Biotechnological Valorisation of Oilseed Cakes in the Formulation of Vegan Yoghurt-like Fermented Beverages

Abstract

The sustainable valorisation of agro-industrial by-products offers a promising pathway to address global protein demand while supporting circular food systems. This study explored the biotechnological potential of pumpkin (Cucurbita pepo), sunflower (Helianthus annuus), and walnut (Juglans regia L.) oilseed cakes as substrates for developing vegan yoghurt-like fermented beverages. Each formulation was fermented with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and comprehensively evaluated for composition, functionality, sensory properties, and bioactivity. The oilseed-based beverages exhibited protein levels between 3.7–4.6%, fibre content up to 1.9%, and reduced syneresis (14–18%) compared with the commercial almond-based product (21.5%). The walnut-based variant (WOCY) showed the highest total polyphenol content (1108.97 mg GAE kg⁻¹) and antioxidant activity (412.54 mg Trolox kg⁻¹ DPPH; 51.5 mg TE g⁻¹ DW ABTS), surpassing both the almond-based vegan yoghurt (238.82 mg GAE kg⁻¹) and dairy reference (96.10 mg GAE kg⁻¹). Preliminary sensory profiling through the Check-All-That-Apply (CATA) method indicated that pumpkin- and walnut-based samples were most associated with “creamy texture,” “nutty aroma,” and “very pleasant” descriptors, achieving acceptance comparable to or higher than conventional yoghurts. Microbiological analyses confirmed product safety and high viable cell counts (<10⁸ CFU mL⁻¹) after 14 days of storage. Oilseed cakes serve as efficient substrates for producing nutrient-dense, antioxidant-rich, and sensorially appealing plant-based fermented beverages, supporting functional food innovation and promoting circular bioeconomy through the sustainable valorisation of agro-industrial by-products.

1. Introduction

The global food system faces increasing challenges in meeting the rising demand for protein, especially of animal origin. Population growth, urbanisation, and changing dietary preferences are expected to intensify pressure on livestock production, which already contributes significantly to environmental degradation. Livestock farming is responsible for approximately 14.5% of total anthropogenic greenhouse gas emissions, in addition to extensive land use, deforestation, and water consumption [1]. These impacts raise concerns about the long-term sustainability of animal protein supply chains. At the same time, the prevalence of non-communicable diseases associated with high-saturated fat, cholesterol-rich diets has heightened consumer concern about healthier alternatives [2]. Together, these considerations have intensified the pursuit of sustainable, healthy, and inexpensive sources of protein that may supplement or, in their place, replace traditional animal-based proteins [3,4]. One of the most promising approaches to addressing the protein shortage is to investigate plant-based alternatives. Various sources, such as legumes, cereals, oilseeds, algae, and microbial biomass, have been explored for their ability to provide key amino acids and functional properties suitable for food-based applications [5]. These options cater to consumer interest in plant-based products, aligning with vegan, vegetarian, and flexitarian diets that are gaining global attention. In particular, the global market for plant-based products will continue to grow due to perceived health benefits, environmental friendliness, and moral concerns about animal welfare [6]. Developing protein-rich plant-based products not only supports dietary diversification but also contributes to the circular bioeconomy by valorising underutilised agricultural and industrial by-products [7,8,9].

Among plant-based innovations, fermented foods occupy a unique niche because of their cultural acceptance, sensory appeal, and health-promoting potential. Fermentation enhances digestibility, improves nutrient bioavailability, and enriches foods with bioactive metabolites, including organic acids, peptides, and exopolysaccharides [10]. Traditional dairy-based fermented beverages, such as yoghurt and kefir, have long been valued for their probiotic content and nutritional attributes [11,12]. Their plant-based analogues, produced from soy, oat, almond, and coconut, are now widely available, offering consumers familiar sensory experiences without animal ingredients [13]. Despite their success, these alternatives are not without limitations. Plant-based milks often exhibit weaker gelling capacity, lower protein content, and off-flavours compared to dairy, which can reduce consumer acceptance [14]. Additionally, reliance on commodity crops such as soy and almonds raises concerns about biodiversity, allergenicity, and water use [15]. Thus, the development of new protein substrates for fermented vegan beverages remains an important research direction.

In this context, oilseed cakes, which are also known as oilseed meals but are obtained by mechanical pressing of seeds and typically contain 8–15% fat, represent an underexploited but up-and-coming resource [16]. These by-products, generated during the extraction of oils from seeds such as hemp, sunflower, pumpkin, rapeseed, flax and so on, are produced in substantial quantities worldwide [17]. Rich in proteins, fibres, and residual bioactive compounds, oilseed cakes combine nutritional density with sustainability advantages, as their valorisation reduces waste streams from the fat-and-oil industry [18,19]. Recent research has demonstrated that proteins extracted from oilseed meals exhibit favourable techno-functional properties, including emulsification, foaming, and water-holding capacity, making them suitable for dairy analogues [20]. Moreover, fermentation of oilseed-based substrates by lactic acid bacteria not only enhances flavour and texture but also reduces anti-nutritional factors, such as phytates and tannins, improving overall digestibility [21].

Several studies highlight the biotechnological potential of oilseed cakes in functional food development. For example, hempseed meal has been shown to provide high-quality protein with a balanced amino acid profile, as well as phenolic compounds with antioxidant activity [22]. Sunflower and rapeseed meals are also rich in bioactive peptides and dietary fibres, which contribute to improved gut health and metabolic regulation [23]. The incorporation of such ingredients into yoghurt-like fermented products aligns with consumer expectations for sustainable, high-protein, and probiotic-rich foods [24]. Furthermore, by valorising these by-products, the food industry can strengthen circular economy practices and reduce dependence on conventional raw materials [25,26].

Given these opportunities, the present study explores the biotechnological valorisation of local oilseed cake in the formulation of a vegan yoghurt-like fermented beverage. The main objective is to evaluate the nutritional, functional, and sensory characteristics of oilseed cake-based substrates after fermentation with selected lactic acid bacteria. This approach not only seeks to develop innovative plant-based alternatives with enhanced protein quality and bioactivity but also contributes to sustainable resource management in agri-food systems.

2. Materials and Methods

2.1. Reagents and Comparative Samples

All biochemical and chemical reagents used in this study were supplied by Sigma-Aldrich (Schnelldorf, Germany), unless indicated otherwise.

