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Article

Development and Characterization of Pistachio Yogurt Analog: A Healthy, Sustainable, and Innovative Plant-Based Alternative

by
Inés M. Ramos
,
Samuel Rodríguez García
and
Justa M. Poveda
*
Departamento de Química Analítica y Tecnología de los Alimentos, Instituto Regional de Investigación Científica Aplicada (IRICA)/Facultad de Ciencias y Tecnologías Químicas, Universidad de Castilla-La Mancha, Avda. Camilo José Cela, s/n, 13071 Ciudad Real, Spain
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(8), 467; https://doi.org/10.3390/fermentation11080467
Submission received: 8 July 2025 / Revised: 28 July 2025 / Accepted: 6 August 2025 / Published: 15 August 2025
(This article belongs to the Special Issue 10th Anniversary of Fermentation: Feature Papers in the "Fermentation for Food and Beverages" Section)
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Abstract

Plant-based yogurts are increasingly recognized as sustainable and health-conscious alternatives to dairy-based products, driven by environmental, ethical, and nutritional motivations. Pistachio milk, derived from an efficient and resilient crop, emerges as a promising raw material for yogurt production, offering unique sensory qualities and a dense nutritional profile. Rich in unsaturated fatty acids, bioactive compounds, and essential nutrients, pistachios are ideal for fermentation with lactic acid bacteria (LAB). In this study, a novel pistachio-based yogurt analog (PBYA) was developed using lactic acid fermentation, with a yogurt commercial starter, of pistachio milk. The production process was optimized to create an additive-free, clean-label formulation without the use of stabilizers or thickeners. The physicochemical, microbiological, and sensory properties of the PBYA were evaluated over refrigerated storage. The final product exhibited high levels of protein (5.6%), fat (5.4–6.8%), and total solids (20.5–21.4%), along with desirable texture and flavor characteristics. Notably, PBYA presented significantly higher concentrations of total free amino acids (754 mg/L) compared to commercial soy (557 mg/L) and cow’s milk yogurts (390 mg/L), particularly in essential amino acids such as lysine, methionine, and tryptophan. This enhanced free amino acid profile contributes to the product’s functional and nutritional value. Sensory analysis revealed good acceptance of the product, with improvements in viscosity and firmness over time, likely due to microbial exopolysaccharide production. Overall, the findings highlight the feasibility and commercial potential of PBYA as a clean-label, plant-based fermented product that meets current consumer demands for sustainability, nutrition, and sensory quality.

1. Introduction

In recent years, growing concerns have driven the search for sustainable alternatives to traditional animal-derived products. Among these alternatives, plant-based dairy analogs, such as yogurt, have gained considerable attention as a sustainable option [1]. Replacing animal-based ingredients with plant alternatives can significantly reduce greenhouse gas emissions, water usage, and land consumption [2,3]. In this sense, raw plant-based materials, such as soy, oats, almonds, and other nuts, have a markedly lower environmental impact compared to animal dairy production. This shift not only addresses environmental concerns but also responds to the growing demand for plant-based diets that support human health and sustainability.
The pistachio tree is known for its resilience to drought, requiring significantly less water than many other crops [4]. The Mediterranean climate in regions like Spain, which is arid and semi-arid, is particularly suited for pistachio cultivation [4], making it an ideal crop for water-scarce areas. Moreover, it thrives with fewer chemical inputs, making them a viable option for environmentally friendly agricultural practices. As a result, pistachio production in Spain has been steadily increasing, and it is currently the leading European producer, reaching approximately 20,000 tons annually [5,6].
Beyond their environmental benefits, pistachios embody circular economy principles, as their by-products, such as shells, can be repurposed for bioenergy, animal feed, or even as biodegradable materials, further reducing waste and improving resource efficiency [7]. Additionally, pistachios are highly valued for their rich nutritional profile, including unsaturated fatty acids, vitamins, minerals, and bioactive compounds like phenolics, phytosterols, and tocopherols [8]. These compounds play an antioxidant, immune, and anti-inflammatory role, promoting the reduction in oxidative stress and having beneficial effects on the prevention of cardiovascular diseases, such as managing cholesterol levels, and preventing type-2 diabetes [9].
Recent studies highlight pistachios as a suitable matrix for fermentation by lactic acid bacteria (LAB), enhancing the release of health-promoting bioactive substances and improving the sensory and shelf-life characteristics of the final product [10,11].
Among the various plant-based options available, pistachio milk stands out as a particularly interesting and underexplored raw material for the production of dairy analogs. While much research has focused on other nut-based products like almond, cashew, and hazelnut yogurts [12,13,14], pistachio-based alternatives have received limited attention. However, the unique nutritional and sensory qualities of pistachios [15] present an exciting opportunity for developing innovative plant-based dairy products.
This study aims to explore the potential of using pistachio milk for plant-based yogurt production. The objective is to design and optimize a production method and evaluate the compositional, microbiological, and sensory properties of additive-free pistachio-based yogurt analog (PBYA). Through this investigation, pistachio is promoted as a sustainable crop with broader applications and pistachio milk is highlighted as a novel, environmentally friendly alternative for plant-based yogurt.

