Sustainable Development of an Innovative Spreadable Plant-Based Product of High Added Value through the Valorization of an Agro-Food By-Product

Featured Application

The product developed herein (“Ελ-yum”) is a vegetable spread rich in nutrients that presents elements of true ecological innovation. The main ingredient of Ελ-yum is purslane, a plant that is actually a weed of the field crop and is considered a by-product of the cultivation process; nevertheless, it is of high nutritional value (e.g., rich in antioxidants, omega-3 and omega-6 fatty acids, vitamins B and C). Ελ-yum can be part of a healthy snack (e.g., served as a dip accompaniment with wholegrain bread sticks) or can be consumed directly as it is. The product follows the trend of circular bio-economy by highlighting the potential of using an agricultural by-product, such as purslane, as a healthy and tasty food choice on a daily basis, with a reduced environmental footprint as a result of the less-intensive agricultural practices followed for the supply of the basic raw material being used.

Abstract

There is an increased demand for healthy foods by the consumer nowadays, while at the same time, circular bio-economy and sustainability in food production represent top priority issues for the food industry. In this context, purslane, a highly nutritious annual plant that grows abundant during the hottest months of the year but is considered a by-product of the agricultural process, was utilized for the development of an innovative, ready-to-eat food product suitable for a vegetarian diet in the form of a bread spread. Following an initial small-scale experimentation for the stabilization of apparent quality attributes, the product’s recipe was finalized on an industrial scale, and the hazard analysis and critical control point (HACCP) for the manufacturing process, the physicochemical (pH, water activity) and sensorial analysis of the end product, together with its nutritional value, shelf-life, and antioxidant capacity, were determined. The results suggested that the acidic (pH 4.3) product had a shelf-life of a minimum of six months to one year and, according to EU legislation, it comprised a source of (dietary) fibers and protein, while being high in poly-unsaturated (e.g., omega-3, omega-6), and mono-unsaturated (e.g., oleic acid) fatty acids. Total phenolic content (TPC) with the Folin–Ciocalteau assay and total antioxidant capacity (TAC) using the Ferric-Reducing Antioxidant Power assay of the product, presented concentrations of 0.95 mg of gallic acid equivalent/g and 0.016 mmol of Fe²⁺/g of dry weight of sample, respectively. However, the predicted bioavailability for TPC and TAC was 28% and 31%, respectively. The product was evaluated positively by a panel of potential consumers without significant differences compared to conventional familiar products. The potential of using agro-food chain by-products, such as purslane, for the development of novel foods representing a healthy and tasty food choice at any time of the day is a promising opportunity for the food industry to meet growing consumer demands for more sustainable, nutritious, and healthy food products with a reduced environmental footprint compared to the traditional plant-originated products of intensive agriculture.

1. Introduction

Food systems strive to adapt to the increasing pressure on sustainability issues and the need for a circular economy perspective in food production [1]. To this end, the valorization of agro-food by-products has been receiving increasing attention worldwide as a means to overcome the current production and consumption prevailing model of conventional agriculture, based on the intensive exploitation of land and resources (e.g., water), towards a swift transition to a more sustainable and ecological approach in primary food production, following less-intensive agricultural practices [2].

More recently and in the last four decades, with the concept of functional foods having emerged in Japan [3], consumers have continuously demanded healthier food choices and products with functional properties [4]. Thus, natural sources that are rich in antioxidants or other bioactive compounds have gained attention as functional food ingredients [5]. From this point of view, the addition of olive oil, edible plants, and seeds could be a good strategy to increase the antioxidant and phytochemical content of such products [6,7,8]. However, owing to the complex mechanisms involved in the digestion process of antioxidants and the various factors that may affect the degree of their biological activity, the study of the predicted bioavailability of these compounds is of vital importance. Therefore, various in vitro digestion models can be used to predict relative bioavailability in a low-cost and less time-consuming manner [9].

Purslane is one of those edible plants rich in nutrients that could offer several desirable properties in the food to which it is added. Egyptian literature from the Pharaoh era describes purslane (Portulaca oleracea); a ubiquitous nutrient-dense vegetable suitable for both animal (fodder) and human consumption [10,11]. It was listed globally as the eighth most common weed and is thought to be a highly invasive plant as it can grow in almost any warm and moist area during the summer and spring seasons, including gardens, crop fields, and waste places [12,13]. Purslane can be eaten raw as a salad or can be consumed cooked and/or as a sauce in soups. According to relevant studies, purslane is a promising natural product with a great nutritional and medicinal potential along with its increased antioxidant capacity [7,14,15]. It is among the most abundant green plant sources of linoleic acid and omega-3 fatty acids [7,16], conferring to the lower incidence of cancer and cardiovascular diseases in the areas where it is consumed [17]. The nutritional and therapeutic qualities of purslane have established it as a “new crop” and as a “power food of the future” [18].