For comparative purposes, two commercial references were included. A control vegan yoghurt (almond-based yoghurt, Terra Bianca SRL, Calaras, Republic of Moldova; cup packaging, 230 g unit, declared shelf-life 15 days) was selected as a representative plant-based product. According to the label, this product contained potable water, sweet almonds (19%), thickener (pectin), and vegan yoghurt starter cultures (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. Bulgaricus). Additionally, a dairy yoghurt (classic yoghurt, Ltd. JLC, Chisinau, Republic of Moldova; 2.5% fat, cup packaging, 140 g unit, declared shelf-life 10 days) was used as the conventional reference. According to its technical sheet, the dairy yoghurt contained cow’s milk and yoghurt starter cultures (S. thermophilus and L. delbrueckii ssp. bulgaricus).

2.2. Materials Collection and Samples Preparation

By-products from the oil and fat industry were sourced to assess their technological potential in fermented plant-based products. In particular, oilseed cakes derived from sunflower (Helianthus annuus), pumpkin (Cucurbita pepo), and walnut (Juglans regia L.) were collected from local oil-processing facilities in the Republic of Moldova (Mira Ltd., Strășeni, Republic of Moldova). Approximately 2 kg of each type of oilseed cake was sampled from different points of the receiving hopper to account for intra-batch variability. The cakes were provided in slab form, vacuum-sealed, and stored at 3 ± 2 °C until further processing.

Before experimental investigations, the oilseed cakes were mechanically ground into fine powders and subsequently sieved through a 150 µm sieve to ensure homogeneity, facilitate hydration, and improve their applicability in developing a vegan yoghurt-like fermented beverage. For fermentation, a commercial vegan yoghurt starter culture containing Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp bulgaricus, Lactobacillus rhamnosus and Streptococcus thermophilus (Cultures for Health, Durham, NC, USA) was used, stored according to the manufacturer’s instructions at refrigeration temperature (4 ± 2 °C) until inoculation.

2.3. Methods

2.3.1. Fermentation and Preparation of Yoghurt-like Samples

For the preparation of yoghurt-like fermented beverages, oilseed cakes (pumpkin, sunflower, or walnut) were first mechanically ground into fine powders and subsequently sieved to obtain a uniform particle size of 150 µm. A portion of 115 g of the sieved material was suspended in 600 mL of distilled water preheated to 80–90 °C and allowed to hydrate for 8 h. The mixtures were homogenised using a high-speed blender, then subjected to thermal treatment by boiling at 100 °C for 20 min, followed by cooling to 30–42 °C. After the addition and complete dissolution of 15 g sucrose, 2 g of freeze-dried vegan yogurt starter cultures were inoculated. Although S. thermophilus and L. delbrueckii ssp. bulgaricus are traditionally associated with lactose metabolism and show weaker growth in lactose-free substrates, the commercial starter culture used in this study also included a broader consortium of strains adapted to plant-based matrices—Bifidobacterium bifidum, Lactobacillus acidophilus, Lb. casei and Lb. rhamnosus. These species are well documented for their ability to efficiently ferment simple carbohydrates such as sucrose, glucose or fructooligosaccharides and for their robustness in non-dairy environments. Thus, the presence of multiple probiotic lactobacilli and bifidobacteria compensated for the limited lactose-fermenting capacity of the classical yoghurt strains, ensuring reliable acidification and desirable aroma development in oilseed-based substrates. The addition of 15 g sucrose was selected based on preliminary optimisation experiments, which showed that this level provided sufficient fermentable substrate to support balanced growth of both yoghurt bacteria and probiotic strains, while preventing excessive sweetness or osmotic stress. Similar sucrose supplementation levels are commonly applied in plant-based fermentations to compensate for the absence of lactose [27,28].

The formulations were portioned into sterile containers and incubated for 6 h at 42 ± 2 °C. Upon completion of fermentation, the yoghurt-like beverages were immediately cooled to 6 ± 1 °C and stored under refrigeration for up to 15 days. The procedure was adapted from methods previously described by Łopusiewicz (2022) for camelina press cake, with minor modifications regarding hydration and thermal treatment steps [20].

The fermentation and preparation method was developed and patented by our group at the Technical University of Moldova [29].

2.3.2. Proximate Composition Analysis

The proximate composition of the yoghurt-like samples and oilseed cakes was determined according to internationally recognised ISO and AOAC standard procedures. Moisture content was determined gravimetrically by oven-drying the samples at 103 ± 2 °C until a constant weight was achieved, following the ISO 6731/IDF 21:2010 method for total solids determination [30]. Ash content was determined by dry mineralisation in a muffle furnace at 550 °C until a white residue was obtained, according to AOAC 923.03 [31]. The obtained mineral fraction was expressed as a percentage on a dry matter basis. Crude protein content was determined by the Kjeldahl method, using a nitrogen-to-protein conversion factor of 6.25, in accordance with AOAC 2001.11 [32]. The results were expressed on a dry matter basis. Crude fat was determined by the gravimetric reference method described in ISO 1211/IDF 1:2010, employing petroleum ether as the extraction solvent in a Soxhlet apparatus [33]. The fat content was reported as a percentage of dry matter. Crude fibre was analysed by the enzymatic-gravimetric method according to AOAC 985.29, which includes sequential enzymatic digestion with α-amylase, protease, and amyloglucosidase, followed by gravimetric quantification of the insoluble residue [34]. The results were expressed as a percentage of dry matter. Total carbohydrates were calculated by difference using the standard proximate analysis approach, as nitrogen-free extract (NFE), according to the equation:

$$\mathrm{Carbohydrates}(\%)=100-\left(\mathrm{moisture}+\mathrm{protein}+\mathrm{fat}+\mathrm{ash}+\mathrm{fibre}\right)$$

(1)

This value represents the soluble carbohydrate fraction, mainly sugars and starches, not included in crude fibre. All determinations were performed in triplicate.

2.3.3. Titrable Acidity

The total acidity of the samples was determined using a potentiometric titration method AOAC 942.15 [35]. A 0.1 M sodium hydroxide solution was gradually added to the sample until the pH reached 8.1. The acidity was calculated as grams of lactic acid per 100 g of yogurt, according to Equation (2). All measurements were carried out in triplicate.

$$TA\left(\%asLacticacid\right)={\displaystyle \frac{V_{NaOH}\times 0.1\times 0.09008\times 100}{V_{sample}}}$$

(2)

where 90.08 g/mol is the molar mass of lactic acid.

2.3.4. Density

The density of the yoghurt samples was determined using the pycnometric method, as described in AOAC 925.22 [36]. A clean and dry 25 mL glass pycnometer was weighed empty and subsequently filled with distilled water at 20 ± 0.1 °C to determine its exact volume. The same pycnometer was then filled with the yoghurt sample, ensuring the absence of air bubbles, and reweighed under identical temperature conditions [36]. The density (ρ) was calculated as the ratio between the mass of the sample and the corresponding volume, using the following equation:

$$\rho ={\displaystyle \frac{m_2-m_0}{V}}$$

(3)

where $m_0$ is the mass of the empty pycnometer (g), $m_2$ is the mass of the pycnometer filled with sample (g) and $V$ is the volume of the pycnometer (mL).

