C until use.
The viability of the encapsulated probiotics was assessed after 24 and 48 hours, and the capsules (n = 15) were crushed in sterile peptone water (0.5%) before being plated onto MRS agar and Petrifilm™ LAB plates for enumeration.
2.6 Development and formulation of macadamia beverages
The macadamia nuts were sorted to remove any damaged or immature kernels, then washed thoroughly. Beverage production followed the protocol shown in Figure 1. Briefly, 100 g of nuts were soaked in distilled water for three or four hours at a nut-to-water ratio of 1:2 or 1:4 (w/v). After soaking, the nuts were rinsed and thermally treated at 90 °C for 15 min. The soaking water was then discarded. Wet grinding was performed by adding distilled water (ratio 1:5, w/v) and processing in a domestic grinder (Philips®) at medium–high speed for 3 min. The slurry was filtered, then washed with 100 mL of water, and finally filtered again. The solid residue was re-suspended in 100 mL of water, filtered through a muslin cloth, and then combined with the liquid fraction. Carboxymethylcellulose was then added to improve colloidal stability. Sweeteners, preservatives, and flavorings were incorporated before pasteurization, while probiotic capsules and omega-3 were added after thermal treatment. The beverages were pasteurized, homogenized, aseptically packaged in 750 mL bottles, and stored at 4 °C. Four formulations were prepared as described in Table 1.
Figure 1
Table 1
| Sample | Extract % | Stevia % | CMC % | Potassium sorbate % | Chocolate flavoring % | Fish oil % | Probiotic % |
|---|---|---|---|---|---|---|---|
| NC | 98.30 | 0.5 | 0.1 | 0.1 | 1 | 0 | 0 |
| NEP | 97.65 | 0.5 | 0.1 | 0.1 | 1 | *E 0.5 | 0.15 |
| NCP | 97.65 | 0.5 | 0.1 | 0.1 | 1 | **C 0.5 | 0.15 |
| NP | 98.15 | 0.5 | 0.1 | 0.1 | 1 | 0 | 0.15 |
Formulation of Macadamia integrifolia nut beverages.
NC means control nut beverage.
NEP means nut beverage with emulsified oil and probiotics.
NCP means nut beverage with encapsulated oil and probiotics.
NP means nut beverage with probiotics.
*E means emulsified.
**C means encapsulated.
CMC, carboximethylcelullose.
During preliminary trials, a tendency toward cream layer formation and phase separation was observed in the beverage matrix. Therefore, the formulation and homogenization conditions were optimized to improve physical homogeneity and reproducibility of the final beverage. The naturally occurring lipids of macadamia nuts were retained in the formulation and contributed to the nutritional profile of the developed product.
2.7 Probiotic properties
Tolerance to acidic conditions was evaluated by adjusting the pH of MRS broth from 2.0 to 5.0 using concentrated HCl. Probiotic suspensions (106–108 CFU/mL) were inoculated into the broth and incubated at 37 °C for up to 4 hours.
Bile salt tolerance was assessed by supplementing the culture medium with bile salts at concentrations ranging from 0.1% to 0.6% (w/v). The cultures were inoculated at a concentration of 106 CFU/mL and incubated under optimal conditions. Viable cell enumeration was then performed using the plate count method.
The viability of the probiotics in the beverage matrix was also monitored using MRS agar and Petrifilm™ LAB plates.
2.8 Microbiological quality
Microbiological quality during refrigerated storage (4 °C) was evaluated on days 1, 4, and 15, according to the methodology described by . Aerobic mesophilic bacteria were enumerated on Plate Count Agar at 30 °C for 48 hours, lactobacilli on Rogosa Agar at 30 °C for 72 hours, Enterobacteriaceae on Violet Red Bile Glucose Agar at 37 °C for 24 hours, fecal coliforms on Coli-ID Chromogenic Medium at 37 °C for 24 hours, and molds and yeasts on Rose Bengal Agar with Chloramphenicol at 25 °C for five days.
Petrifilm™ plates (3M) were also used to enumerate Escherichia coli, lactic acid bacteria, molds, and yeasts (MY), following the manufacturer’s instructions.
2.9 Statistical analysis
All experiments were performed in duplicate. For physicochemical, nutritional, and microbiological analyses, measurements were carried out in duplicate and results were expressed as mean ± standard deviation. If the coefficient of variation (CV) between duplicate measurements exceeded 5%, the analysis was repeated to improve analytical precision.
Data were analyzed using InfoStat software (). Prior to analysis of variance (ANOVA), model assumptions were evaluated through residual diagnostics, including graphical assessment of residual normality using normal probability (Q–Q) plots and evaluation of variance homogeneity. When significant differences were detected (p < 0.05), treatment means were compared using Duncan’s multiple range test.
3 Results and discussion
3.1 Chemical composition of the raw material
Proximate analysis of macadamia nut batches revealed a low moisture content of 1.62 ± 0.39% and an ash content of 1.28 ± 0.19%. The protein content was 8.01% ± 0.82%, while lipids represented the largest fraction at 69.56% ± 3.96%. Total carbohydrate content accounted for 15.28% ± 3.23%, while dietary fiber content was 4.27% ± 1.36%. These values are consistent with the compositional data reported by the , which confirms the high lipid density and moderate protein content of macadamia nuts.
The fatty acid profile was dominated by monounsaturated fatty acids (MUFAs), representing approximately 70% of the total, primarily oleic and palmitoleic acids (see Figure 2). Among saturated fatty acids, palmitic acid was predominant (approximately 17%), followed by stearic acid. Polyunsaturated fatty acids (PUFAs) were present in lower proportions, with linoleic acid being the main component, followed by arachidonic and linolenic acids. Long-chain omega-3 fatty acids, namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were not detected.