The aim of the present study was to develop a new spreadable plant-based product of high added value with functional properties, exploiting purslane’s nutritional and phytochemical components and having it as the main raw material in the product. To the best of our knowledge, no such widely distributed product with purslane as the main ingredient is on the market so far. Its industrial production is expected to be a sustainable solution to the ever-demanding trend of minimizing the environmental footprint by using less-intensive agricultural practices and crops [19].

2. Materials and Methods

2.1. Raw Materials

All but one of the raw materials being used were of plant origin and were supplied from the Greek market by certified suppliers working closely with the collaborating company (GAEA Products S.M.S.A.). The raw materials used are presented in

Table 1. Raw materials used as ingredients for the recipe of a spreadable plant-based product.

Ingredient	Quantity (in Grams per 125 g Portion)	Percentage (%) 1
Purslane	14.0	11.2
Purslane (in pieces)	7.5	6.0
Green pitted olives	7.0	5.6
Olive oil	7.5	6.0
Sunflower seeds (soaked)	35.125	28.1
Pistachio (powder)	1.0	0.8
Linseeds (powder)	3.5	2.8
Garlic (powder)	0.125	0.1
Tomato flakes (powder)	1.5	1.2
Yellow hard cheese	5.0	4.0
Black pepper	0.25	0.2
Agave syrup	2.5	2.0
Mustard	7.5	6.0
Thyme	0.125	0.1
Lemon juice	17.0	13.6
Lemon zest	0.125	0.1
Water	15.0	12.0

¹ Proportion of ingredient per 125 g of product’s portion.

. The only animal-originated ingredient of the product was Kefalotyri, a traditional Greek yellow hard cheese made from pasteurized fresh cow’s milk. Kefalotyri cheese that was used in the product’s recipe came from local cheese manufacturing companies in the wider Aitoloakarnania region near Agrinio, Western Greece. Green olives and olive oil were kindly provided by the company. Due to seasonality in the availability of purslane (Portulaca oleracea L.; Figure 1) in the Aitoloakarnania region during the warm spring and summer months, producers on Crete island at the south of Greece with a warm climate all year long were contracted, providing a steady supply of the plant at all months of experimentation.

2.2. Product Development and Manufacture

2.2.1. Initial Small-Scale Experimentation

An initial three-month (February to April 2022) small-scale experimentation was conducted in the Department of Food Science and Technology of the University of Patras in Agrinio Campus. Experiments were set up using a thermomixer (K/CY033, Karamco). Quality and safety determinants (pH, a_w) as well as the product’s final recipe were finalized in that manner.

2.2.2. Industrial Large-Scale Manufacture and Packaging of Vegetable Spread

Following the initial small-scale experimentation, the formulated spreadable plant-based end product (bread spread) was manufactured at an industrial level in the company’s facilities. The vegetable spread was packaged in glass jars containing 125 g of the product’s portions and stored at ambient temperature. The production process is summarized in the flowchart in Figure 2. Briefly, the raw materials are checked during their receipt to ensure that they meet the specifications of the product and are stored as appropriate (e.g., dry, chilled, frozen). There is a separate designated area in the facility which is marked appropriately and where the allergens, like pistachio and mustard, are being stored until use. Before use, the raw materials are pre-treated accordingly (e.g., sieved, soaked, de-frozen), if necessary, for further processing. In particular, sunflower seeds are hydrated using acidified water (pH 3.5), whereas all processes involving the use of allergenic ingredients are carried out in a remote area of the facility using different utensils (e.g., bowls, sieves), before entering the main production area. Following pre-treatment of the raw materials, all of the product’s ingredients are mixed in a homogenizer with the addition of water, and the mixture is heated to a temperature above or equal to 72 °C to achieve hot filling of jars with the product. The open jars filled with the product are continuously passing through the metal detector before being capped with the metal lids, and then the product is transferred into the steam pasteurization chamber, where it is kept and monitored using a data logger so as to obtain a temperature of 80 °C for at least 5 min at its thermal center. After pasteurization, the jars are checked with a lid vacuum detector, and the end product is finally labelled and stored at ambient temperature until its distribution. It is worthy to comment that the final product containing allergens is stored in a different marked area from other non-allergenic products produced in the facility. Cleaning and disinfection of the production line in such case is followed by swab testing for checking allergen presence (AllerSnap™, Hygiena LLC, Camarillo, CA, USA) to avoid cross-contamination.