All measurements were performed in triplicate, and results were expressed in g·cm⁻³ at 20 °C.

2.3.5. Viscosity

The apparent viscosity of the yoghurt samples was measured using an IKA ROTAVISC me-vi rotational rheometer (IKA Werke, Staufen im Breisgau, Germany) equipped with spindle No. 8. Measurements were carried out at 21 ± 2 °C, and the viscosity was determined at a constant rotational speed of 40 rpm for 5 min. The apparent viscosity was calculated automatically by the rheometer software based on the torque measured during rotation. All analyses were performed in triplicate, and the results were expressed in Pascal-seconds (Pa·s) [37].

2.3.6. Syneresis

Syneresis was determined after 120 h of refrigerated storage (2 ± 1 °C) by centrifuging in a Hettich EBA 20S centrifuge (Hettich, Tuttlingen, Germany) 10× g of yoghurt at 1600 rpm for 10 min. The expelled serum was subsequently collected and weighed on a laboratory analytical scale to quantify the amount of separated liquid [37].

The syneresis index was expressed as the percentage of serum separated relative to the initial sample weight, calculated as:

$$\mathrm{S}\mathrm{y}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{i}\mathrm{s}(\%)={\displaystyle \frac{m_{serum}}{10}}\times 100$$

(4)

where m_serum is the mass (g) of the separated whey.

2.3.7. Preliminary Sensory Evaluation by the Check-All-That-Apply (CATA) Method

The preliminary sensory evaluation of the yoghurt-like fermented beverages was conducted using the Check-All-That-Apply (CATA) method, a consumer-oriented technique designed to capture descriptive sensory information from spontaneous perceptions. A panel of 58 untrained assessors (aged 20–55, both genders) who regularly consume fermented dairy and plant-based products participated voluntarily in the evaluation. The sensory study is preliminary due to the small number of consumers in the test. This can result in the population being non-representative and may reduce the ability to differentiate among samples. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Technical University of Moldova (project identification code and name: 23.70105.5107.06T “Valorization of vegetable proteins from secondary products of the local fat and oil industry (ProVeg)”, 2 January 2024).

Before assessment, the samples were coded with random three-digit numbers and presented in a randomised, balanced order to minimise positional bias. Each participant received five coded samples, corresponding to the three experimental formulations—POCY (pumpkin oilseed cake), WOCY (walnut oilseed cake), and SOCY (sunflower oilseed cake)—and two commercial references: VYC (vegan almond-based yoghurt) and DYC (dairy yoghurt). All samples were served at 7 ± 1 °C, in identical transparent plastic cups (50 mL per portion), under controlled laboratory conditions (artificial daylight, 21 ± 2 °C, odour-free environment).

Participants were instructed to taste each sample sequentially and to check all sensory terms they considered appropriate from a predefined list of attributes (

Table 1. CATA attributes for yoghurt-like beverages.

Attribute Category	CATA Attributes
Visual Aspect	homogeneous appearance, visible particles, creamy texture, dull appearance, phase separation, sediment formation, unappealing look
Colour	light beige, yellowish tone, greyish hue, brownish tint, natural colour, artificial colour, uniform colour, non-uniform colour
Odour	nutty aroma, vegetal odour, earthy odour, fermented odour, sour odour, pleasant odour, unpleasant odour
Flavour	mild taste, bitter taste, sour taste, sweet taste, unpleasant flavour
Aftertaste	clean finish, nutty aftertaste, bitter aftertaste, acidic aftertaste, lingering flavour, unpleasant residual taste
Mouthfeel	smooth, creamy, viscous, watery, thick, grainy, pleasant mouthfeel, unpleasant mouthfeel
Overall Acceptability	very pleasant, acceptable, neutral, slightly unpleasant, unpleasant
Emotional Response	comforting, natural, surprising, familiar, strange, unappealing

). The list included eight attribute categories: visual aspect, colour, odour, flavour, aftertaste, mouthfeel, overall acceptability, and emotional response. These attributes were developed based on preliminary focus group discussions, product-specific sensory lexicons, and previous research on plant-based fermented beverages.

The frequency of citation for each descriptor was recorded and subsequently used for statistical processing. The significance of differences in attribute selection among samples was tested using Cochran’s Q test (p < 0.05). Correspondence Analysis further analysed attributes showing significant variation to explore the sensory positioning of each formulation relative to the reference products. All statistical analyses were performed using XLStat software (version 7.5.2, Addinsoft, Paris, France), integrated into Microsoft Excel.

2.3.8. Total Polyphenols

The total polyphenolic content (TPC) of raw materials and yoghurt-like beverages was determined using the Folin–Ciocalteu colorimetric method [38]. Briefly, 1 mL of appropriately diluted sample extract was mixed with 5 mL of 10% (v/v) Folin–Ciocalteu reagent and incubated at room temperature for 5 min. Subsequently, 4 mL of 7.5% (w/v) sodium carbonate solution was added, and the mixture was incubated in the dark for 30 min. Absorbance was measured at 765 nm using a UV-Vis spectrophotometer (Shimadzu UV-1800, Kyoto, Japan). Results were calculated against a calibration curve prepared with gallic acid and expressed as mg gallic acid equivalents per 1000 g of sample (mg GAE 100 kg⁻¹).

2.3.9. DPPH Radical Scavenging Activity

Antioxidant capacity was evaluated by measuring the ability of the samples to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, according to the method described by Lin and Zhou (2018) [39] with slight modifications. A 0.1 mM methanolic DPPH solution was freshly prepared, and 1 mL of the sample was added to 3 mL of the DPPH solution. After incubation for 30 min in the dark at room temperature, absorbance was measured at 517 nm [39]. Results were expressed as mg Trolox equivalents per kg of sample.

2.3.10. ABTS Radical Cation Scavenging Activity

Antioxidant activity was evaluated using the ABTS•⁺ radical cation decolourisation assay according to Arnao (2001) [40], with minor adaptations for yoghurt matrices. The ABTS•⁺ solution was prepared by reacting 7 mM ABTS with 2.45 mM potassium persulfate, incubating the mixture in the dark for 16 h, and diluting to an absorbance of 0.700 ± 0.02 at 734 nm. An aliquot of 0.1 mL of yoghurt was mixed with 3.9 mL of ABTS•⁺ working solution, and absorbance was recorded at 734 nm after 6 min [40]. Results were expressed as mg Trolox equivalents per g dry weight (mg TE g⁻¹ DW), based on a Trolox calibration curve (0–500 μmol L⁻¹, R² = 0.9996).