2. Material and Methods

2.1. Raw Materials

The raw material used was pistachio (Pistacia vera L., Kerman variety). It was purchased in its natural state—unpeeled, unsalted, and untoasted—from the local market.
Commercial yogurt starter culture (Ferlac, Type I yogurt culture, Abiasa, S.L., Pontevedra, Spain), containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus was used for the yogurt making.
Two commercial samples of plant-based yogurts were used to compare the results of the experimental PBYA in terms of free amino acids (FAA) profile and sensory characteristics. For this purpose, a soy-based yogurt analog (Hacendado, Valencia, Spain)—selected as it represents the most widely marketed plant-based alternative—and a traditional cow’s milk yogurt (Hacendado, Valencia, Spain) were analyzed. The main composition of soy-based yogurt was 3.0% protein, 3.0% fat, 120 mg calcium/100 g, and 9% total solids, and for cow’s milk yogurt, it was 3.5% protein, 3.5% fat, 120 mg calcium/100 g, and 12% total solids.

2.2. Production of Pistachio Milk and Pistachio-Based Yogurt Analog Manufacture

Pistachio milk was prepared following the method described by Al Zahrani and Shori [16] with modifications. Pistachios were soaked in water for 24 h to facilitate moisture absorption and subsequent processing. Following this, the seeds were blanched at 100 °C for 1 min and peeled. To obtain the pistachio milk, the nuts were ground with water at a 1:1.5 seed-to-water ratio using a countertop blender (Cecotec brand, Power Black Titanium 2600MAX Pro model, Valencia, Spain). This ratio was selected as it exhibited the best results in previous trials. The resulting mixture was filtered through a double fine-mesh cloth to remove solid residues. The pistachio milk obtained was then pasteurized at 70 °C for 1 min, cooled to room temperature, and stored refrigerated at 4 °C. The composition of pistachio milk obtained was 9.4% fat, 4.3% protein, 59 mg of calcium per 100 g, and 18% total solids. No additives, sugars, or stabilizers were added to the pistachio-based milk for yogurt manufacture.
For fermentation, the pistachio milk was heated at 42 °C and inoculated with 2% (w/v) of the commercial yogurt starter culture (Section 2.1). This culture was precultured in the pistachio milk following the manufacturer’s instructions (5 g/100 L) and incubated for approximately 16 h at 37 °C until a population of 6 log CFU/mL was reached.
The inoculated milk was transferred to 30 mL glass containers with airtight lids and incubated at 42 °C until a pH of 4.5 ± 0.1 was reached. The fermentation process was finalized by rapidly cooling the yogurt to 4 °C, and then it was stored refrigerated for 21 days. PBYA manufacture was carried out in duplicate one week later. A flowchart summarizing the entire pistachio yogurt production process is presented in Figure 1.
Physicochemical, microbiological, and individual free amino acids and sensory analyses of the PBYA were conducted at different times during the 21-day refrigerated storage period. All the analyses were performed in duplicate, except the microbiological counts, which were carried out in triplicate.

2.3. Fermentation Kinetics

During the milk fermentation process, pH levels were initially monitored at 1 h intervals using a pH meter (Crison, Barcelona, Spain). As the pH approached 5.0, measurements were taken every 30 min. The maximum acidification rate (Vm), expressed as absolute values (mpH units/min), was determined from the pH-time curves using the following formula:
Vm = max{d(ph)/dt}
Additionally, key kinetic parameters were calculated to describe the fermentation process: Tm, the time when the maximum acidification rate occurred, and Te, the time required to reach a pH of 4.6.

2.4. Microbiological Counts

Counts of total aerobic mesophilic bacteria were performed on plate count agar (PCA; Condalab, Madrid, Spain) under aerobic conditions at 37 °C for 48 h.
For the determination of the cell population of L. delbrueckii subsp. bulgaricus and S. thermophilus from the commercial starter, the pour plate method was used, and serial dilutions of the samples were spread onto MRS agar (Condalab, Madrid, Spain; pH = 5.42, adjusted with HCl 0.1 M) or M17 agar (Scharlab, Barcelona, Spain) enriched in lactose (10%) [17], respectively. Both cultures were incubated for 72 h at 37 °C [17].
Microbial counts in the three culture media were performed in triplicate.