2.3. HACCP Plan Development and Implementation

2.3.1. Preliminary Steps of HACCP

There are five preliminary steps for the development and proper implementation of a Hazard Analysis Critical Control Point (HACCP) plan [20,21]. The company should first assemble the HACCP food safety team which at a later stage will be responsible for describing the product in detail and identifying its intended use, constructing the detailed flow diagram that should cover all steps in the manufacturing process of the specific product under study (Figure 2), and finally confirming this flow diagram with an on-site inspection.

2.3.2. Application of HACCP Principles—Hazard Analysis and CCP Determination

The Codex Alimentarius Commission (CAC) and the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) have adopted seven principles regarding the successful implementation of the HACCP system in food operations [20,21]. Of these seven principles, listing of all potential hazards associated with each step of the manufacturing process, conducting the hazard analysis, and considering any measures to control the identified hazards should be the first priority of the HACCP team. Then, the team must determine which steps in the manufacturing process are critical in terms of effectively controlling each identified hazard. In this context, the determination of a Critical Control Point (CCP) in the HACCP system can be facilitated by the application of a decision tree [20,21].

2.4. Sensory Evaluation

The manufactured bread spread (“Ελ-yum”) was evaluated by a panel of 25 student consumers from the Department of Food Science and Technology of the University of Patras in Agrinio Campus. The product was evaluated in its fourth month of storage against five main attributes: taste, odor, color, consistency, and packaging of the product. Acceptability of the product was based on the rating of sensory attributes using a 5-point hedonic scale, where 1 = dislike extremely, 2 = dislike slightly, 3 = neither like nor dislike, 4 = like slightly, and 5 = like extremely. The final sensory profile of the product was determined by the average scoring for each attribute compared to the acceptable limit value (i.e., 3 based on the above scale).

2.5. Chemical Analyses for Determination of Nutritional Value

All chemical analyses of the product regarding its nutritional value were kindly provided and conducted by Eurofins Athens Analysis Laboratories, as described by Karantonis et al. [22]. Briefly, the analyses included the determination of moisture and volatile compounds in the product’s samples after drying in an air oven, solid residue (ash) determination, protein content and dietary fibers (crude) determination after digestion with concentrated sulfuric acid using the Kjeldahl method (proteins), followed by the use of a concentrated solution of sodium hydroxide when it comes to fibers. Total fat was determined after acid hydrolysis of samples and extraction in an organic solvent, while fatty acid profile was performed with a gas chromatography–flame ionization detector (GC–FID). Sodium was measured with inductively coupled plasma mass spectrometry (ICP-MS), whereas sodium chloride in the product was calculated from sodium [23]. The carbohydrates and energy were calculated from proximate analysis values [23].

2.6. Microbiological Analyses for Shelf-Life Determination

Microbiological analyses along with organoleptic control of the vegetable spread took place for determination of its microbiological quality and the verification of the product’s expected shelf-life. While sensory evaluation described in Section 2.4. focuses on consumer perception of attributes, like taste and odor, the organoleptic control described herein primarily aims to assess shelf-life stability of the product. Analyses for the enumeration of total viable count (TVC), aerobic and anaerobic bacterial spores, Escherichia coli, coagulase-positive staphylococci, sulfite-reducing bacteria, Clostridium perfringens, Bacillus cereus, yeasts and molds count (YMC), as well as for the detection of Salmonella spp. and Listeria monocytogenes and the organoleptic evaluation of the product’s five sensory attributes (i.e., taste, odor, color, consistency, packaging) by a panel of 5 analysts, were performed on days 1, 5, 25, 45, 90, 120, 150, and 180 at Eurofins Athens Analysis Laboratories. Microbiological analyses were performed according to standard methods published by the International Organization for Standardization (ISO) and the American Public Health Association (APHA) in an ISO 17025-accredited microbiology laboratory [24], depending on the target microorganism for enumeration or detection in food, whereas culture media and incubation conditions were all supplied by Merck (Darmstadt, Germany) and were the same as described by Andritsos et al. [25,26]. Additionally, dextrose tryptone agar was utilized for the enumeration of aerobic and anaerobic bacterial spores after incubation at 35 °C for 48 h, tryptose sulfite cycloserine agar for the enumeration of C. perfringens following incubation at 37 °C for 24 h, and mannitol egg yolk polymyxin agar was used for the enumeration of B. cereus after incubating at 30 °C for 24 h. For the enumeration methods, three serial decimal dilutions were plated onto a Petri dish with the appropriate culture medium. Microbiological counts were calculated from the weighted mean of two plates from two countable dilutions, containing 30–300 or 15–150 colonies for general purpose and selective media, respectively.