Figure 2
Overall, these results suggest that macadamia nuts are a valuable nutritional raw material characterized by significant protein and fiber content, a favorable MUFA-rich lipid profile, and an absence of endogenous LCω3 PUFAs. This compositional profile supports their suitability as a plant-based matrix for the formulation of functional beverages, for enrichment with probiotic microorganisms, and for the incorporation of omega-3 fatty acids derived from fish oil.
3.2 Preparation and formulation of macadamia nut beverages
Although the production of plant-based beverages generally follows common processing steps, the specific protocol may vary depending on the raw materials used and the intended product’s characteristics (). In this study, a laboratory-scale process was developed with potential industrial scalability in mind. This included soaking, draining, thermal treatment, wet grinding, filtration, fortification, and pasteurization.
Two substrate-to-water ratios (1:2 and 1:4, w/v) were evaluated. While no significant processing issues were encountered between formulations, the 1:4 ratio yielded a notably higher extraction yield (approximately 66 mL/100 g) than the 1:2 ratio (approximately 50 mL/100 g). This difference can be attributed to the hydration and softening of the nut matrix induced by soaking, which facilitates cellular disruption during grinding and improves extract recovery. As expected, increasing the proportion of water enhanced the extraction efficiency.
The thermal treatment step reduced the microbial load and inactivated enzymes, particularly lipases, which compromise lipid stability (). During wet grinding, both water content and processing intensity influenced beverage concentration, in line with previous findings regarding plant-based drinks (). Filtration yielded a smooth extract free of coarse solids.
Macadamia-based beverages can be described as oil-in-water emulsions consisting of oil droplets and protein particles dispersed within an aqueous phase. These emulsions are inherently thermodynamically unstable and therefore susceptible to creaming, flocculation, coalescence, and phase separation (). The stability of plant-based emulsions is mainly influenced by particle size distribution, interfacial properties, and protein solubility ().
Preliminary formulation trials revealed visible phase separation in beverages containing higher oil concentrations, resulting in the formation of two or three distinct phases during refrigerated storage. Therefore, the final formulation was selected based on its visual physical stability, as no visible phase separation or sedimentation was observed during 15 days of storage at 4 °C.
Although these observations indicate acceptable colloidal stability under the evaluated conditions, future studies should include quantitative physical stability measurements, such as sedimentation index determination, particle size analysis, zeta potential evaluation, and rheological characterization.
Hydrocolloid stabilizers are commonly used to improve the physical stability of plant-based beverages. Gellan gum, locust bean gum, guar gum, carrageenan, sodium carboxymethylcellulose, and xanthan gum are among the most widely employed stabilizers in commercial formulations (). In the present study, sodium carboxymethylcellulose was selected because of its thickening and stabilizing properties, which increase the viscosity of the continuous phase and reduce the mobility of dispersed droplets, thereby reducing phase separation ().
Beverage homogeneity was qualitatively evaluated by visual inspection of 10 mL samples during refrigerated storage, and the minimum concentration of stabilizer required to maintain physical stability was selected.
After pasteurization for 15 min, the beverages were cooled to room temperature, enriched with encapsulated probiotics and omega-3 fatty acids, sonicated for 2 min to improve dispersion, bottled, and stored under refrigeration for further analyses.
Finally, chocolate flavor and stevia were incorporated to improve palatability and may contribute to masking marine off-flavors associated with fish-oil fortification; however, sensory validation is required.
3.3 Physicochemical characterization of beverages
The physicochemical properties of the macadamia beverages developed are summarized in Table 2. The crude protein content ranged from 2.12% to 3.18%, with the highest average being observed in beverages prepared with a substrate-to-water ratio of 1:4 and a soaking time of 4 hours. The moisture content ranged from 87.29% to 97.90%, which is consistent with the added aqueous phase during processing. The density values ranged from 0.96 to 0.99 g/mL, and the pH values from 5.5 to 6.6, both within the acceptable range for plant-based beverages. Titratable acidity ranged from 0.015% to 0.1%. Based on these results, the 1:4 substrate-to-water ratio with a 4-hour soaking time was selected for further enrichment due to its favorable protein content and extract yield.
Table 2
| Sample | Protein (%) | Yield protein (%) | Moisture (%) | Density (g/mL) | pH | Acidity (%) |
|---|---|---|---|---|---|---|
| 1:2, 3h | ||||||
| NC | 2.12aA | 26.46 aA | 92.25bB | 0.97aAB | 6.51aB | 0.070aB |
| NEP | 2.40bA | 30.02 bA | 87.77aA | 0.98aA | 6.21aAB | 0.088bC |
| NCP | 2.40bA | 30.02bA | 94.32bAB | 0.97aA | 6.46aB | 0.035aB |
| NP | 2.40bA | 30.2 bA | 92.18bA | 0.99aA | 6.31aB | 0.050bB |
| 1:2, 4h | ||||||
| NC | 2.38aAB | 29.71 aAB | 91.80aAB | 0.98aB | 6.60aB | 0.05aB |
| NEP | 2.91aA | 36.26 aA | 92.22aA | 0.99aA | 6.30aB | 0.07aB |
| NCP | 3.18aB | 39.70 aB | 91.70aA | 0.98aA | 6.50aB | 0.06aC |
| NP | 2.65aA | 33.14 aA | 91.90aA | 0.97aA | 6.46aB | 0.1bC |
| 1:4, 3h | ||||||
| NC | 3.13bB | 39.07bB | 89.45aAB | 0.97aAB | 5.7aA | 0.016aA |
| NEP | 2.86aA | 35.64aA | 92.27aA | 0.96aA | 5.9aA | 0.02aA |
| NCP | 3.0aB | 37.45aB | 97.90bC | 0.98aA | 6.0aA | 0.02aA |
| NP | 2.65aA | 33.10 aA | 87.32aA | 0.97aA | 5.5aA | 0.015aA |
| 1:4, 4h | ||||||
| NC | 2.75aAB | 34.33aAB | 88.11aA | 0.96aA | 6.41aB | 0.017aA |
| NEP | 2.88aA | 35.95aA | 94.02bB | 0.96aA | 6.24aAB | 0.016aA |
| NCP | 3.18aB | 39.70aB | 94.33bBC | 0.98aA | 6.25aAB | 0.018aA |
| NP | 2.92aA | 36.45aA | 87.20aA | 0.99aA | 6.43aB | 0.015aA |
Chemical characterization of nut beverages formulations and soaking varied protocol.