2.5. Compositional Analysis

The pH was measured by using a digital pH-meter (Crison, Barcelona, Spain). Total solids (TS) were determined gravimetrically after oven drying, and titratable acidity (TA) was measured according to AOAC method [18]. Fat was assessed by the Gerber butyrometric method [18]. Total protein was determined by the Kjeldahl method [18], applying a conversion factor of 6.25 to convert total nitrogen to total protein, and calcium content was measured using a complexometric method [19]. Protein and calcium values were only determined in the samples of 1 day, as these components are generally stable throughout refrigerated storage and are not significantly affected by the fermentation process or storage duration.

2.6. Color Measurements

The color characteristics of the pistachio yogurts were assessed using a Minolta Chroma Meter CR-400 (Konica Minolta, Osaka, Japan), configured with a D65 light source and a 10° observation angle. The device was calibrated against a white reference plate, and the CIELab system was employed to evaluate the color coordinates L*, a*, and b*. The L* value, ranging from 0 to 100, represents lightness, with 0 being black and 100 being white. The a* value denotes the transition from red (+a*) to green (−a*), while the b* value indicates the shift from yellow (+b*) to blue (−b*).

2.7. Free Amino Acid Content Analysis

For sample preparation, proteins were precipitated by mixing 3 mL of each yogurt sample with 3 mL of 12% trichloroacetic acid (TCA). The mixtures were then homogenized using an Ultra-Turrax T18 Basic (IKA-Werke GmbH & Co. KG, Staufen, Germany) at 14,000 rpm for 2 min to ensure complete protein denaturation. Following homogenization, samples were centrifuged at 15,000× g for 15 min at 4 °C. The resulting supernatants were collected and stored at −20 °C until further analysis by reversed-phase high-performance liquid chromatography (RP-HPLC).
The quantification of 19 amino acids was performed by RP-HPLC using diethyl ethoxymethylenemalonate (DEEMM) derivatisation method [20]. Briefly, a mixture of 400 μL of the sample, 900 μL of 1 mol/L borate buffer pH 9.0, 300 μL of methanol, 20 μL of internal standard (1.0 g/L), and 30 μL of DEEMM was incubated at 30 °C in an ultrasonic bath for 30 min. The samples were then heated in an oven at 70 °C for 2 h to allow the complete degradation of excess DEEMM and reagent byproducts. After derivatization, the samples were filtered through regenerated cellulose esters 0.2 μm membranes (Agilent Technologies, Barcelona, Spain) coupled to a syringe into conical vials.
The analysis was performed on an Agilent 1200 HPLC (Agilent Technologies, Madrid, Spain) comprising an Agilent binary pump, an Agilent autosampler, and an Agilent photodiode array detector. Chromatographic separation was performed using a Zorbax Eclipse XDB C18 column particle (Agilent Technologies, Santa Clara, CA, USA) size 5 μm (250 mm × 4.6 mm) and an Agilent guard cartridge C18 particle size 5 μm (12.5 mm × 4.6 mm) thermostatted at 16 °C in an Agilent thermostatted column compartment. The mobile phase consisted of a 25 mmol/L acetate buffer pH 5.8 with 0.02% sodium azide (eluent A) and an 80:20 mixture of acetonitrile and methanol (eluent B). Samples (50 μL) were applied to the column and eluted at a flow rate of 0.9 mL/min according to the binary gradient used by Poveda [21].
The compounds of interest were identified based on their retention times and spectral characteristics at 280 nm, and they were quantified using the internal standard method.

2.8. Sensory Evaluation

Quantitative descriptive analysis (QDA) was conducted to evaluate the appearance, aroma, flavor, and texture attributes of the PBYA, following the methodology outlined by ISO 13299 standard [22] and as described in Ramos and Poveda [23]. The sensory panel consisted of eight trained assessors, aged 24 to 60, selected from the university staff. Their training in evaluating plant-based milks and yogurts was conducted in accordance with ISO 8586 standard [24], and they also had prior experience in assessing dairy products, particularly yogurt.
This study was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants involved in the study.
The panelists were asked to score the sample attributes, organized into four categories in the following order: visual (color and firmness); odor (dairy yogurt odor, nut odor, odor intensity and odor quality); flavor (dairy yogurt taste, sour, nut taste, bitter, flavor intensity and flavor quality); and texture in mouth (viscosity), using an unstructured 0 to 10 scale.
The samples were presented to the tasters coded with three-character tasting keys composed of two numbers and a central letter and were served randomly in 30 mL glass containers with airtight lids, each containing 20 mL of yogurt. The evaluations were conducted in duplicate across two separate sessions in a tasting room designed according to the recommendations of ISO 8589 standard [25].