2.3.11. Microbiological Analyses of Yoghurt-like Beverages

Sample Preparation, Dilutions and Plate Counts

Test portions of 10 g were aseptically transferred into sterile diluent (0.1% peptone water) and homogenised. Decimal dilutions were prepared according to ISO 6887-1:2017 [41]. Plate selection, countability ranges, confirmation rules (where applicable), and calculation of results followed ISO 7218:2007 [42]. The counts were expressed as colony-forming units per millilitre of product (CFU mL⁻¹).

Total Viable Counts

Total aerobic mesophilic microorganisms were enumerated by the colony-count technique at 30 °C on Plate Count Agar (pour plate method), following ISO 4833-1:2013 [43]. Plates were incubated for 48 ± 3 h, and countable plates (10–300 colonies) were used to calculate the number of colony-forming units per millilitre of product (CFU mL⁻¹).

Coliforms

Coliform bacteria were enumerated on Violet Red Bile Lactose (VRBL) agar using the colony-count technique, according to ISO 4832:2006. Plates were incubated at 37 °C for 24 ± 2 h, and typical purplish-red lactose-fermenting colonies, with or without a precipitated bile halo, were counted [44].

Yeasts and Moulds

Yeasts and moulds were enumerated by the colony-count technique according to ISO 21527-1:2008, using Potato Dextrose Agar (PDA), suitable for products with water activity greater than 0.95. Plates were incubated at 25 ± 1 °C for 5 days [45]. Results were expressed as colony-forming units per millilitre of product (CFU mL⁻¹).

2.3.12. Statistical Analysis

All data were expressed as mean ± standard deviation (SD). Statistical significance was determined using a two-way analysis of variance (ANOVA) followed by an NIR Fisher test. The values were considered significantly different when p < 0.05. All analyses were performed with Statistica version 10 (StatSoft Polska, Kraków, Poland).

3. Results

3.1. Physicochemical Characteristics and Composition of Oilseed Cakes

The proximate composition of pumpkin, sunflower and walnut oilseed cakes was determined to establish their technological potential as substrates for vegan yoghurt-like fermented beverages. The results are presented in

Table 2. Physicochemical characteristics and composition of oilseed cakes used as raw materials.

Parameter	POC	WOC	SOC	Reference Values (Literature Range)
Moisture (%)	7.2 ± 0.2 a	8.0 ± 0.3 b	8.1 ± 0.3 b	6–9
Ash (% d.m.)	1.42 ± 0.10 a	5.54 ± 0.15 bc	5.0 ± 0.20 b	4.5–6.0
Acidity (degrees)	6.60 ± 0.25 a	13.5 ± 0.4 c	12.5 ± 0.3 b	12–14
Crude protein (% d.m.)	59.8 ± 1.8 c	49.9 ± 1.5 b	44.0 ± 1.3 a	43–52
Crude fat (% factual)	14.6 ± 0.7 c	7.9 ± 0.5 a	9.0 ± 0.6 b	8–12
Crude fat (% d.m.)	15.7 ± 0.8 c	8.6 ± 0.6 a	9.5 ± 0.7 b	8–12
Fibre (% d.m.)	10.5 ± 0.4 a	12.1 ± 0.5 b	14.2 ± 0.6 c	10–15
Carbohydrates (NFE, % d.m.)	12.7 ± 0.5 a	23.9 ± 0.6 b	27.2 ± 0.7 c	18–25

Results indicate the mean value of three independent assays and are expressed as mean ± standard deviation (SD). In each row, different letters (a–c) denote significant differences (p ≤ 0.05). Abbreviations: POC—samples of pumpkin oilseed cake; WOC—samples of walnut oilseed cake; SOC—samples of sunflower oilseed cake.

, where all values except for moisture are expressed on a dry matter basis (% d.m.) to ensure comparability between samples. Carbohydrates are presented as nitrogen-free extract (NFE), as described in the Methods section. For reference, values from the literature are also included, providing typical ranges reported for oilseed cakes [46,47,48].

The results indicate that all three oilseed by-products exhibit promising nutritional characteristics for use in plant-based fermented beverages. According to Table 2, the protein and fat values of POC and WOC fall within the ranges reported in the literature, whereas SOC showed lower values, which may be attributed to technological aspects of oil pressing and its comparatively higher ash content. Pumpkin oilseed cake showed the highest protein concentration (59.8% d.m.) combined with moderate lipid levels, providing a well-balanced substrate with strong gelling and water-binding potential attributed to its fibre fraction. Walnut oilseed cake, although containing slightly less protein (49.9% d.m.), displayed higher ash content (5.5%), which may reflect its mineral-rich composition and potential functional contribution to buffering capacity during fermentation. Sunflower oilseed cake had the lowest protein value (44.0% d.m.) but contained moderate lipid fractions (~9.0%), which may enhance flavour and creaminess, although excessive fat could negatively influence syneresis and microbial fermentation.

Overall, the proximate composition of all three cakes supports their suitability as fermentation substrates. Their high protein levels exceed those of many cereal-based raw materials, while moderate fat and fibre contents provide functionality for structure formation and bioactive enrichment. These properties indicate that pumpkin, sunflower, and walnut oilseed cakes represent valuable resources for the development of vegan yoghurt- and kefir-like beverages, aligning with circular bioeconomy principles by valorising agro-industrial by-products.

3.2. Proximate Composition of Yoghurt-like Beverages

The proximate composition of the vegan yoghurt-like beverages formulated with different oilseed cakes is presented in

Table 3. Comparative proximate composition of yoghurt-like beverages and reference samples.

Parameter	POCY	WOCY	SOCY	VYC *	DYC *
Moisture (%)	88.5 ± 0.3 b	88.0 ± 0.2 b	87.2 ± 0.4 a	89.35	86.6
Protein (%)	4.6 ± 0.1 c	3.7 ± 0.2 a	4.2 ± 0.1 b	2.1	3.4
Fat (%)	2.3 ± 0.1 c	1.4 ± 0.1 b	1.2 ± 0.1 a	8.7	2.7
Carbohydrates (%)	3.3 ± 0.2 b	3.9 ± 0.2 c	4.8 ± 0.3 e	1.9	4.6
Fibre (%)	1.8 ± 0.1 bc	1.5 ± 0.15 a	1.9 ± 0.1 c	1.7	–
Ash (%)	0.21 ± 0.01 a	0.73 ± 0.02 c	0.80 ± 0.02 d	0.58	0.75

Results indicate the mean value of three independent assays and are expressed as mean ± standard deviation (SD). In each row, different letters (a–e) denote significant differences (p ≤ 0.05). Abbreviations: POCY—yoghurt sample based on pumpkin oilseed cake; WOCY—yoghurt sample based on walnut oilseed cake; SOCY—yoghurt sample based on sunflower oilseed cake; VYC—commercial vegan yoghurt (reference sample); DYC—commercial dairy yoghurt (reference sample). * The values were taken from the technical specification of the finished product.