Values are expressed as mean values (n = 2). Different lowercase letters within the same soaking condition indicate significant differences among beverage formulations (p < 0.05), according to Duncan’s multiple range test. Different uppercase letters within the same formulation indicate significant differences among soaking conditions (p < 0.05). NC, control beverage; NEP, beverage containing emulsified fish oil and probiotics; NCP, beverage containing encapsulated fish oil and probiotics; NP, beverage containing probiotics.
3.4 Physicochemical and oxidative characterization of the refined fish oil
The baseline oxidative and hydrolytic quality of the refined fish oil that was utilized for the calcium-alginate delivery core is summarized in Table 3. The batch satisfied the strict safety criteria outlined by international frameworks. The acid value (1.11 ± 0.08 mg KOH/g oil) indicates a well-preserved matrix with low hydrolytic cleavage. In terms of oxidative parameters, the primary hydroperoxides, as determined by the peroxide value (4.50 ± 0.16 meq O2/kg), and the secondary volatile precursors, as determined by the p-anisidine value (8.88 ± 0.21), were both within the regulatory limits set out in the Codex Alimentarius CXS 329-2017 () (≤ 5.0 and ≤ 20.0, respectively). Consequently, the calculated TOTOX index (17.88) remains well below the voluntary GOED guideline standard () of 26.0. Using a thoroughly characterized and uniform lipid batch is crucial for the technological control of our parallel macro-bead formulation trials. Ensuring this specific baseline freshness guarantees the absence of early volatile aldehydes, thereby preventing sensory cross-contamination or masking defects when the batch is integrated into a mild-flavored plant suspension, such as macadamia nut milk. Furthermore, the density and Gardner color scales matched our previously reported regional refining profiles ().
Table 3
| Parameter | Experimental value |
|---|---|
| Acid Value (mg KOH/g oil) | 1.11 ± 0.08 |
| Peroxide Value (meq O2/kg oil) | 4.50 ± 0.16 |
| p-Anisidine Value | 8.88 ± 0.21 |
| TOTOX Value | 17.88 |
| Gardner Color Scale | 7 – 8 |
| Relative Density (g/mL) | 0.918 ± 0.07 |
Physicochemical and oxidative quality parameters of the refined fish oil.
Values are expressed as mean values (n=2) ± sd. The physicochemical and oxidative parameters correspond to the initial characterization of the refined fish oil batch used as the lipid source for the omega-3 encapsulation system applied in different plant-based beverage formulations.
3.5 Characterization and efficiency of LCω3PUFAs alginate beads
As shown in Figure 3, morphological characterization of the dry, encapsulated matrices revealed an average bead size of 3 mm and a sphericity index of 0.97, confirming fully uniform, spherical geometry (). The encapsulation efficiency of fish oil in alginate-based systems was 93.4%, consistent with previous reports (). Recovery of LC ω-3 PUFA in freshly prepared beverages was 96% for fish oil in emulsion and 98% for encapsulated fish oil, exceeding the recoveries reported for free (94%), microencapsulated (59%), and nanoliposomal fish oil (75%) (). These high retention rates are particularly relevant given that incorporating omega-3s into plant milks often causes physical instability and formulation defects if suitable delivery vehicles are not used (). A 240 mL serving of the enriched beverage is estimated to provide 425 mg (emulsion) and 446 mg (encapsulated) of EPA plus DHA, representing approximately 85% and 89% of the recommended daily intake, respectively ().
Figure 3
3.6 Enrichment of beverage
To ensure probiotic viability, L. plantarum was encapsulated before incorporation into the beverage. Viable cell counts performed on Petrifilm confirmed the presence of live bacteria within the capsules and beverage matrix. Probiotic functionality was further assessed under simulated gastric conditions. The strain exhibited high tolerance to acidic media at pH 3 and 4 for up to 4 h, while no colonies were detected at pH 2 (Table 4), confirming that the encapsulation effectively protected the cells during exposure to stomach-like conditions ().
Table 4
| Gastrointestinal stress factor | Testing condition | Probiotic viability (CFU/mL) | Viability (log CFU/mL) |
|---|---|---|---|
| Medium pH (4 h at 37 °C) | pH 2.0 | ND | ND |
| pH 2.5 | 2.18 × 108 | 8.34 ± 0.1 | |
| pH 3.0 | 4.30 × 108 | 8.63 ± 0.1 | |
| pH 3.5 | 4.43 × 108 | 8.65 ± 0.2 | |
| pH 4.0 | 6.75 × 108 | 8.83 ± 0.1 | |
| Bile salts concentration (% w/v) | 0.1 | 2.05 × 106 | 6.31 ± 0.1 |
| 0.2 | 1.12 × 106 | 6.05 ± 0.2 | |
| 0.3 | 5.10 × 105 | 5.71 ± 0.1 | |
| 0.4 | 2.19 × 105 | 5.34 ± 0.1 | |
| 0.5 | 8.00 × 104 | 4.90 ± 0.3 | |
| 0.6 | 5.20 × 104 | 4.72 ± 0.2 |
Viability of Lactiplantibacillus plantarum under simulated gastrointestinal conditions of pH and bile salts concentrations (% w/v) values during 4 h of incubation at 37°C.