2.9. Statistical Analysis

A one-way analysis of variance (ANOVA) was applied to the results, using Tukey’s test for the comparison of means (p < 0.05). Normality, heteroscedasticity, and the independence of samples had been previously checked. These statistical treatments were performed using the IBM SPSS statistics package version 26.0 (SPSS Inc., Chicago, IL, USA).

3. Results and Discussion

3.1. Fermentation Kinetics

The changes in pH of PBYA during fermentation can be observed in Figure 2. During the first 100 min, the pH remains relatively stable at approximately 6.5, suggesting an adaptation phase in which microorganisms acclimate to the medium and available nutrients. Subsequently, between 100 and 200 min, a gradual decline in pH is observed as LAB begin metabolizing the naturally occurring monosaccharides in pistachio nuts more efficiently, leading to lactic acid production.
Between 200 and 250 min, the acidification process accelerates significantly, as evidenced by a pronounced drop in pH, reflecting the peak metabolic activity of LAB and the rapid lactic acid synthesis. Beyond 250 min, the acidification rate slows down, with the pH approaching 4.6. This marks the end of fermentation. The observed acidification trend aligns with that reported for both dairy-based yogurts [26] and plant-based analogs [27]. This fermentation pattern is primarily driven by the metabolic interactions between the starter cultures, L. delbrueckii subsp. bulgaricus and S. thermophilus. L. delbrueckii exhibits strong proteolytic activity, hydrolyzing proteins into peptides and amino acids that promote S. thermophilus growth. In return, S. thermophilus produces formic, folic, and pyruvic acids, as well as CO2, enhancing the metabolic activity of L. delbrueckii [28]. This behavior has also been previously observed in other studies on the production of sheep’s and cow milk yogurts [23,29].
The acidification kinetics parameters are summarized in Table 1. The maximum acidification rate (Vm) indicated a moderately high metabolic efficiency of LAB during the peak fermentation phase. This parameter is critical, as it dictates the rate at which pH decreases in the most active stage of the process.
The time required to reach the maximum acidification rate (Tm) was influenced by key fermentation variables, including bacterial strain selection, inoculum size, temperature, and overall fermentation duration, all of which significantly impact the pH dynamics of the final product [30].
Finally, the total fermentation time (Te) required to reach the final pH of 4.6 was 342 min. This intermediate duration is considered optimal, as shorter fermentation times may adversely affect the textural properties of the yogurt [31], while extended fermentation increases the risk of microorganism spoilage [30]. Notably, these values are comparable to those reported by Pereita et al. [28] for a fermented soy-based product and with those shown for cow’s milk yogurts [29], underscoring the efficiency and stability of fermentation process in pistachio-based yogurt. These findings support the feasibility of PBYA as a high-quality plant-based alternative with a well-balanced acidification profile and fermentation time.

3.2. Microbial Counts

To ensure health benefits and support gut microbiota, it is essential to maintain an appropriate viable load of bacterial cultures in yogurt throughout shelf life. According to regulatory guidelines, yogurt must contain a minimum starter culture count of 7 log CFU/mL [32]. In this study, Streptococcus thermophilus (M-17) and Lactobacillus bulgaricus (MRS) counts in PBYA samples were monitored throughout storage (Table 2). The results indicate that cell counts in both MRS and M17 showed a slight decrease during the storage period but remained above the minimum-recommended viable cell levels for dairy yogurt (107 UFC/g) throughout. The viability of bacterial cells during yogurt storage is influenced by factors such as acid accumulation, nutrient depletion, and the buildup of metabolic by-products. The observed decline in cell counts can be attributed to the increasing acidity of the medium and the nutrient limitation, leading to bacterial growth inhibition [33]. This trend is consistent with findings in other plant-based yogurts, such as those made from almonds or cashews [34], as well as for dairy-based yogurts [35]. Moreover, the final counts at the end of storage (8.1 ± 0.2 log CFU/mL) exceed the minimum legal threshold, indicating that the product remains viable for market introduction.
Additionally, to assess the hygienic quality of the pistachio milk prior to yogurt manufacture, counts on PCA were performed, yielding a value below 2.18 log CFU/mL. This indicates the good hygienic quality of the raw milk. Furthermore, counts on the same medium were carried out in the produced yogurts throughout the storage period. The results were very similar to those obtained using the selective media (Table 2), suggesting that the total aerobic bacteria predominantly correspond to the inoculated LAB, supporting the absence of significant contamination from external sources.