2.7. Antioxidant Capacity

The in vitro bioactivity of total antioxidants (total antioxidant capacity; TAC) and total phenolic compounds (TPC) were assessed using the ferric-reducing antioxidant power (FRAP) assay and the Folin–Ciocalteau assay, respectively. The predicted bioavailability of these bioactive compounds was determined, performing an in vitro digestion process, through simulation of the gastrointestinal digestion. Chemicals used in the assays were attained by Sigma-Aldrich (St. Louis, MO, USA) and Merck.

2.7.1. Preparation of Vegetable Spread Solution

For the preparation of the vegetable spread solution, 1.5 g of the spread were dissolved in 15 mL of deionized water. The resulting mixture underwent homogenization through vortex mixing, followed by centrifugation at 4500 rpm for 15 min at 4 °C. Subsequently, the supernatant fraction was extracted and utilized for all the following analyses.

2.7.2. Determination of TPC Using Folin–Ciocalteau Assay

The Folin–Ciocalteau method was employed to quantify the TPC of the sample [27]. This assay assesses the reductive capacity of the Folin–Ciocalteau reagent. In each well of a 96-well plate, 20 μL of Folin–Ciocalteau reagent, 20 μL of Na₂CO₃ solution, and 50 μL of the vegetable spread solution were gradually mixed. The 96-well plate was kept in the dark for 30 min, and afterwards, the absorbances were measured at 765 nm using a spectrophotometer (SPARK, TECAN, Mennedorf, Switzerland). The TPC was quantified using a standard gallic acid (GAE) curve, and the results were expressed in milligrams of gallic acid equivalents per gram of dry weight of sample (mg GAE/g dw). The analysis was conducted in triplicate.

2.7.3. Assessment of TAC via FRAP Assay

To evaluate TAC, the FRAP assay was applied [28,29,30]. This assay relies on the reduction of TPTZ-Fe³⁺ to TPTZ-Fe²⁺. In a 96-well plate, 80 μL of FRAP Reagent and 20 μL of the sample solution were combined. The 96-well plate was kept in the dark for 30 min, and the absorbances were measured at 595 nm using a spectrophotometer (SPARK, TECAN, Männedorf, Switzerland). The TAC was determined using a standard FeSO₄ curve, and the results were expressed in millimoles of Fe²⁺ per gram of dry weight of sample (mmol Fe²⁺/g dw). The analysis was conducted in triplicate.

2.7.4. In Vitro Digestion Analysis

An adapted static in vitro digestion model, based on the Kapsokefalou et al. [29] method as previously described by Kaloteraki et al. [31], was performed to simulate the gastrointestinal digestion process and to predict the bioavailability of total antioxidants and total phenolics. Specifically, 2 mL of the sample solution was dispensed into 6-well plates, along with 100 μL of porcine pepsin solution (0.2 g of pepsin, 5 mL of HCl 0.1 M). The samples were incubated in a shaking incubator (Shaking Incubator SKI-4, China) at 37 °C for 2 h. After incubation, the appropriate membrane was introduced into each well, followed by the addition of 2 mL of piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) buffer reagent (0.15 M), adjusting the pH of the sample to 6.3. Subsequently, the samples were further incubated (1 h, 37 °C). Afterwards, 0.5 mL of a pancreatin and bile salts solution (0.02 g of pancreatin, 0.12 g of bile salts, 10 mL of 0.1 M NaHCO₃) was added to each well, followed by additional incubation (2 h, 37 °C). The supernatant fraction that was released during the simulated digestion and contained the high-molecular-weight compounds was centrifuged at 4500 rpm (15 min, 4 °C). The TAC and TPC were determined using the FRAP and Folin–Ciocalteau assays, respectively, as previously described. Finally, the predicted bioavailabilities of the TAC and the TPC were calculated according to Equation (1):

% predicted bioavailability = (C_f/C_i) × 100

(1)

where C_f: post-digestion concentration of a compound in the supernatant fraction, and C_i: pre-digestion, initial concentration of the same compound, as suggested by Mihaylova et al. [32].