ND, Not detected (below the detection limit, <10 CFU/mL). Values are expressed as mean counts. CFU, colony-forming units.
The isolate’s resistance was observed after exposure to acidified media at all pH levels except pH 2, where no colonies were present. No significant differences were observed at pH 3 and 4 compared to the control (pH 7) during the incubation period, which ranged from 0 to 4 hours.
To select isolates with probiotic properties, resistance to bile salts is an important factor for bacterial survival and growth in the gastrointestinal tract (). The results presented in Table 4 indicate that the isolated strain is bile-tolerant, surviving at all tested concentrations (from 0.1% to 0.6%). At a bile salts concentration of 0.3%, critical for screening human probiotics, 90% of L. plantarum strains are resistant. A reduction in the number of viable cells from 106 CFU/mL to 105 CFU/mL within 4 hours is considered to be bile salt tolerance ().
Formulated beverages must contain sufficient numbers of viable bacteria (>106 CFU/ml or g) at the time of consumption to provide the claimed health benefit. Fortification with encapsulated probiotics enables L. plantarum counts to be maintained above the required level. In terms of survival under storage conditions, L. plantarum viability was monitored for 15 days at refrigerated temperatures, yielding positive results. Therefore, it has been demonstrated that this drink contains the necessary levels of microorganisms to have a beneficial effect, and that it is highly tolerant of acidic environments and bile salts, as well as being resilient to storage conditions.
The selected technological parameters played a critical role in preserving product quality throughout processing and storage. Pasteurization (90 °C, 10 min) ensured microbial stabilization of the beverage matrix prior to probiotic addition, while post-cooling incorporation of the encapsulated probiotic and omega-3 ingredients minimized thermal degradation of these bioactive compounds. Under refrigerated storage (4 °C), the beverage maintained probiotic viability above the recommended threshold for functional foods and preserved the targeted EPA and DHA concentrations, demonstrating the suitability of the selected processing conditions.
The possible release of probiotic cells from the calcium-alginate beads into the beverage matrix was not specifically quantified in this study. However, no visible deterioration of the beads was observed during refrigerated storage, suggesting that the encapsulation structure remained physically stable under the evaluated conditions. Future studies should include a quantitative assessment of probiotic release kinetics and the distribution of free and encapsulated cells during storage.
3.7 Microbiological quality
The microbiological quality of plant-based beverages affects both their shelf life and the biochemical changes that occur during storage (). The composition of the beverages, coupled with a pH close to neutral, could encourage microbiological growth. Therefore, it is necessary to apply preservation treatments to maintain quality over time. In the current study, the pH value ranged from 5.5 to 6.5, creating an environment ideal for molds and yeasts. Heat treatment not only extends the shelf life of plant-based beverages but has also been shown to improve taste and overall acceptability ().
The samples were analyzed for the microbial groups most commonly found in this type of product. A microbiological count of 3 CFU was found for the MS samples on the first day after the drinks were made. Total coliforms and E. coli are often used as indicators of hygiene in food handling environments (). EB, FC, and E. coli were not detected in any of the samples. MY was 1 UFC after four days, and LB counts were ≥100 and ≥300 CFU after one and four days, respectively. After 15 days of storage, none of the indigenous microbiota populations exceeded the maximum limit for microorganisms in minimally processed foods as set out in Spanish legislation ().
The macadamia nut beverage developed in this study exhibited a protein content comparable to commercial cow’s milk and substantially higher than that of the analyzed commercial almond-based beverage (Table 5). The USDA reports a reference protein value of 3.28 g/100 g for cow’s milk, which is slightly higher than the values obtained in the present study. Although protein quality and biological value were not experimentally assessed, literature data allow a preliminary comparison of the protein fraction. Macadamia proteins are characterized by a relatively balanced amino acid composition but contain tryptophan as the first limiting amino acid relative to human requirements (). Consequently, their biological value is expected to be lower than that of dairy proteins, which are recognized as high-quality proteins with excellent digestibility and amino acid balance (). Nevertheless, macadamia proteins are particularly rich in arginine and exhibit a low lysine-to-arginine ratio, characteristics that have been associated with cardiovascular benefits and favorable lipid metabolism (; ). Therefore, while the developed beverage may not match the protein quality of dairy milk, it provides a nutritionally relevant plant protein source with potential functional advantages.
Table 5
| Parameter | 1:4 4h NEP | 1:4 4h NCP | Cows milk | Almond milk |
|---|---|---|---|---|
| Protein% | 2.88b | 3.18bc | 2.90b | 0.48a |
| Fat% | 1.17a | 0.99a | 1.00a | 1.10a |
| Carbohydrates% | 1.12a | 0.91a | 4.39b | 0.34a |
| Ash% | 0.56b | 0.41a | 0.75c | 0.58b |
| Fiber% | 0.25b | 0.18ab | 0.00 | 0.13a |
| Moisture% | 94.02b | 94.33b | 90.96a | 97.37c |
| Energy Kcal/100 mL | 26.53b | 25.27b | 38.16c | 13.18a |
| Energy Kcal/240 mL | 63.67b | 60.65b | 91.58c | 31.63a |
Chemical characterization of macadamia nut developed beverage, commercial cow’s milk and almond drinks.
Values are expressed by mean value (n=2). The same letter in the row means values of each beverage are not different by the Duncan test (p < 0.05).
ait means the minor value, cit means the major value.