3.3. Compositional Analysis

The compositional analysis of the assessed PBYAs is presented in Table 3. The pH values decreased significantly during storage, ranging from 4.62 to 4.24, due to the residual metabolic activity of LAB strains, which continued producing organic acids, particularly lactic acid even at 4 °C. This ongoing acidification was reflected in a corresponding increase in TA, which rose significantly from 0.54 g lactic acid/100 g at the start of storage to 0.64 g lactic acid/100 g on day 21. These values of TA meet the Codex Alimentarius standards [32] for samples at 14 and 21 days of storage, which stipulate a minimum acidity of 0.6 g lactic acid/100 g. A similar trend in pH and TA was observed by Soumya et al. [34] on various plant-based yogurts, showing values comparable to those of PBYA. For instance, after 21 days of storage, coconut yogurt reached a TA of 0.58 g lactic acid/100 g, while oat yogurt exhibited a TA of 0.81 g lactic acid/100 g. However, these values remain lower than those reported for commercial dairy yogurts (1.035–1.38 g lactic acid/100 g) [36,37].
Total solids, which reflect the dry matter content of yogurt, play a crucial role in determining product quality. A high total solids content reduces syneresis, thus improving structural stability. Additionally, it enhances viscosity, contributing to a creamier texture and better overall consistency [38]. The total solids content in PBYA ranged from 20.5 to 21.4% during cold storage. Compared to other plant-based yogurts, PBYA exhibits a similar total solids content to walnut yogurt (19.4%) [39] and groundnut yogurt (18.15%) [40]. Notably, it contains higher total solids than dairy yogurts, such as those made from cow’s milk (11.39–16.49%) [40,41] or buffalo milk (14.48%) [38].
The results of the fat content of PBYAs showed no significant differences (p < 0.05), although the highest result was found 21 days after storage (6.8%), being higher when compared to the content of cow (3.33%), sheep (4.33%) and goat (2.81%) yogurts [42]. High fat content is a desirable attribute as it improves the taste and texture of the final product [12]. Furthermore, pistachio fat is predominantly composed of monounsaturated and polyunsaturated fatty acids, particularly oleic and linoleic acids, which are known to support cardiovascular health, reduce inflammation, and contribute to a favorable lipid profile [43].
PBYAs had a protein content of 5.6%, higher than those found in dairy yogurts (4.4%) or other plant-based yogurts, such as soy (4%) or cashew (2%), and lower than oat yogurt (9%) or lentil yogurt (12%) [12]. This is to be expected, as pistachios contain a high amount of protein, resulting in a yogurt analog product with substantial protein levels. Proteins play a key role in determining physicochemical properties, including water retention and gel formation [12]. The calcium content in PBYA was 71.9 mg/100 g. This amount is lower compared to dairy yogurts (153.80 mg/100 g) [44], but similar to other plant-based products such as coconut yogurt (85 mg/100 g) [45]. As can be seen, calcium, a vital micronutrient, is present in significant and highly bioavailable quantities in dairy products. However, relying on plant-based products can result in deficiencies of this essential nutrient. To address this issue, supplementation with critical nutrients is recommended [12]; however, as the primary objective of this study was to obtain a clean label product, the addition of calcium was not considered, and addressing this deficiency will be considered in a future study.

3.4. Color Measurement

Color plays a crucial role in food, as it is the primary characteristic that consumers perceive initially, affecting their acceptance, sensory attributes, and perceived safety, quality, and freshness of the product [39]. In the case of yogurt, color is a key indicator that affects quality, freshness, flavor expectations, commercial value, and product acceptability [46].
Table 4 displays the results of instrumental color analysis of the PBYA during the storage period. The obtained L* values are high, indicating greater luminosity in the yogurts. Grasso et al. [36], who analyzed the color parameters of various plant-based yogurts, including soy, almond, coconut, and hemp yogurts, reported much lower L* values, ranging from 60.2 to 64.2. According to these results, the PBYA exhibits a brighter color, appealing to consumers who prefer lightness (L*) in such type of product [47]. The values of L*, and b* reflect a slight tendency towards a loss of brightness and a decrease in the intensity of the yellow color over the 21 days of storage of the PBYA. These changes could be related to the oxidation of the natural pigments present in pistachio, as it has yellowish pigments such as flavonoids, which, as they are soluble in water, could be present in the final product [43]. However, the a* values show stability in the green hue, suggesting that there were no significant alterations in the perception of the green freshness of the product during this period. Nevertheless, although measuring instruments can accurately detect slight variations in color parameters, these changes are often imperceptible to the human eye. This suggests that, from a practical and commercial perspective, the color of the yogurt remains stable during this time, which is a positive aspect for its acceptance and overall visual quality.