2.8. Statistical Analysis

Statistical analysis was conducted using SPSS v21.0 (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) through SPSS software was used to investigate differences between the digestion process in total phenolics (TPC) and antioxidants (TAC). A probability value of less than 0.05 (p < 0.05) was defined as statistically significant. Microbial counts (cfu/g) were converted to log cfu/g. Energy values, carbohydrates, and microbial counts are reported as mean ± laboratory standard uncertainty (expressed as intralaboratory reproducibility standard deviation), whereas all other chemical constituents are reported as % expanded uncertainty (U = k × laboratory standard uncertainty; where k = 2 for 95% confidence interval) [33]. TAC and TPC are expressed as mean ± standard deviation (SD).

3. Results

3.1. Basic Ingredients

The two basic ingredients of the product, comprising ca. 45% of the product’s portion, were purslane and soaked sunflower seeds (Table 1). However, purslane should be considered the main ingredient since it confers the special attributes and gives the special character to the product, although it is not the ingredient participating in the highest absolute numbers in the product’s recipe.

3.2. Physicochemical Analysis

After numerous trials in small-scale experiments, the pH of the product was standardized to 4.3 ± 0.1 by measuring acidity with a portable pH meter (pH-Star, Matthäus GmbH, Pottmes, Germany). The water activity (a_w) of the product was measured at 0.974 by using a LabTouch-a_w device (Novasina, Lachen, Switzerland).

3.3. CCPs of the Manufacturing Process

After conducting the hazard analysis for each step of the manufacturing process, application of the HACCP principles led to the determination of six CCPs for product preparation. Those CCPs are depicted in Figure 2. The established critical limits were: absence of foreign matter and waxes for filtering of olive oil, absence of foreign matter for sieving of spices and powdered raw materials, non-detectable levels of protein residues (i.e., below 3 μg of protein) for effective allergen control, temperature above or equal to 72 °C for hot filling of jars with the product, absence of foreign metal bodies (no metal traces detected) through the use of a metal detector before capping of product with the metal jar lids, steam pasteurization for at least 5 min at 80 °C in the center of the product and cooling of the end product through water sprinkling at ambient temperature, presence of vacuum with the use of a vacuum detection device.

The company implements an ISO 22000 food safety management system, thus operational prerequisites programs (OPRPs) (Figure 2) have been established in order to better safeguard the product’s integrity in terms of its safety [34]. To this end, efficient washing in the jar washer that adds to the product’s hygiene, as well as proper labeling of the product’s allergens (e.g., pistachio) to avoid unwanted consumption by people showing allergic reactions, are two of the most important OPRPs in the manufacturing process.

3.4. Sensory Evaluation of Ελ-Yum

Figure 3 summarizes the results obtained from the consumer acceptability test conducted on the final product of Ελ-yum bread spread samples (Figure 4).

Most sensory attributes of the product were neither liked nor disliked by the panelists. On average, however, packaging material and the product’s size (4.70) were liked more than the other characteristics. The other attributes, namely taste (3.00), consistency (3.40), odor (3.40), and color (3.60), were averagely neither liked nor disliked by the consumers. Considering taste, the most decisive criterion for selection of a product, 52% of the panelists (13 out of 25) were neutral (3.00), 28% (7 out of 25) liked it slightly (4.00), while 20% of the consumers (5 out of 25) disliked slightly (2.00) the product’s taste.

According to the company’s evaluation protocol for product acceptability, Ελ-yum vegetable spread is acceptable for manufacture since the average score of the product (3.60) is higher than the limit score (3.00).

3.5. Nutritional Value and Nutrition Claims for Product Labelling

The nutritional composition of the product is presented in

Table 2. Nutritional composition of a vegetable spread with purslane as its main ingredient.

Parameter (Unit)	Result	Uncertainty
Energy value (Kcal/100 g)	230	11 kcal
Energy value (Kj/100 g)	950	48 kj
Moisture and volatiles (g/100 g)	63.3	1%
Proteins (g/100 g)	7.1	2%
Ash (g/100 g)	1.8	2.7%
Dietary fibers (g/100 g)	4.1	22.1%
Carbohydrates (g/100 g)	4.0	1.4 g/100 g
of which sugars (g/100 g)	2.8	8.7%
Galactose (g/100 g)	ND 1	-
Glucose (g/100 g)	1.3	17.7%
Lactose (g/100 g)	ND	-
Maltose (g/100 g)	ND	-
Sucrose (g/100 g)	ND	-
Fructose (g/100 g)	1.5	8.7%
Fat (g/100 g)	19.7	5.4%
of which saturated (g/100 g)	3.0	11.7%
of which saturated % (g/100 g fat)	15.4	7.4%
of which mono-unsaturated (g/100 g)	10.3	11.7%
of which mono-unsaturated % (g/100 g)	52.4	2.5%
of which poly-unsaturated (g/100 g)	6.3	11.7%
of which poly-unsaturated % (g/100 g)	32.2	2.5%
Sum of trans (C18:1T) and (C18:2T) acids	ND	-
Sodium (mg/Kg)	2220	11.4%
Sodium chloride (g NaCl/100 g)	0.56	11.4%

¹ ND: Not Detected.

