Impact of Incubation Conditions and Addition of Red Beet and Leek Powders as Natural Nitrate Sources on the Physicochemical and Sensory Properties of Cooked Sausages

Abstract

Nitrite remains a central component in industrial cured meat processing for its role in providing colour stability, oxidative protection, and microbial safety. However, synthetic nitrite is associated with the formation of nitrosamines, leading to increased health concerns and negative consumer perception of synthetic additives, thereby increasing demand for healthier meat products produced with natural nitrite sources. This study employed a two-stage design to assess microbial nitrate curing in cooked sausages and its extension to vegetable powders. In Stage 1, sodium nitrate (100 mg/kg) combined with Staphylococcus carnosus was incubated at 30 or 40 °C for 90 or 180 min. Incubation at 30 °C yielded residual nitrite concentrations of 18–29 mg/kg, corresponding to 35–40% of those in nitrite controls, and resulted in equivalent colour (CIE ΔE* < 2) and oxidative stability (0.07–0.09 mg MDA/kg versus 0.08 mg MDA/kg in the control). In Stage 2, application of red beet (2%) and leek (1%) powders supplying 100 mg/kg NO_3⁻ produced adequate curing but induced substantial compositional and sensory deviations, including higher redness (CIE a* ≈ 23 versus 15), fourfold higher lipid oxidation (0.35–0.42 mg MDA/kg), and intensified vegetable aroma and sweetness. These findings demonstrate that microbial nitrate reduction at 30 °C effectively reproduces the technological performance of direct nitrite addition, whereas vegetable-based nitrate curing introduces significant colour, oxidative, and sensory differentiation, highlighting both potential and limitations of microbial nitrate curing.

1. Introduction

Nitrites have long been considered indispensable in cured meat processing due to their multiple technological functions. They stabilize colour through nitrosylmyoglobin formation, inhibit lipid oxidation thereby influencing odour and flavour, and provide antimicrobial protection against Clostridium botulinum [1,2]. Concerns regarding the potential formation of carcinogenic N-nitroso compounds [3,4,5], combined with negative consumer perception of synthetic additives [6,7], have intensified research efforts toward identifying natural alternatives [8,9].

The use of nitrate-rich vegetables in combination with nitrate-reducing starter cultures such as Staphylococcus carnosus represents a promising strategy [3,8,9]. Celery, spinach, radish, lettuce, and cabbage have been identified as suitable alternatives because they provide natural sources of nitrates as well as pigments and antioxidant compounds [10,11,12]. However, nitrate levels in these vegetables vary considerably with season and cultivation practices, resulting in inconsistent curing outcomes [13,14,15]. Genetic and agronomic factors, such as nitrogen dosage, chemical form, and nutrient availability, as well as environmental conditions including soil temperature, rainfall, and light intensity, influence nitrate reductase activity and, consequently, nitrate accumulation [4,15]. These variations have led to the establishment of regulatory limits for nitrate content in certain vegetables in several European countries [15]. Previous studies have primarily focused on celery powder and juice, which, after adequate incubation, can generate cured colour and sensory traits comparable to sodium nitrite-treated controls [16,17,18]. Conversely, spinach or radish powders have sometimes generated higher TBARS values and less stable colour than nitrite controls [11,14,19].

Two technical approaches are commonly used for incorporating vegetable nitrates in meat curing: (i) direct addition of powders or extracts and (ii) use of pre-converted nitrite preparations. Powders align with consumer expectations for “natural” products [20] and provide additional bioactive compounds, but their effectiveness depends on controlled incubation and starter culture activity [10,13,21]. Pre-converted nitrite, produced through prior fermentation of vegetables, ensures consistent curing performance with antimicrobial and colour-stabilizing effects comparable to synthetic nitrite [8,22,23]. Yet, since pre-converted nitrite is chemically identical to synthetic counterpart, concerns regarding nitrosamine formation remain unresolved [6,7].

Red beet (Beta vulgaris L., Amaranthaceae) and leek (Allium ampeloprasum L., Amaryllidaceae) are notable vegetable nitrate sources with distinct compositional profiles. Red beet is classified among vegetables with very high nitrate content (>2500 mg/kg) [15] and contains betalains and phenolic compounds that contribute to its strong colour and antioxidant activity [24,25,26]. Its application in fermented sausages has maintained redness and sensory quality for up to 56 days [27], while fermented beet extracts supplemented with ascorbic acid achieved colour and oxidative stability comparable to synthetic nitrite [23]. However, its intense pigmentation may obscure the typical cured hue, and elevated sugar content can promote lipid oxidation [28]. Leek is classified among vegetables with moderate to high nitrate concentrations (1000–2500 mg/kg) [15] and contains sulphur- and flavonoid-based compounds that contribute to its antimicrobial and antioxidant activity [29]. The inclusion of freeze-dried leek powder enabled a 50% nitrite reduction without sensory loss [30], and fresh leek improved nitrite retention and quality stability in camel sausages [31]. However, its colour-forming capacity is limited due to lower nitrate and pigment levels compared with vegetables such as celery or beetroot, resulting in a paler cured appearance [4,10,15,28]. Moreover, leek’s high content of sulphur volatiles, including thiosulfinates and disulphides [29], may generate atypical odours and off-flavours during heating or prolonged storage. These drawbacks suggest that leek may be more effective when used in combination with other nitrate-rich vegetables to balance colour development and flavour stability. Considering these characteristics, red beet and leek were selected as model vegetable sources representing root and allium types with distinct pigment and volatile profiles, enabling evaluation of both colour enhancement and flavour modification under standardised microbial nitrate reduction conditions.