NEP sample with emulsified oil and probiotics.
NCP sample with encapsulated oil and probiotics.
Values are expressed as mean value (n = 2). Means in the same row with different superscript letters (a, b, c) are significantly different according to Duncan’s test (p < 0.05), where 'a' indicates the lowest value, 'b' indicates intermediate values, and 'c' indicates the highest value. NEP: beverage with emulsified oil and probiotics; NCP: beverage with encapsulated oil and probiotics.
Although the protein concentration of the developed macadamia beverage was comparable to or higher than that reported for several commercial plant-based beverages, a complete nutritional assessment would require determination of amino acid composition, protein digestibility, and protein quality indices such as PDCAAS or DIAAS. Soy and dairy proteins generally present a more balanced essential amino acid profile and higher biological value than most tree nut proteins. Therefore, further studies are required to fully characterize the nutritional quality of the protein fraction in the developed beverage.
Despite the naturally high lipid content of macadamia nuts (69.56%), the final beverage contained approximately 1% total lipids. During processing, a cream layer formed due to phase separation of part of the endogenous oil fraction, and processing conditions were adjusted to improve beverage homogeneity and formulation reproducibility. Similar reductions in lipid content have been reported for other nut-based beverages, including Brazil nut beverages (). Even after omega-3 fortification, the lipid concentration remained low and no significant differences were observed between enriched formulations and the non-enriched control beverage (NC, 1:4, 4 h), which contained 1.01% lipids. These results suggest that omega-3 enrichment can improve the nutritional profile of plant-based beverages without substantially increasing total fat content.
The most pronounced compositional differences among the beverages were observed for carbohydrate content. The macadamia beverages contained lower carbohydrate levels than cow’s milk but higher levels than the analyzed almond beverage. This observation agrees with previous reports indicating that carbohydrate concentrations in plant-based beverages can vary considerably depending on the botanical source and formulation strategy (). In addition, the dietary fiber content of the formulated macadamia beverages exceeded that of the commercial beverages evaluated. According to USDA reference values, macadamia nuts contain approximately 12.5 g/100 g dietary fiber, whereas fiber is negligible in cow’s milk ().
From an energy perspective, a 240 mL serving of the developed macadamia beverage provides approximately 60 kcal while supplying a higher protein concentration than many commercially available nut-based beverages, which typically contain between 0.3 and 2.2% protein (; ).
3.8 Quality framework, critical process parameters, and technological risk assessment
To provide a technological perspective on the development of the macadamia beverage, the main processing stages potentially affecting product safety, stability, and functional quality were identified, and their associated technological risks and mitigation strategies are summarized in Table 6.
Table 6
| Processing stage | Potential risk | Impact on final product | Mitigation strategy |
|---|---|---|---|
| Raw material handling | Microbial contamination and lipid deterioration | Reduced safety and quality | Selection of good-quality raw materials and appropriate storage conditions |
| Thermal treatment | Insufficient microbial reduction | Reduced microbiological safety | Controlled pasteurization conditions |
| Incorporation of omega-3 | Lipid oxidation | Development of off-flavors and loss of nutritional quality | Encapsulation and reduced exposure to heat, oxygen and light |
| Probiotic incorporation | Loss of viable cells | Reduced functional efficacy | Addition after cooling and refrigerated storage |
| Homogenization and storage | Phase separation, sedimentation, possible structural instability of encapsulated systems | Loss of physical stability and product acceptability. Possible alteration of bioactive protection | Optimization of stabilizer concentration, mild mixing conditions and refrigerated storage |
Critical processing stages, potential technological risks and mitigation strategies during macadamia beverage production.
3.8.1 Critical processing parameters
Two-unit operations were identified within the manufacturing line as critical processing checkpoints that directly govern product safety and functional efficacy:
Thermal pasteurization (90 °C for 10 min): Thermal treatment represents a key processing step for reducing the microbial load and improving the microbiological quality of the beverage satisfying international processing safety guidelines (). Additionally, heating contributes to the inactivation of endogenous enzymes and anti-nutritional compounds naturally present in the macadamia matrix.
Aseptic Cold Filling and Storage (4 °C): The incorporation of probiotics and omega-3 ingredients after cooling, followed by refrigerated storage at 4 °C, represents another critical processing stage to prevent thermal damage to bioactive compounds and minimize post-processing contamination ().
3.8.2 Technological risk assessment
Distinct from biological safety boundaries, the manufacturing of a dual-fortified matrix entails multiple structural vulnerabilities regarding compound preservation (Table 6). The primary technological risks involve lipid auto-oxidation of the highly unsaturated fish oil fraction, mechanical shear disruption of the calcium-alginate delivery beads, and osmotic or thermal cell death of the L. plantarum strains.
To minimize these risks, bioactive compounds were incorporated after cooling the beverage to temperatures compatible with probiotic survival and lipid stability. Introducing the macro-beads under mild mechanical stirring after cooling the fluid is expected to reduce thermal stress on the probiotic cells and limit the oxidative deterioration of highly unsaturated fatty acids during processing.
In addition to physicochemical and microbiological characteristics, the overall product quality was considered from a technological perspective (Table 6). The selected formulation exhibited a homogeneous appearance and no visible phase separation or sedimentation throughout 15 days of refrigerated storage. Preliminary observations did not reveal unacceptable odor or sensory defects; however, a standardized sensory evaluation was not performed and should be included in future studies to confirm consumer acceptance.