. Notable differences were observed among the formulations, particularly in protein, fat, and fibre contents, reflecting the nutritional composition of the respective oilseed cakes. Compared to the commercial vegan yoghurt (based on almond kernels), enrichment with pumpkin, walnut, and sunflower oilseed cakes resulted in significantly higher protein levels. At the same time, fat content was markedly lower than in the commercial vegan sample. Compared with commercial dairy yoghurt, the oilseed-based beverages exhibited a higher fibre content, which may contribute to improved digestive function and enhanced nutritional value. Moreover, the obtained formulations demonstrated protein concentrations comparable to, or even exceeding, those of commercial dairy yoghurt, indicating their potential to compete with traditional dairy products in terms of protein quality.

Although pumpkin oilseed cake contained the highest total protein on a dry matter basis (59.8%), the corresponding yoghurt-like beverage also retained the highest protein concentration (4.6%), followed by the sunflower-based formulation (4.2%) and the walnut-based beverage (3.7%). This distribution reflects differences in protein extractability and solubility, which vary markedly among oilseed sources. In pumpkin oilseed cake, globulins and albumins are partially associated with insoluble dietary fibre and phenolic compounds, yet a significant fraction remains recoverable into the aqueous phase during preparation and fermentation [49,50]. By contrast, walnut oilseed cake proteins—although present at a moderate level in the raw material (49.9% d.m.)—show limited extractability due to their compact structure and intense interactions with tannins and phenolics, which restrict their transfer into the liquid matrix [51,52]. Sunflower oilseed cake proteins, dominated by storage globulins such as helianthinin, display only moderate solubility under aqueous conditions [53], resulting in an intermediate protein level in the final beverage (4.2%). The obtained results further demonstrate that oilseed cake–based formulations provide a nutritionally enriched alternative to standard commercial vegan yoghurts produced from whole seeds or nuts. Protein levels exceeded those of the vegan reference sample. They were comparable to, or in some cases higher than, those of commercial dairy yoghurt, while the dietary fibre fraction—absent in conventional yoghurt—was significantly increased, particularly in pumpkin- and sunflower-based formulations. Fat levels remained moderate, indicating a favourable lipid profile for functional food development. These characteristics confirm that oilseed cake matrices are suitable substrates for lactic fermentation, supporting microbial growth and contributing to desirable textural and sensory properties. Therefore, the incorporation of oilseed by-products into fermented beverages is not only technologically feasible but also nutritionally advantageous, aligning with current strategies of sustainable valorisation of agro-industrial residues.

3.3. Physicochemical Characteristics of Yoghurt-like Beverages

The physicochemical and technological parameters of the yoghurt-like beverages formulated with pumpkin, sunflower, and walnut oilseed cakes are presented in

Table 4. Physico-chemical and technological parameters of yoghurt-like beverages.

Parameter	POCY	WOCY	SOCY	VYC	DYC
Acidity (% lactic acid)	0.87 ± 0.01 a	0.98 ± 0.02 b	0.77 ± 0.03 a	0.81 ± 0.02 a	0.83 ± 0.02 a
Density (g·cm−3)	1.033 ± 0.003 a	1.019 ± 0.002 a	1.012 ± 0.002 a	0.981 ± 0.001 a	0.985 ± 0.004 a
Viscosity (Pa·s, 20 °C)	0.91 ± 0.02 b	0.97 ± 0.01 bc	0.87 ± 0.03 b	0.63 ± 0.06 a	1.30 ± 0.02 c
Syneresis, 120 h storage (%)	14.0 ± 1.0 b	16.5 ± 0.5 c	18.0 ± 1.0 d	21.5 ± 0.5 e	11.0 ± 1.0 a

Results indicate the mean value of three independent assays and are expressed as mean ± standard deviation (SD). In each row, different letters (a–e) denote significant differences (p ≤ 0.05). Abbreviations: POCY—yoghurt sample based on pumpkin oilseed cake; WOCY—yoghurt sample based on walnut oilseed cake; SOCY—yoghurt sample based on sunflower oilseed cake; VYC—commercial vegan yoghurt (reference sample); DYC—commercial dairy yoghurt (reference sample).

. These characteristics—ash, acidity, density, viscosity, and syneresis—are critical indicators of nutritional quality, fermentation dynamics, and product stability. Comparing oilseed-enriched yoghurts with control vegan and dairy yoghurts allows for the assessment of how effectively these by-products can serve as functional substrates for lactic acid fermentation.

It should be noted that acidity, density and viscosity presented in Table 4 were determined at time 0, immediately after fermentation, whereas the syneresis values correspond to measurements performed after 120 h of refrigerated storage, as defined in the Methods section. The technological parameters of the yoghurt-like beverages presented in Table 4 revealed certain inconsistencies with the compositional characteristics of the respective oilseed cakes, particularly regarding acidity, viscosity, and syneresis. Although the beverage based on sunflower oilseed cake contained the highest amount of fermentable carbohydrates (≈4.8%) and exhibited a relatively low protein solubility, its final acidity (0.77% lactic acid) was the lowest among all samples. This apparent deviation can be attributed to the high buffering capacity of sunflower proteins (mainly globulins such as helianthinin) and their strong interactions with phenolic compounds and minerals, which tend to stabilise the medium pH and limit the accumulation of free lactic acid during fermentation. Moreover, the abundant lignocellulosic fibre fraction in sunflower cake contributed to a weaker gel structure, which explains the highest syneresis value (18%) despite its relatively high total fibre content. Similar structural effects have been previously reported for plant matrices rich in insoluble fibre, which hinder water retention and create micro-channels facilitating serum separation during fermentation [50,54].

In contrast, the yoghurt-like beverage obtained from walnut oilseed cake displayed the highest titratable acidity (0.98%) and viscosity (0.97 Pa·s), even though its protein content was lower than in the pumpkin-based formulation. This behaviour can be explained by the hydrophobic and phenolic-rich nature of walnut proteins, which promote extensive protein–polyphenol aggregation and gel densification, leading to a more viscous, spoonable texture. However, the exact mechanism may induce localised protein over-coagulation, resulting in partial whey expulsion and a slightly higher syneresis (16.5%) than theoretically expected.