.

According to Regulation (EC) No. 1924/2006 on nutrition claims [23], as amended by Regulation (EU) No. 116/2010 and currently in effect [35], Ελ-yum spread is a “source of (dietary) fiber” since it contains 4.1 g fibers/100 g of product (Table 2), which is more than the limit value of 3 g fibers/100 g of product required for the claim to be made. Furthermore, the product is a “source of protein” as 28.4 Kcal (calculated from Equation (2)) out of the 230 Kcal/100 g of product (Table 2) corresponds to marginally more than 12% of the energy value of the food being provided by protein (12.4%).

% energy from proteins = [(g proteins/100 g × 4 Kcal/g)/total energy Kcal/100 g] × 100

(2)

Moreover, the manufactured bread spread is “high in unsaturated fat” because 84.6% of the fatty acids present in the product derive from unsaturated fat (i.e., mono- and poly-unsaturated fat; Table 2), which is more than the minimum required 70% of unsaturated fatty acids to be present in the product, while also unsaturated fat provides more than 20% of the product’s energy (i.e., 66.1%; calculated from Equation (3)).

% energy from unsaturated fat = [((g mono-unsaturated fat/100 g + g poly-unsaturated fat/100 g) × 9 Kcal/g)/total energy Kcal/100 g] × 100

(3)

Finally, Ελ-yum spread is “high in monounsaturated fat” since 52.4% of the fatty acids present in the product derive from monounsaturated fat (Table 2), which is more than the minimum required 45% of monounsaturated fatty acids to be present in the product, while at the same time, monounsaturated fat provides more than 20% of energy of the product (i.e., 40.3%; calculated from Equation (4)).

% energy from unsaturated fat = [(g mono-unsaturated fat/100 g × 9 Kcal/g)/total energy Kcal/100 g] × 100

(4)

3.6. Shelf-Life Assessment of Ελ-Yum

The microbiological analysis at each different day of storage for the developed new product is given in

Table 3. Monitoring of microbiological parameters for Ελ-yum spread during three months of product storage at room temperature (20–25 °C).

Microbiological Parameter (Unit)	Day 1	Day 5	Day 25	Day 45	Day 90	Day 120	Day 150	Day 180
TVC 1 (log cfu/g)	2.04 ± 0.06	2.60 ± 0.06	2.48 ± 0.06	3.52 ± 0.06	5.45 ±0.06	5.32 ± 0.06	5.54 ± 0.06	5.49 ± 0.06
Aerobic spores 2 (log cfu/g)	<1.00	<1.00	<1.00	<1.00	<1.00	<1.60	<1.60	<1.60
Anaerobic spores 2 (log cfu/g)	<1.60	<1.00	<1.00	<1.60	<1.00	<1.60	<1.60	<1.00
E. coli 3 (log cfu/g)	<1.00	<1.00	<1.00	<1.00	<1.00	<1.00	<1.00	<1.00
Staphylococci 4 (log cfu/g)	<1.00	<1.00	<1.00	<1.00	<1.00	<1.00	<1.00	<1.00
SR anaerobes 5 (log cfu/g)	<1.00	<1.00	<1.00	<1.00	<1.00	<1.00	<1.00	<1.60
C. perfringens (log cfu/g)	<1.00	<1.00	<1.00	<1.00	<1.00	<1.00	<1.00	<1.00
B. cereus (log cfu/g)	<2.00	<2.00	<2.00	<2.00	<2.00	<2.00	<2.00	<2.60
YMC 6 (log cfu/g)	<2.00	<2.00	<2.00	<2.00	<2.00	<2.00	<2.00	<2.00
Salmonella spp. (cfu/25 g)	ND 7	ND	ND	ND	ND	ND	ND	ND
L. monocytogenes (cfu/25 g)	ND	ND	ND	ND	ND	ND	ND	ND

¹ TVC: Total Viable Count. ² It refers to bacterial spores. ³ β-glucuronidase-positive E. coli. ⁴ Coagulase-positive staphylococci. ⁵ Sulfite-Reducing (SR) anaerobic bacteria. ⁶ YMC: Yeasts and Molds Count. ⁷ ND: Not Detected.