3.5. Free Amino Acid Profile

Table 5 shows significant differences (p < 0.05) in free amino acid (FAA) concentrations among pistachio-based yogurt, commercial soy yogurt, and commercial cow milk yogurt. PBYA exhibited the highest total FAA content (754 ± 8 mg/L), markedly surpassing that of soy (557 ± 5 mg/L) and cow milk yogurt (390 ± 2 mg/L). This superior FAA content in PBYA highlights its potential as a nutrient-dense, plant-based fermented product.
Among the essential amino acids (EAAs), PBYA showed significantly higher levels of lysine, methionine, threonine, tryptophan, and valine as compared to soy yogurt and cow’s milk yogurt. Notably, the lysine content in PBYA was over 16-fold higher than that in cow’s milk yogurt and more than double that found in soy yogurt. Given that lysine is often the limiting amino acid in plant-based diets [46], its abundance in PBYA may offer a valuable nutritional advantage. Similarly, the elevated levels of methionine and tryptophan—critical for methylation processes and serotonin synthesis, respectively—suggest enhanced functional properties compared to commercial counterparts.
Soy yogurt showed higher isoleucine and leucine levels than the other two matrices. Nevertheless, PBYA presented a more balanced EAA profile overall. Conversely, cow’s milk yogurt consistently exhibited the lowest values for nearly all EAAs, including lysine and methionine, aligning with previous findings that suggest reduced FAA accumulation in dairy-based yogurts post-fermentation [46].
The non-essential amino acids (NEAAs) profile further supports the superior bioactivity of PBYA. It contained the highest levels of γ-aminobutyric acid (GABA), a non-protein amino acid recognized for its hypotensive, anxiolytic, and neuroprotective properties [48]. In comparison, GABA levels in soy and cow yogurts were significantly lower. The elevated GABA in PBYA is likely attributed to the microbial decarboxylation of glutamate during fermentation, a process well-documented in plant-based substrates rich in glutamic acid [49].
In addition, PBYA exhibited significantly higher values of glycine, tyrosine, and arginine, all of which contribute to metabolic regulation and antioxidant activity. Although soy yogurt contained more glutamate, which enhances umami flavor perception, PBYA followed closely, suggesting favorable sensory properties as well.
The superior amino acid profile of PBYA aligns with recent reports on the enhanced nutritional quality of nut-based fermented products [50,51]. For instance, Akbarian et al. [50] found comparable increases in bioactive amino acids in almond-based kefir, while Borrego-Ruiz et al. [51] reported lower FAA levels in oat-based yogurts compared to legume and nut fermentations. The results of this study reinforce the suitability of pistachio matrices for developing functional plant-based yogurts that not only meet but exceed the nutritional benchmarks set by conventional dairy alternatives.

3.6. Sensory Evaluation

Yogurt, as a fresh ready-to-eat product, is valued for its sensory characteristics, which play a key role in consumer preference. Traditional yogurt is characterized by a smooth, creamy texture, mild tanginess, and refreshing flavor. Its consistency is typically viscous and homogeneous, free from syneresis, ensuring a pleasant mouthfeel [46]. These initial properties define the quality of yogurt and serve as a reference point before any changes occur during storage. In order to assess the acceptability of the PBYA, a sensory evaluation was performed, comparing the experimental PBYA of this study to that of the commercial cow yogurt and soy-based yogurt analog.
First, the three samples of PBYA showed a similar profile, with some small changes during the storage period (Figure 3). The samples were characterized by low firmness, moderate yogurt odor and flavor quality, and high color and nutty flavor scores. While some attributes in PBYA such as yogurt flavor and sourness declined over time—likely due to lipid oxidation and pH variation [52]—nut odor and nut flavor intensified, enhancing the characteristic profile of the pistachio matrix. Although the viscosity in mouth of PBYA was initially low, it is noteworthy that it increased significantly (p < 0.05) with storage time, reaching the same value as for the soy yogurt analog at 21 days.
The three sensory profiles are clearly differentiated in Figure 3, with the profiles of PBYAs being very different from those of the commercial dairy and soy yogurts. Cow’s milk yogurt clearly had the highest scores (p < 0.05) for yogurt odor and flavor, as well as firmness and viscosity in the mouth. Soy yogurt was characterized by low odor and flavor intensity and the lowest (p < 0.05) odor and flavor quality. It presented intermediate firmness and viscosity and very low yogurt odor and flavor. Compared to the commercial samples, the PBYA generally displayed intermediate characteristics (p < 0.05). Notably, bitterness was completely absent in all PBYA samples, in contrast to the soy yogurt, which exhibited perceptible bitter notes. In terms of overall impression, the PBYA samples scored intermediate between the cow’s milk yogurt and the soy milk yogurt analog, which received the lowest (p < 0.05) scores.
These results underscore the potential of the obtained PBYA as a clean-label product with good sensory quality, even superior to that of commercial soy yogurt analog, with a good sensory acceptance, with odor and flavor characteristics close to those of dairy yogurt, and a viscosity similar to that of soy yogurt analog.