3.8.3 Critical quality attributes
In order to establish a systematic quality framework, the critical quality attributes (CQAs) determining the safety, functionality, and technological acceptability of the developed macadamia-based functional beverage were identified (Table 7). Among these attributes, probiotic viability is essential and must remain above the minimum level generally associated with probiotic efficacy (≥ 6.0 log CFU/mL) throughout the intended storage period. The content and preservation of long-chain omega-3 polyunsaturated fatty acids (LCω3PUFAs), particularly EPA and DHA, represent key nutritional quality indicators of the developed formulation. In addition, oxidative stability, evaluated through lipid oxidation indicators such as peroxide value, p-anisidine value, and the TOTOX index, is considered a critical quality attribute because it reflects the preservation of the lipid fraction and the prevention of undesirable oxidative deterioration. Finally, physical homogeneity and colloidal stability, assessed through the absence of visible phase separation or sedimentation during refrigerated storage.
Table 7
| Critical quality attribute (CQA) | Acceptance criterion | Evaluation method | Relevance |
|---|---|---|---|
| Appearance and physical stability | Homogeneous beverage without visible phase separation or sedimentation during 15 days at 4 °C | Visual inspection | Ensures consumer acceptance and colloidal stability |
| Odor and sensory acceptability | No evident unacceptable odor during preliminary observations* | Informal sensory observation | Preliminary indication of product acceptability |
| pH | 6.71–7.54 | pH meter | Influences protein stability and microbial development |
| Probiotic viability | ≥ 106 CFU/mL at the end of storage | Plate count | Required to maintain probiotic functionality |
| Microbiological safety | Absence of indicator microorganisms during storage | Standard microbiological analyses | Ensures product safety |
| EPA + DHA content | 425–443 mg per 240 mL serving | Fatty acid analysis | Determines nutritional functionality |
| Oxidative quality | TOTOX value within recommended limits | Peroxide value, p-anisidine value and TOTOX | Indicates preservation of the lipid fraction |
Critical quality attributes (CQAs), acceptance criteria and analytical methods for the developed macadamia-based functional beverage.
*Formal sensory analysis was not performed; these observations should be considered preliminary.
3.8.4 Preservative compatibility and impact of technological parameters
Potassium sorbate was incorporated into the formulation at a concentration of 0.1% (w/v) as a technological barrier against fungal and bacterial spoilage. Although weak-acid preservatives may negatively affect bacterial membrane function and cellular metabolism, the viability results obtained during refrigerated storage demonstrated that the encapsulated L. plantarum population remained above the generally accepted minimum level required for probiotic functionality, reaching 6.0 ± 0.1 log CFU/mL after 15 days at 4 °C (from 7± 0.1 log CFU/mL at 0 day). The maintenance of probiotic viability suggests that the calcium-alginate encapsulation system contributed to protecting bacterial cells from adverse environmental conditions within the beverage matrix, including potential interactions with potassium sorbate. Therefore, under the conditions evaluated in this study, the selected concentration of 0.1% (w/v) provided microbiological protection without reducing probiotic viability below the recommended threshold for functional foods. Nevertheless, further studies comparing formulations with and without potassium sorbate would be necessary to determine the specific contribution of the preservative to probiotic survival.
4 Conclusions and limitations
This study demonstrates the feasibility of producing a functional probiotic-enriched beverage containing long-chain omega-3 polyunsaturated fatty acids from Macadamia integrifolia nuts. The proposed processing protocol yielded a stable beverage matrix, with the highest extraction efficiency achieved at a substrate-to-water ratio of 1:4 during soaking. From a nutritional standpoint, the developed beverages exhibited a protein content comparable to cow’s milk and higher than several commercial almond-based alternatives.
A 240 mL serving of the enriched beverage provides at least 85% of the minimum recommended daily intake of EPA and DHA. Additionally, Lactiplantibacillus plantarum demonstrated tolerance to simulated gastrointestinal conditions, maintaining viability above the generally accepted threshold for probiotic functionality for up to 15 days of refrigerated storage.
The identification of critical quality attributes (CQAs), critical process parameters (CPPs), and the main technological risks associated with the formulation provides a preliminary quality framework that supports process reproducibility and future product development.
Overall, the developed macadamia beverage represents a promising non-dairy functional food combining nutritional value, effective omega-3 delivery, and probiotic stability. The results demonstrate technological feasibility and provide a foundation for future pilot-scale evaluation and industrial development.
Some limitations of the present study should be acknowledged. Sensory acceptance, flavor perception, and the effectiveness of flavoring agents in masking potential marine off-notes were not experimentally evaluated. Storage stability was assessed for only 15 days under refrigerated conditions; therefore, longer-term studies are required to establish commercial shelf-life potential. In addition, probiotic leakage from the alginate capsules into the beverage matrix was not quantified, and the specific contribution of potassium sorbate to probiotic survival was not independently assessed. Protein quality was discussed based on literature data, but amino acid composition, protein digestibility, and biological value were not experimentally determined. Future studies should address these aspects together with pilot-scale validation and extended storage evaluations to further support commercial application of the developed beverage.
Statements
Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.
Author contributions
SÁ: Investigation, Funding acquisition, Formal Analysis, Writing – review & editing, Methodology, Data curation. VAE: Writing – review & editing, Methodology, Formal Analysis. DL: Conceptualization, Writing – original draft, Project administration, Formal Analysis, Writing – review & editing, Methodology, Funding acquisition, Supervision, Validation, Investigation, Visualization, Data curation, Resources.
Funding
The author(s) declared that financial support was not received for this work and/or its publication.