. No deviations from normal and expected findings were observed in the organoleptic characteristics of the product during the analysis, since the product scored more than the average (3.0) in all sensory attributes tested (Supplementary Figure S1). Apart from TVC, all other microbiological parameters were below the limit of detection (e.g., <1.00 or <1.60 log cfu/g) or were not detected per 25 g of product during storage at ambient temperature (Table 3). Thus, the microbiological quality of the product is deemed satisfactory as compared to other ready-to-eat vegetarian spreads [36], whereas TVC does not exceed the critical value of 10⁶ cfu/g [37] at any time of storage (Table 3). Those findings reveal that the shelf-life of Ελ-yum is more than three months. Based on previous research for similar products, plant-based spreads have a minimum shelf-life of six months from the date of production when stored at room temperature (20–25 °C) [38,39,40,41]. After opening, they can remain in the refrigerator for up to three days. Given the above, the combined shelf-life of the developed new product is at least six months to one year [39,41].

3.7. Evaluation of TPC and TAC

Table 4. TPC and TAC values for a vegetable spread and their predicted bioavailability. TPC is expressed as mg of gallic acid equivalents (GAE) per gram of dry weight of sample. TAC is expressed as mmol of Fe²⁺ per gram of dry weight of sample. Results are expressed as mean ± standard deviation (SD).

	TPC (mg GAE/g dw)	TAC (mmol Fe2+/g dw)
Before digestion	0.95 ± 0.06	0.016 ± 0.00
After digestion	0.27 ± 0.03	0.005 ± 0.00
Predicted bioavailability %	28.03%	30.58%

presents the estimated mean values for TPC and TAC before and after in vitro digestion, including their predicted bioavailability.

Prior to the in vitro digestion process, the mean TPC value determined by the Folin–Ciocalteau assay was 0.95 mg of GAE/g dw, with a standard deviation of 0.06. Post-digestion, the mean TPC of the vegetable spread decreased significantly (p < 0.05) to 0.27 mg of GAE/g dw, with a standard deviation of 0.03 (Table 4).

The mean TAC of the vegetable spread before digestion was found to be 0.016 mmol of Fe²⁺/g dw, with a standard deviation of 0.00. After subjecting the sample to the in vitro digestion process, the TAC of the vegetable spread decreased significantly (p < 0.05) to 0.005 mmol of Fe²⁺/g dw with a standard deviation of 0.00 (Table 4). The observed reduction in FRAP values indicates a substantial alteration in the TAC of the product following the simulated digestion.

The predicted bioavailability for TAC of the product was 30.58%, indicating that approximately 31% of the initially measured antioxidant compounds was retained after the simulated digestion. Similarly, the predicted bioavailability for TPC of the product was 28.03%, signifying the preservation of approximately 28% of the initial phenolics through the digestion process (Table 4). The observed reduction in TPC and TAC values after the in vitro digestion process along with the predicted bioavailability for the components of the product with antioxidant capacity are also schematically presented in Figure 5.

4. Discussion

The innovation of the product developed herein lies in the utilization of purslane as the main ingredient in the product. The latter is considered a by-product of the agricultural practice and has minimum to zero application today in conventional foods sold in the market. Purslane is an annual plant rich in nutrients (e.g., antioxidants, omega-3 and omega-6 fatty acids), whose exploitation by its inclusion in the human diet could lead to environmentally friendly and sustainable food choices. Ελ-yum is one such healthy and tasty food choice for any time of the day, provided to the consumer in the form of a bread spread containing the portulaca plant (P. oleracea). The first syllable of the product name comes from the word “Ελλάδα” (Greece) as it reflects the Greek Mediterranean diet, while the word “yummy” used as a synthetic in the product’s name emphasizes the delicious taste and the special savor the product should offer to its consumers. Ελ-yum spread is also suitable for a vegetarian diet as it does not include meat, poultry, and fish in its ingredients.

Several studies have highlighted the functional properties of purslane in developing food products with high nutritional value, such as ice cream, cookies, yogurt, bread, and spaghetti [42,43,44,45,46,47,48,49]. TAC and TPC appeared to be higher after purslane supplementation to these products. Particularly, TAC in plants like purslane could be attributed to their high total phenol and flavonoid content [50]. More specifically, according to Uddin et al. [7], the TPC of P. oleracea cultivars ranges from 127 ± 13 to 478 ± 45 mg of GAE/100 g fresh weight of plant, and the addition of purslane could lead to increased TPC in food commodities. Furthermore, as the predicted bioavailability is crucial for assessing polyphenol release from the food matrix, studies that determine the TPC of plant products should also evaluate their predicted bioavailability [9]. In this study, the relative bioavailability of a vegetable spread having purslane as its main ingredient was assessed using an in vitro static digestion model and was determined to be 28% and 31% for TPC and TAC, respectively (Table 4).