Among the studied samples, the pumpkin oilseed cake beverage showed the most balanced profile—moderate acidity (0.87%), relatively high density (1.033 g·cm⁻³), and low syneresis (14%)—reflecting an optimal ratio between soluble protein fractions (albumins and globulins) and functional dietary fibre. The cohesive network formed during fermentation ensured good water retention and stability, approaching that of commercial dairy yoghurt (reference sample), which remained the most viscous (1.30 Pa·s) and least prone to whey separation (11%).

Compared with the commercial vegan yoghurt (reference sample) based on almond kernels, all oilseed-cake formulations exhibited higher density, improved viscosity, and markedly lower syneresis, confirming that the incorporation of protein- and fibre-rich by-products enhances the rheological stability and water-holding capacity of plant-based fermented systems. These findings emphasise that the technological performance of oilseed cakes depends not only on the quantitative levels of proteins and fibres but also on their qualitative attributes—including solubility, molecular conformation, and the extent of phenolic cross-linking—which collectively define acidification kinetics and gel microstructure during lactic fermentation. Consequently, the pumpkin-based formulation appears the most promising substrate for developing functional, clean-label, and sustainable yoghurt-like products with improved textural and sensory characteristics.

3.4. Preliminary Sensory Evaluation of Yoghurt-like Beverages

The preliminary sensory characterisation of the yoghurt-like fermented beverages aimed to assess the influence of different oilseed cakes (pumpkin, walnut, and sunflower) on consumer perception and product acceptability. The Check-All-That-Apply (CATA) approach was applied to capture qualitative sensory differences among the experimental formulations and commercial references. This analysis provides an integrated understanding of how the type of plant matrix affects the visual, aromatic, gustatory, and emotional attributes of the vegan yoghurt-like products. From the full list of sensory descriptors defined for the CATA questionnaire, a total of 23 attributes were selected for statistical processing based on their citation frequency and relevance to consumer perception.

The results of the CATA sensory evaluation were analysed through correspondence analysis, revealing a well-structured sensory space, with the first two principal dimensions (F1 and F2) accounting for 87.37% of total variance (Figure 1). This high explanatory power indicates a robust differentiation between the tested yoghurt-like beverages and their reference counterparts. Among the experimental samples, the pumpkin-based yoghurt (POCY) and walnut-based yoghurt (WOCY) were most positively associated with desirable sensory attributes such as “very pleasant,” “nutty aroma,” “creamy texture,” and “fermented odour.” These descriptors suggest that the fermentation of these oilseed matrices resulted in a harmonious flavour profile and favourable mouthfeel, likely due to their intrinsic lipid and protein content contributing to enhanced emulsification and volatile compound development. Comparable results have been observed in dairy yoghurts, where aroma and textural uniformity are recognised as primary sensory drivers of consumer preference [55,56].

Notably, the walnut-based formulation was also linked to “surprising” and “grainy” attributes, indicating a distinct sensory character that could polarise consumers but may also serve as a point of differentiation in the plant-based dairy alternatives market. In contrast, the sunflower-based yoghurt (SOCY) was perceived as the least favourable, clustering near negative attributes such as “vegetal odour,” “greyish hue,” “visible particles,” and “watery.” These results are consistent with literature describing challenges related to coarse texture and astringent flavours in products based on sunflower meal, primarily due to higher insoluble fibre and residual phenolics [57].

The commercial dairy yoghurt (DYC), serving as a reference standard, was aligned with “light beige,” “uniform colour,” “pleasant mouthfeel,” and “mild taste,” confirming its sensory stability and expected consumer familiarity, in agreement with previous sensory benchmarks for fermented milks [58]. Meanwhile, the commercial almond-based vegan yoghurt (VYC) occupied a neutral position, close to “clean finish,” “natural,” and “acceptable,” reflecting its moderate sensory appeal but lack of standout attributes—a finding also reported by Grasso (2020) and Gupto (2022), who identified the sensory neutrality and flavour masking strategies as common limitations of commercial vegan yoghurts [59,60].

From the initial pool of 52 sensory attributes grouped into eight descriptive categories, 23 terms were retained for final analysis based on their significant discriminatory power as determined by Cochran’s Q test (p < 0.05). This refinement ensured that only the most perceptually relevant descriptors contributed to the construction of the sensory space. The resulting configuration clearly shows that the oilseed-derived yoghurt-like formulations—particularly those based on pumpkin (POCY) and walnut (WOCY) oilseed cakes—exhibit notable sensory advantages, with favourable descriptors such as a very pleasant, nutty aroma and a creamy texture. These findings demonstrate their high potential to rival or even exceed the hedonic and textural appeal of commercial plant-based yoghurt products.

3.5. Bioactive Profile of Yoghurt-like Beverages

The incorporation of oilseed cakes into fermented plant-based formulations aimed not only to enhance their protein and fibre content but also to valorise their inherent phytochemical richness. In this context, the total polyphenol content (TPC) and antioxidant activity (AA) of the yoghurt-like beverages were quantified using complementary analytical methods (Folin–Ciocalteu, DPPH•, and ABTS•⁺ assays), to assess their potential functional benefits.

Table 5. Comparative bioactive profile of yoghurt-like beverages and reference samples.

Parameter	POCY	WOCY	SOCY	VYC	DYC
Total polyphenol content (mg GAE kg−1)	564.85 ± 2.78 c	1108.97 ± 3.87 e	856.43 ± 4.42 d	238.82 ± 3.14 b	96.10 ± 2.80 a
DPPH (mg Trolox kg−1)	278.49 ± 4.33 c	412.54 ± 5.38 e	338.49 ± 4.54 d	125.05± 1.75 b	48.72 ± 2.24 a
ABTS, mg TE g−1 DW	37.6 ± 0.3 c	51.5 ± 0.6 e	44.9 ± 0.3 d	25.0 ± 0.4 b	12.3 ± 0.8 a

Results indicate the mean value of three independent assays and are expressed as mean ± standard deviation (SD). In each row, different letters (a–e) denote significant differences (p ≤ 0.05). Abbreviations: POCY—yoghurt sample based on pumpkin oilseed cake; WOCY—yoghurt sample based on walnut oilseed cake; SOCY—yoghurt sample based on sunflower oilseed cake; VYC—commercial vegan yoghurt (reference sample); DYC—commercial dairy yoghurt (reference sample).

presents the comparative bioactive profiles of the experimental samples and two commercial references.