4. Conclusions

The development of PBYA represents a significant innovation in plant-based alternatives, offering a unique, nutritious, and additive-free option for health-conscious consumers. Pistachio serves as an excellent base for the manufacture of plant-based yogurt, providing a rich, creamy texture, and distinct flavor in the final product. Nutritionally, PBYA is notable for its high content of unsaturated fats and its protein levels, which can even surpass those of conventional cow’s milk yogurts, further enhancing its appeal as a functional food. The lactic fermentation process ensured optimal acidification and a stable, high-quality product, with lactic acid bacteria adapting well to the non-dairy base. Compositional analysis showed that PBYA is rich in total solids, fat, and protein, meeting the nutritional needs of consumers. Its bright color remained attractive throughout its shelf life, further enhancing its visual appeal, which is an important factor for consumer acceptance. Importantly, FAA analysis revealed significantly higher total FAA content in PBYA compared to commercial soy and cow milk yogurts. In particular, essential amino acids were present at significantly higher levels than in cow’s milk yogurt, with lysine concentrations up to six times higher. Additionally, the elevated levels of γ-aminobutyric acid (GABA), glycine, tyrosine, and arginine highlight the functional and bioactive potential of this formulation. Sensory analysis revealed that, despite low firmness and moderate viscosity in the mouth, the product maintained an overall appealing flavor profile throughout the entire storage period, being better valued than a commercial soy yogurt analog. Further work is warranted to improve the texture of this product by using exopolysaccharide-producing LAB, thus avoiding the addition of texturizers or stabilizers, and also selected LAB strains, in order to improve the functional properties of the product.
These findings position pistachio-based yogurt analog as a competitive and health-promoting plant-based fermented product, suitable for consumers with dietary restrictions and for those seeking enhanced nutritional value in dairy alternatives. These results highlight the commercial viability of pistachio-based yogurt as a nutritious, visually attractive, and additive-free plant-based option, making it a strong contender in the growing market for clean-label alternatives.

Author Contributions

Conceptualization, data curation, formal analysis, investigation, methodology, and visualization, I.M.R. and J.M.P.; writing—original draft preparation, I.M.R., S.R.G., and J.M.P.; funding acquisition, project administration, resources, supervision, and writing—review and editing, J.M.P. All authors have read and agreed to the published version of the manuscript.