Acknowledgments
This work was supported by the National Council for Scientific and Technical Research, Argentina (CONICET), the National Institute of Fisheries Research and Development, Argentina (INIDEP), and FACEN CONACYT, Paraguay. MSc. Yadira Parra from the Department of Biotecnología Industrial and Bioprocesos at FACEN UNA (Asunción, Paraguay) is acknowledged for providing the probiotic strain used in this study, obtained from the “Evaluación de la producción de bacteriocinas a partir de Lactobacilos aisladas de productos lácteos” CONACYT project. Also, we would like to thank Omega Sur for providing the samples of crude fish oils.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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References
1
AlcortaA.PortaA.TárregaA.AlvarezM. D.VaqueroM. P. (2021). The role of plant-based foods in human nutrition. Foods10, 293. doi: 10.3390/foods10020293
2
Association of Official Analytical Chemists (2005). Official Methods of Analysis. 18th ed (Washington, DC: AOAC International).
3
AydarE. F.TutuncuS.OzcelikB. (2020). Plant-based milk substitutes: Bioactive compounds, conventional and novel processes, bioavailability, and health effects. J. Funct. Foods70, 103975. doi: 10.1016/j.jff.2020.103975
4
BakariD.TatsadjieuN. L.MbawalaA.MbofungC. M. (2011). Physiological properties of lactic acid bacteria isolated from chicken intestine. Innov. Rom. Food. Bio/Technol.8, 33–40.
5
BlighE.DyerW. J. (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol.37, 911–917. doi: 10.1139/o59-099
6
Chalupa-KrebzdakS.LongC. J.BohrerB. M. (2018). Nutrient density of milk and plant-based milk alternatives. Int. Dairy J.87, 84–92. doi: 10.1016/j.idairyj.2018.07.018
7
Codex Alimentarius Commission (2017). Standard for Fish Oils (CXS 329-2017) (Roma, Italia: FAO/WHO Food Standards Programme). Available online at: https://www.fao.org/fao-who-codexalimentarius/ (Accessed June 21, 2026).
8
Codex Alimentarius Commission (2020). General Principles of Food Hygiene (CXC 1-1969) (Rome: Food and Agriculture Organization/World Health Organization). Available online at: https://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXS%2B329-2017%252FCXS_329e.pdf](https://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXS%2B329-2017%252FCXS_329e.pdf (Accessed June 21, 2026).
9
Codina-TorrellaI.GuamisB.ZamoraA.QuevedoJ. M.TrujilloA. J. (2018). Microbiological stabilization of tiger nuts’ milk beverage using ultra-high pressure homogenization. Food Microbiol.69, 143–150. doi: 10.1016/j.fm.2017.08.002
10
DavarcıF.TuranD.OzcelikB.PonceletD. (2017). The influence of solution viscosities and surface tension on calcium-alginate microbead formation using dripping technique. Food. Hydrocolloid62, 119–127. doi: 10.1016/j.foodhyd.2016.06.029
11
DhankharJ. (2023). Dairy milk alternatives. J. Microbiol. Biotechnol. Food Sci.12, e9543. doi: 10.55251/jmbfs.9543
12
DickinsonE. (2003). Hydrocolloids at interfaces and their influence on dispersed systems. Food. Hydrocoll.17, 25–39. doi: 10.1016/S0268-005X(01)00120-5
13
Di RienzoJ. A.CasanovesF.BalzariniM. G.GonzalezL.TabladaM.RobledoC. W. (2017). Infostat Software, Version 2017 (Córdoba: Universidad Nacional de Córdoba). Available online at: https://www.infostat.com.ar (Accessed May 05, 2024).
14
El-ShafeiK.AbdallahN. A.TawfikN. F.El-SayedH. S.MahmoudM. (2018). Effect of different microencapsulating materials on survivability of Streptococcus thermophilus under simulated food processing and gastrointestinal conditions. Middle East. J. Appl. Sci.8, 259–271.
15
FayedB.El-SayedH. S.AboodA.HashemA. M.MehannaN. S. H. (2019). Application of multiparticulate microcapsules containing probiotic bacteria and inulin nanoparticles to enhance probiotic survivability in yoghurt. Biocatal. Agric. Biotechnol.22, 101391. doi: 10.1016/j.bcab.2019.101391
16
FehérA.GazdeckiM.VéhaM.SzakályM.SzakályZ. (2020). A comprehensive review of the benefits of and the barriers to the switch to a plant-based diet. Sustainability12, 4136. doi: 10.3390/su12104136
17
FlockM. R.HarrisW. S.Kris-EthertonP. M. (2013). Long-chain omega-3 fatty acids: Establishing dietary reference intakes. Nutr. Rev.71, 692–707. doi: 10.1111/nure.12071
18
GlennA. J.AuneD.FreislingH.MohammadifardN.KendallC. W. C.Salas-SalvadóJ.et al. (2023). Nuts and cardiovascular disease outcomes: A review of the evidence and future directions. Nutrients, 15(4), 911. doi: 10.3390/nu15040911
19
Global Organization for EPA and DHA Omega-3s (GOED) (2022). Goed Voluntary Monograph (Salt Lake City, UT, USA: GOED). Available online at: https://goedomega3.com/storage/app/media/Monograph/GOED%20Monograph%20-%202022%2001%2006%20-%20FINAL.pdf (Accessed June 21, 2026).
20
Grau-FuentesE.RodrigoD.GarzónR.RosellC. M. (2023). Understanding marketed plant-based beverages: Ingredients, technological function, and nutritional value. J. Funct. Foods106, 105609. doi: 10.1016/j.jff.2023.105609
21
LamasD. L.ÁlvarezS. T. (2023). Advances in omega-3 fortification strategies for functional foods. Food Chem. Adv.3, 100564. doi: 10.1016/j.focha.2023.100564
22
LamasD. L.MassaA. E. (2019). Ray liver oils obtained by different methodologies: characterization and refining. J. Aquat. Food. Product Tech.28, 555–569. doi: 10.1080/10498850.2019.1605554
23
McClementsD. J. (2004). Food Emulsions: Principles, Practices, and Techniques. 2nd Ed (Boca Raton, FL: CRC Press). doi: 10.1201/9781420039436
24
Ministerio de la Presidencia (2001). Real Decreto 3484/2000, De 29 De Diciembre, Por El Que Se Establecen Las Normas De Higiene Para La Elaboración, Distribución Y Comercio De Comidas Preparadas (Madrid, Spain: Boletín Oficial del Estado). Available online at: https://www.boe.es (Accessed September 06, 2023).