The comparative analysis of the bioactive profiles of the experimental yoghurt-like formulations (POCY, WOCY, SOCY) reveals a significantly higher antioxidant potential than both the commercial vegan (VYC) and dairy-based (DYC) references. Among the plant-based prototypes, WOCY (walnut oilseed cake-based yoghurt) displayed the highest total polyphenol content (1108.97 ± 3.87 mg GAE kg⁻¹), followed by SOCY (856.43 ± 4.42 mg GAE kg⁻¹) and POCY (564.85 ± 2.78 mg GAE kg⁻¹). These values far exceed the polyphenolic concentrations recorded in VYC (238.82 ± 3.14 mg GAE kg⁻¹) and DYC (96.10 ± 2.80 mg GAE kg⁻¹), demonstrating a 4.6- to 11.5-fold increase, respectively. Such enhancement can be directly attributed to the inherent polyphenolic richness of oilseed cakes, especially those derived from walnut and sunflower, which are well-documented sources of flavonoids, phenolic acids, and lignans.

Comparable findings were reported by Bayram (2025), who demonstrated that polyphenol-enriched strained yoghurts supplemented with medicinal plant extracts could reach polyphenol concentrations exceeding 950 mg GAE kg⁻¹, significantly improving antioxidant capacity and sensory profile during storage [61]. Similarly, Al-Quwaie (2023) emphasised that fortifying yoghurt with Portulaca oleracea elevated its total phenolic content up to 770 mg GAE kg⁻¹, highlighting the feasibility of biotechnological enhancement of dairy analogues using plant-based materials [62].

In terms of radical scavenging activity, DPPH and ABTS assays confirmed the superior antioxidant status of the experimental samples. The DPPH radical scavenging capacity reached 412.54 ± 5.38 mg Trolox kg⁻¹ in WOCY, followed by 338.49 ± 4.54 mg kg⁻¹ in SOCY and 278.49 ± 4.33 mg kg⁻¹ in POCY. These levels are substantially higher than those recorded for commercial references (125.05 ± 1.75 mg kg⁻¹ for VYC and 48.72 ± 2.24 mg kg⁻¹ for DYC). This aligns with the data reported by Şanlıdere Aloğlu and Öner (2011), who noted that the DPPH activity of bioactive peptide fractions isolated from standard yoghurts typically ranges from 70 to 130 mg Trolox kg⁻¹, depending on fermentation time and the extent of protein hydrolysis [63].

The ABTS results mirrored the DPPH trend, confirming the antioxidant hierarchy: WOCY > SOCY > POCY. Notably, the WOCY sample exhibited an ABTS value of 51.5 ± 0.6 mg TE g⁻¹ DW, which is more than four times higher than that of DYC (12.3 ± 0.8 mg TE g⁻¹ DW) and twice that of VYC (25.0 ± 0.4 mg TE g⁻¹ DW). These data indicate that oilseed cake-derived formulations exhibit markedly elevated antioxidant potential, likely due to the synergistic presence of polyphenols, residual oils rich in unsaturated fatty acids, and possibly minor peptides formed during fermentation.

It is worth noting that the fermentation process, as well as the specific oilseed matrix, may have contributed to increased bioaccessibility of phenolic compounds and the formation of bioactive metabolites. Previous studies (e.g., Bulut et al., 2022; Gupta et al., 2022) have shown that plant-based protein matrices can not only preserve but, in some cases, enhance antioxidant potential when subjected to controlled fermentation with lactic acid bacteria [57,59].

The antioxidant and polyphenolic hierarchy WOCY > SOCY > POCY ≫ VYC > DYC is consistent with the biochemical composition of the oilseed cakes (Table 2) and the physicochemical profiling of the fermented beverages (Table 3 and Table 4). Walnut oilseed cake is known to contain significant amounts of ellagitannins, ellagic acid, gallic acid, flavonoids and proanthocyanidins, compounds with strong redox potential [64]. In fact, Brück et al. (2022) demonstrated that, during solid-state fermentation of walnut press cake, ellagitannins are hydrolysed, releasing free ellagic acid, thereby enhancing antioxidant availability [64]. This suggests that WOCY could leverage similar biochemical pathways during lactic fermentation to increase extractable phenolics.

In our data, WOCY’s higher fat content (1.4%) and moderate acidity (0.98% lactic acid) may have supported the solubilization and mobility of hydrophobic antioxidant compounds, improving performance in both DPPH and ABTS assays. Meanwhile, sunflower oilseed cake—rich in chlorogenic acid and caffeic acid—is constrained by its lignocellulosic matrix, whose binding interactions may limit phenolic release and explain its intermediate antioxidant metrics (SOCY). Studies of seed and nut cakes show that fibre matrices can trap phenolic compounds, reducing extractability [63]. Pumpkin oilseed cake, though highest in protein (59.8% d.m.) and favourable for gel structure, naturally contains fewer phenolic acids and thus exhibits the lowest antioxidant responses among the experimental formulations, albeit still markedly above the commercial references.

Technologically, the positive coupling between functional potential and product stability emerges clearly. Our oilseed-based beverages (POCY, SOCY, WOCY) demonstrated higher viscosity (0.87–0.97 Pa·s) and lower syneresis (14–18%) compared to the commercial vegan yoghurt (0.63 Pa·s; 21.5%), indicating that the protein–fibre network contributed to structural integrity while retaining phenolic compounds within the hydrogel matrix. This dual functionality—structural stabilisation plus bioactive retention—is essential for functional fermented beverages.

The dairy yoghurt (DYC), despite its microbial richness, showed minimal antioxidant activity, likely because milk lipids and the lacteal environment do not supply significant polyphenols, and are more prone to oxidative deterioration. This reinforces the functional advantage of plant-based formulations enriched with phenolic-rich by-products.

Comparisons with the literature further support these observations. Łopusiewicz et al. (2022) successfully used camelina press cake in yoghurt-like beverages [20]. They reported that fermentation boosted polyphenol levels and antioxidant activity over time, confirming that plant press cakes serve as effective functional enrichment agents [20]. Similarly, evaluation of seed and nut cakes showed that walnut and other oilseed cakes have significantly higher baseline polyphenolic content than many agricultural residues, suggesting promising substrates for bioactive fortification [65].

3.6. Microbiological Parameters of Yoghurt-like Beverages

The microbiological quality of yoghurt-like beverages enriched with pumpkin, sunflower, and walnut oilseed cakes was evaluated to assess the viability of starter cultures and the safety of the final products. Commercial dairy and vegan yoghurts were used as reference samples. The results are summarised in

Table 6. Microbiological profile of yoghurt-like beverages during storage.