Funding

This work is part of the research project SBPLY/23/180225/000026, funded by the European Union through the European Regional Development Fund (Fondo Europeo de Desarrollo Regional, FEDER) and the Regional Government of Castilla-La Mancha (Junta de Comunidades de Castilla-La Mancha, JCCM) under the INNOCAM initiative.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Fermentation 11 00467 g001
Figure 1. Flowchart of the PBYA production process.
Figure 1. Flowchart of the PBYA production process.
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Figure 2. Acidification profile of the PBYA.
Figure 2. Acidification profile of the PBYA.
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Figure 3. Sensory profile of PBYAs during the refrigerated storage period (1, 14, and 21 days), and cow and soy yogurts. PBYA: pistachio-based yogurt analog; SY: soy yogurt; CY: cow yogurt.
Figure 3. Sensory profile of PBYAs during the refrigerated storage period (1, 14, and 21 days), and cow and soy yogurts. PBYA: pistachio-based yogurt analog; SY: soy yogurt; CY: cow yogurt.
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Table 1. Kinetic acidification parameters of the PBYA.
Table 1. Kinetic acidification parameters of the PBYA.
Parameters
V max (m unit pH/min) 6.4
Tm (min)240
Te (min)342
V max: maximum acidification rate; Tm: time to reach Vm; Te: time to reach the end of fermentation (pH 4.6).
Table 2. Microbial counts of the PBYAs (log CFU/mL; n = 3).
Table 2. Microbial counts of the PBYAs (log CFU/mL; n = 3).
Microbial GroupPeriod of Storage
11421p Value
Streptococcus thermophilus (M-17)8.6 a ± 0.28.4 b ± 0.38.1 c ± 0.20.0007
Lactobacillus bulgaricus (MRS)8.4 a ± 0.18.2 b ± 0.18.2 b ± 0.20.0085
Total aerobic mesophilic bacteria (PCA)8.8 a ± 0.38.1 b ± 0.28.2 b ± 0.30.0041
a–c: Means in the same row with different letters indicate statistically significant differences between the period of storage (p < 0.05).
Table 3. Physico-chemical composition of PBYAs during refrigerated storage (n = 2).
Table 3. Physico-chemical composition of PBYAs during refrigerated storage (n = 2).
TimepHTA (%)TS (%)Fat (%)Protein (%)Calcium (mg/100 g)
1 4.62 a ± 0.010.54 b ± 0.0320.5 ± 0.15.8 b ± 0.35.6 ± 1.771.9 ± 3.2
144.40 b ± 0.010.64 a ± 0.0121.4 ± 0.85.4 b ± 0.6ndnd
214.34 c ± 0.010.64 a ± 0.0220.6 ± 0.06.8 a ± 0.3ndnd
p value<0.00010.00210.0800.0067--
a–c: Means in the same column with different letters indicate statistically significant differences between the period of storage (p < 0.05). nd: not determined.
Table 4. Color parameters of the PBYAs during refrigerated storage (n = 2).
Table 4. Color parameters of the PBYAs during refrigerated storage (n = 2).
SampleL*a*b*
PBYA 1 d 83.1 b ± 0.1−4.5 ± 0.636.9 a ± 3.4
PBYA 14 d83.7 a ± 0.1−4.1 ± 0.133.6 b ± 0.2
PBYA 21 d82.1 c ± 0.1−4.3 ± 0.133.5 b ± 0.1
p value0.0020.180.0005
a–c: Means in the same column with different letters indicate statistically significant differences between the period of storage (p < 0.05).
Table 5. Free amino acid content (mg/L) in PBYA at 14 days of storage and commercial soy yogurt analog and cow milk yogurt.
Table 5. Free amino acid content (mg/L) in PBYA at 14 days of storage and commercial soy yogurt analog and cow milk yogurt.
Free Amino AcidPBYACommercial Soy YogurtCommercial Cow Yogurtp Value
Essential amino acids  
Histidine11.2 a ± 1.612.2 a ± 4.84.15 b ± 0.810.017
Isoleucine9.15 c ± 0.9821.6 a ± 0.815.3 b ± 0.5<0.001
Leucine13.0 b ± 0.420.3 a ± 0.59.35 c ± 0.38<0.001
Lysine67.8 a ± 2.328.7 b ± 1.44.03 c ± 0.07<0.0001
Methionine25.7 a ± 1.56.73 b ± 0.584.11 c ± 0.21<0.0001
Phenylalanine9.91 b ± 0.1511.7 a ± 0.25.12 c ± 0.97<0.001
Threonine67.5 a ± 1.418.9 b ± 1.218.0 b ± 1.2<0.0001
Tryptophan54.9 a ± 2.616.8 c ± 1.920.3 b ± 1.1<0.0001
Valine46.7 a ± 3.923.7 b ± 2.528.9 b ± 1.0<0.001
Nonessential amino acids 
Alanine47.8 b ± 3.051.9 a ± 1.917.3 c ± 0.8<0.001
Arginine25.2 b ± 0.432.0 a ± 0.21.57 c ± 0.01<0.0001
Aspartic acid + Serine51.5 a ± 1.338.1 b ± 0.725.6 c ± 0.11<0.0001
Cystine27.9 a ± 1.617.2 b ± 0.926.4 a ± 0.230.002
GABA112 a ± 171.4 c ± 1.183.6 b ± 1.3<0.0001
Glutamate129 b ± 2134 a ± 191.3 c ± 1.7<0.0001
Glycine8.34 a ± 1.35.18 b ± 0.693.17 c ± 0.080.001
Proline31.7 b ± 0.237.2 a ± 0.628.8 c ± 0.9<0.001
Tyrosine15.6 a ± 0.89.87 b ± 0.473.55 c ± 0.14<0.0001
Total754 a ± 8557 b ± 5390 c ± 2<0.0001
a–c: Means in the same row with different letters indicate statistically significant differences between the different yogurts (p < 0.05).
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Ramos, I.M.; Rodríguez García, S.; Poveda, J.M. Development and Characterization of Pistachio Yogurt Analog: A Healthy, Sustainable, and Innovative Plant-Based Alternative. Fermentation 2025, 11, 467. https://doi.org/10.3390/fermentation11080467

AMA Style

Ramos IM, Rodríguez García S, Poveda JM. Development and Characterization of Pistachio Yogurt Analog: A Healthy, Sustainable, and Innovative Plant-Based Alternative. Fermentation. 2025; 11(8):467. https://doi.org/10.3390/fermentation11080467

Chicago/Turabian Style

Ramos, Inés M., Samuel Rodríguez García, and Justa M. Poveda. 2025. "Development and Characterization of Pistachio Yogurt Analog: A Healthy, Sustainable, and Innovative Plant-Based Alternative" Fermentation 11, no. 8: 467. https://doi.org/10.3390/fermentation11080467

APA Style

Ramos, I. M., Rodríguez García, S., & Poveda, J. M. (2025). Development and Characterization of Pistachio Yogurt Analog: A Healthy, Sustainable, and Innovative Plant-Based Alternative. Fermentation, 11(8), 467. https://doi.org/10.3390/fermentation11080467

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