25
Mohd NawaweeN. S.Abu BakarN. F.ZulfakarS. S. (2019). Microbiological safety of street-vended beverages in Chow Kit, Kuala Lumpur. Int. J. Environ. Res. Public Health16, 4463. doi: 10.3390/ijerph16224463
26
ÖzerB.KirmaciH. A.ŞenelE.AtamerM.HayaloğluA. (2009). Improving the viability of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 in white-brined cheese by microencapsulation. Int. Dairy J.19, 22–29. doi: 10.1016/j.idairyj.2008.07.001
27
PandeyA.KamranF.ChoudhuryM.LiL.RahmanM. S.HussainM. A. (2025). Omega-3 fatty acid fortification of plant-based beverages to enhance their nutritional profile. Foods14, 1602. doi: 10.3390/foods14091602
28
PapamanoliE.TzanetakisN.Litopoulou-TzanetakiE.KotzekidouP. (2003). Characterization of lactic acid bacteria from Greek dry-fermented sausage. Meat Sci.65, 859–867. doi: 10.1016/S0309-1740(02)00292-9
29
PenhaC. B.SantosV. D. P.KurozawaL. E. (2021). Plant-based beverages: Ecofriendly technologies in the production process. Innov. Food. Sci. Emerg. Technol.72, 102760. doi: 10.1016/j.ifset.2021.102760
30
RastiB.ErfanianA.SelamatJ. (2017). Nanoliposomal encapsulation of omega-3 fatty acids and food applications. Food Chem.230, 690–696. doi: 10.1016/j.foodchem.2017.03.089
31
Sahye-PudaruthS.MaD. W. L. (2023). Omega-3 fatty acids and human health. Nutrients15, 1001. doi: 10.3390/nu15041001
32
SaritaB.SamadhanD.HassanM. Z.KovalevaE. G. (2025). A comprehensive review of probiotics and human health-current prospective and applications. Front. Microbiol.15, 1487641. doi: 10.3389/fmicb.2024.1487641
33
Scholz-AhrensK. E.AhrensF.BarthC. A. (2020). Nutritional and health attributes of milk and milk imitations. Eur. J. Nutr.59, 19–34. doi: 10.1007/s00394-019-01936-3
34
Serrano ZavalaJ.Hernández-MartínezD.Osorio-RevillaG. (2023). Elaboración de una bebida de nuez de macadamia. Investig. Desarro. Cienc. Tecnol. Alimentos8, 471–476. doi: 10.29105/idcyta.v8i1.63
35
SethiS.TyagiS. K.AnuragR. K. (2016). Plant-based milk alternatives: An emerging segment of functional beverages. J. Food Sci. Technol.53, 3408–3423. doi: 10.1007/s13197-016-2328-3
36
TamjidiF.NasirpourA.ShahediM. (2012). Physicochemical and sensory properties of food products enriched with omega-3 fatty acids. Food Sci. Technol. Int.18, 381–390. doi: 10.1177/1082013211428212
37
UkwuruM. U.OgbodoA. C. (2010). Effect of processing treatments on tigernut milk quality. Pak. J. Nutr.10, 95–100. doi: 10.3923/pjn.2011.95.100
38
U.S. Department of Agriculture. USDA (2019). Fooddata Central (Formerly National Nutrient Database for Standard Reference) (Washington, DC: USDA). Available online at: https://fdc.nal.usda.gov (Accessed September 06, 2023).
39
Vasquez-RojasW. V.MartínD.MirallesB.RecioI.FornariT.CanoM. P. (2021). Composition of Brazil nut, its beverage and by-products. Foods10, 3007. doi: 10.3390/foods10123007
40
VenkatachalamM.SatheS. K. (2006). Chemical composition of selected edible nut seeds. J. Agric. Food. Chem.54, 4705–4714. doi: 10.1021/jf0606959
41
XieA.DongY.LiuZ.LiZ.ShaoJ.LiM.et al. (2023). A review on plant-based drinks addressing nutrients, flavor, and processing technologies. Foods12, 3952. doi: 10.3390/foods12213952
Summary
Keywords
Macadamia integrifolia, alginate encapsulation, fish oil, functional foods, Lactiplantibacillus plantarum, nut-based beverage, omega-3 fatty acids, probiotic viability
Citation
Álvarez S, Alcolea Ersinger VF and Lamas DL (2026) Novel functional macadamia nuts (Macadamia integrifolia) beverage: omega-3 and probiotic lactobacilli incorporated into alginate edible coatings. Front. Ind. Microbiol. 4:1888197. doi: 10.3389/finmi.2026.1888197
Received
22 May 2026
Revised
25 June 2026
Accepted
29 June 2026
Published
22 July 2026
Volume
4 - 2026
Edited by
Marta Laranjo, Universidade de Évora, Portugal
Reviewed by
Malgorzata Ziarno, Warsaw University of Life Sciences, Poland
Liliya Ibragimova, Kazakh National Medical University, Kazakhstan
Updates
Copyright
© 2026 Álvarez, Alcolea Ersinger and Lamas.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Daniela Lorena Lamas, dlamas@mdp.edu.ar
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.