Parameter	POCY	WOCY	SOCY	VYC	DYC
Total counts after fermentation (CFU mL−1)	5.4 × 105	4.9 × 105	4.1 × 105	4.6 × 105	3.6 × 105
Total counts after 14 days of storage (CFU mL−1)	9.7 × 106	8.6 × 107	8.3 × 107	7.9 × 107	1.2 × 107
Coliforms	NF	NF	NF	NF	NF
Yeasts and moulds	NF	NF	NF	NF	NF

Results indicate the mean value of three independent assays and are expressed as mean ± standard deviation (SD). Abbreviations: POCY—yoghurt sample based on pumpkin oilseed cake; WOCY—yoghurt sample based on walnut oilseed cake; SOCY—yoghurt sample based on sunflower oilseed cake; VYC—commercial vegan yoghurt (reference sample); DYC—commercial dairy yoghurt (reference sample); NF—not found.

.

A substantial presence of viable microorganisms was observed in all fermented samples, particularly in those containing oilseed cakes, which exhibited microbial profiles comparable to or slightly higher than those of the dairy yoghurt reference.

The microbial analysis of the yoghurt-like beverages provided valuable insights into their microbiological stability, safety, and potential shelf life. The compositional properties of the substrates and processing techniques likely influenced differences in microbial loads observed across the samples.

The viable counts recorded after fermentation indicated that all samples contained an adequate population of lactic acid bacteria, confirming successful fermentation [66]. The counts in plant-based yoghurt samples were moderately higher than in the dairy yoghurt reference immediately after fermentation, ranging between 4.1 × 10⁵ and 5.4 × 10⁵ CFU mL⁻¹. In contrast, the commercial vegan yoghurt exhibited 4.6 × 10⁵ CFU mL⁻¹ and the dairy yoghurt 3.6 × 10⁵ CFU mL⁻¹. The counts in plant-based yoghurt samples were moderately higher because the oilseed cake substrates provided additional nutrients, such as proteins and fermentable carbohydrates, that supported enhanced bacterial growth during fermentation [67].

After 14 days of refrigerated storage (2 ± 1 °C), microbial loads increased in all samples, ranging between 9.7 × 10⁶ and 8.6 × 10⁷ CFU mL⁻¹ in plant-based yoghurt samples and reaching 1.2 × 10⁷ CFU mL⁻¹ in DYC. Despite the observed increases, all values remained within the acceptable microbiological limit of 1 × 10⁸ CFU mL⁻¹, as recommended by the European Commission and the U.S. FDA for fermented milk and yoghurt products [68,69].

The increase in microbial counts during storage may be attributed to the nutrient-rich composition of oilseed cake substrates, which promotes microbial proliferation even under refrigerated conditions. Similar observations were reported by Asante et al. (2025), who demonstrated that the nutrient profile of plant-based substrates such as tiger nut milk supports microbial growth during yoghurt storage [70]. Importantly, no spoilage indicators such as coliforms, yeasts, or moulds were detected in any of the samples throughout the study, confirming good hygienic practices and effective fermentation. The slightly higher microbial load observed in the dairy yoghurt at day 14 may be explained by the greater availability of fermentable substrates in milk or differences in buffering capacity and microbial dynamics [70].

While all yoghurt samples demonstrated microbiological stability and safety throughout the 14-day storage period, the results highlight the importance of consistent temperature control, proper hygiene, and optimised formulation to maintain quality in both plant-based and dairy-based yoghurts. Coliforms were not detected during the 14 days of storage, confirming the hygienic quality of the processing environment [69].

Based on the obtained data and considering the potential microbiological and sensory risks identified during storage of the plant-based yoghurt samples, a graphical model was developed to summarise the recommended monitoring points for maintaining product quality (Figure 2). The overall suggested storage period for such products is up to 15 days under refrigerated conditions.

From a microbiological standpoint, day 14 proved to be the maximum acceptable storage limit for the yoghurt-like beverages. At this time point, all samples remained free of spoilage microorganisms (coliforms, yeasts and moulds), in compliance with the safety requirements of Regulation (EC) 2073/2005 [71] for ready-to-eat foods, while the viable lactic acid bacteria counts were still below the technologically accepted threshold of 1 × 10⁸ CFU mL⁻¹, a limit commonly referenced to prevent excessive post-acidification and textural destabilisation in fermented products. These findings are consistent with the FAO/WHO “Guidelines for the Evaluation of Probiotics in Food” (2001) [72] and the Codex Alimentarius Standard for Fermented Milks (CXS 243-2003) [73], which emphasise that probiotic viability and microbiological safety must be ensured throughout the declared shelf life. The concomitant preservation of high microbial viability and the absence of contamination therefore support the establishment of day 14 as the end of shelf life for the oilseed cake-based yoghurt-like beverages.

4. Conclusions

The present study demonstrated that oilseed cakes from pumpkin, sunflower, and walnut can be effectively biotransformed through lactic fermentation into yoghurt-like beverages with enhanced nutritional and functional quality. The formulated samples showed significant improvements over commercial plant-based and dairy yoghurts in terms of protein, polyphenol content, and antioxidant activity.

The formulations exhibited higher protein levels than both commercial plant-based and dairy yoghurts (e.g., up to 4.6% protein in the pumpkin-based sample), along with a leaner lipid profile and favourable density, viscosity and syneresis values.

The incorporation of oilseed cakes substantially enhanced the bioactive potential of the beverages. Total polyphenol content reached over 1100 mg GAE kg⁻¹ in the walnut-based sample, with correspondingly high antioxidant activity. When ranked by bioactive potential, the formulations followed a descending order: walnut > sunflower > pumpkin, reflecting the intrinsic phenolic richness of the respective oilseed matrices.

The CATA preliminary sensory analysis further validated the consumer appeal of these formulations. Both pumpkin- and walnut-based yoghurts were rated highest for “creamy texture,” “nutty aroma,” and “very pleasant taste,” achieving sensory acceptability levels comparable to traditional dairy yoghurt. In contrast, the sunflower variant showed more vegetal notes and a coarser texture, yet still achieved acceptable hedonic scores.

Microbiological assessment confirmed product safety and preservation of viable lactic acid bacteria within internationally accepted limits throughout storage, with no detection of coliforms, yeasts or moulds, supporting day 14 as the maximum microbiologically acceptable shelf-life.

These obtained results confirm that biotechnological valorisation of oilseed cakes represents a sustainable and nutritionally effective strategy for producing high-quality fermented plant-based beverages. The developed products not only exhibit 2–5 times higher polyphenol content and 3–4 times greater antioxidant capacity than commercial analogues but also maintain desirable technological, sensory, and microbiological profiles. Such innovations directly contribute to circular bioeconomy objectives and to the development of next-generation functional foods that address health and environmental challenges.

5. Patent

The fermentation and preparation method was developed and patented by our group at the Technical University of Moldova (Short-term Patent No. 1821, issued by the State Agency on Intellectual Property of the Republic of Moldova, 15 July 2024).