Although high in moisture (ca. 63%; Table 2) and a_w (0.974), the acidic character of Ελ-yum (pH 4.2–4.4) adds to the enhanced safety and microbiological stability of the product during storage (Table 3). Findings suggest that the shelf-life of the manufactured bread spread is 6–12 months, which is an acceptable shelf-life of the product for the food market and facilitates its distribution in distant markets. The microbiological stability of the unopened spread, due to the thermal processing treatment (pasteurization) it receives (Figure 2), renders the use of “Best before date” more appropriate for this type of product.

The newly developed vegetable spread of Ελ-yum was evaluated extremely positively by the consumers for its packaging and the product’s offered marketable portion (Figure 3). The product was evaluated positively by a panel of potential consumers without significant differences (p > 0.05) compared to conventional similar products with which the consumers are more familiar. In total, four out of five people (80%) were neutral or positive towards the taste as they neither liked nor disliked the product (52% scored 3.00 in the evaluation form) or liked it slightly (28% scored 4.00 in the evaluation form). At this point, it should be noted though that this apparently poor acceptability of the product in terms of its taste is due to the tasting of product without any accompaniment (e.g., bread, bread sticks, cracker). This lack of accompaniment happened on purpose in order to be able to evaluate the pure form of the product and also to prevent tainting the taste and obtaining biased results. Finally, this should be enough to explain why the majority of panelists admitted not liking or disliking the product, mainly because of its taste and consistency (i.e., texture and appearance). Overall, improvement of the product’s organoleptic characteristics could be inferred upon the company’s continuous effort and commitment towards this direction, by conducting numerous tests through modifications in the product recipe and the use of the trial-and-error method for evaluating the manufactured vegetable spread. Moreover, the company could consider this strategy for altering the nutritional profile of Ελ-yum and expanding its consumer base on different markets.

Compared to other similar products in the field, existing literature data are in favor of Ελ-yum in terms of shelf-life, sensory characteristics, salt content, calorie and fat intake. The purslane spread manufactured in this study has a shelf-life of more than six months and can be preserved for an extended period of time without the addition of chemical preservatives in its formula. Indeed, the microbiological quality of Ελ-yum is better, and the product shows a noteworthy microbial stability during its recommended shelf-life, while the latter is much longer than other vegetarian spreads (e.g., chickpea-, sesame seed-based spreads) and dips (e.g., pesto, tapenade, salsa, guacamole) sold in the retail market [36,51]. Recently, Kostyra et al. [52] have shown that the addition of vegetable purees and spices significantly improves the sensory properties of soy-based spreads (p < 0.05), while earlier sensory evaluations by Kirse et al. [53] have demonstrated that in several developed bean spreads, the ones using sun-dried tomatoes were generally more preferable compared to others (p < 0.05), which are deemed as advantageous elements for the application of purslane and Ελ-yum’s formula (Table 1) in the aforementioned spreads. When it comes to salt, Ελ-yum shows a reduced salt content compared to other marketable bread spreads, for instance when compared to chickpea spreads [51]. As far as energy uptake and fat intake are concerned, compared to Ελ-yum, there is an increased calorie and fat intake from nut- and seed-based spreads [54], whereas carrot- and parsnip-based spreads provide more or less the same energy as Ελ-yum but with a reduced amount of fat [55]. Fat is also minimally provided from flaxseed-based spreads; however, those spreads are mostly rich in carbohydrates [56].

5. Conclusions

A novel functional vegetable spread was successfully manufactured by incorporating purslane in the product’s recipe and scaling up the production at an industrial large-scale level. The new product comprises a source of (dietary) fibers and protein, while being high in unsaturated and mono-unsaturated fat. It is distinguished by the ubiquitous character of purslane and its increased antioxidant power compared to other aromatic plants rich in antioxidants (e.g., mountain tea, oregano, lavender) [57], whereas its predicted bioavailability in TPC and TAC was estimated at approximately one-third of its original content in phenolics and antioxidants. The product is expected to maintain its good sensory attributes during storage at room temperature (20–25 °C) for at least six months. Conclusively, the valorization of an abundant agro-food by-product (purslane) for the sustainable development of an innovative spreadable plant-based product with a nutraceutical potential, like Ελ-yum described herein, is expected to boost the sales of vegetarian spreads as mid-day snacks and facilitate food exports of such products.