In this study it was hypothesised that microbial nitrate curing could replicate the technological effects of conventional nitrite curing, whereas vegetable-derived nitrates would introduce distinct compositional and sensory characteristics. To test this hypothesis, this research was designed in two stages: first (Stage 1), to validate the efficiency of microbial nitrate-to-nitrite conversion using Staphylococcus carnosus under controlled incubation conditions; and second (Stage 2), to apply the validated process to cooked sausages cured with red beet and leek powders as natural nitrate sources. This design isolated the effects attributable to the vegetable powders from those related to microbial conversion or incubation parameters. To our knowledge, this study is the first to combine these two vegetable sources within a unified curing system to directly compare their technological and sensory effects under identical processing parameters.

2. Materials and Methods

2.1. Sausage Production and Treatments

Cooked sausages were produced from pork meat, fatty tissue, water, and selected seasonings, with formulations varying according to treatment. The formulation of the Control (A) treatment is presented in

Table 1. Sausage production processes for Control (A), Stage 1—Nitrate Conversion Validation (B), and Stage 2—Vegetable Nitrate Application (C) treatments.

Formulation, %	Stage
	Control (A)	1—Nitrate Conversion Validation (B)				2—Vegetable Nitrate Application (C)
Pork shoulder	58.4	58.4				54.4
Pork belly	24.3	24.3				20.4
Water	14.6	14.6				19.5
NaCl	1.9 *	1.9 **				1.9
Black pepper	0.3	0.3				0.3
Garlic granules	0.2	0.2				0.2
Dextrose	0.15	0.15				0.15
Smoke flavour	0.1	0.1				0.1
Ascorbic acid	0.05	0.05				0.05
Red beet powder	-	-				2
Leek powder	-	-				1
Starter culture BITEC S 10	-	0.5 g/kg meat batter				0.5 g/kg meat batter
Incubation/temperature treatments	-	B-30-90	B-30-180	B-40-90	B-40-180	C-30-90	C-30-180
Incubation temperature, °C	-	30	30	40	40	30	30
Incubation time, min	-	90	180	90	180	90	180

* NaCl including nitrites (0.526%) as NaNO₂ (providing 100 mg/kg in the meat batter); ** NaCl including nitrates (0.526%) as NaNO₃ (providing 100 mg/kg meat batter).

. No phosphates were added to avoid influencing myofibrillar protein activation which is important for texture definition and oxidative stability. Two independent batches of each treatment were produced.

Boneless pork was sourced from a commercial supplier, stored at 2.5 °C under chilled conditions, and processed within 24 h of delivery. Production began with trimming and cutting the meat into smaller pieces, followed by mincing through an 8 mm plate using electrical mincing machine (Tre Spade, Facem, Forno Canavese, Italy; model TC-22 Elegant). Ingredients were weighed according to the formulation and mixed in a 70 L horizontal meat mixer (VI-JA, Petrovče, Slovenia), filled into polyamide casings using a 25 L hydraulic filler (VI-JA, Petrovče, Slovenia) and cooked in a convection oven (UNOX Cheftop Mind. Maps ONE XEVC-0511, Cadoneghe, Italy) at 82 °C until the internal temperature reached 70 °C, held for 15 min, cooled in ice water to 15 °C, and stored at 2 °C until analysis. All analyses were carried out within two days of sampling, except for the elemental analysis, for which samples were vacuum-packed and stored at −20 °C for 30 days.

Stage 1—Nitrate Conversion Validation (B treatments): Sausages were prepared identically to the Control treatment (A) (Table 1), with sodium nitrite replaced by sodium nitrate (NaNO₃, 100 mg/kg meat batter). The starter culture BITEC S 10 (Staphylococcus carnosus, 8 × 10⁹ CFU/g; Frutarom Savory Solutions, Salzburg, Austria) was added at 0.5 g/kg after rehydration (12 h, 20 °C). Incubation was conducted at 30 °C or 40 °C for 90 or 180 min, starting when the product reached the target temperature. Cooking followed the same procedure as for the Control. Starter performance was evaluated based on the following: (i) nitrate-to-nitrite conversion, (ii) colour equivalence to the control (ΔE < 2), (iii) oxidative stability (TBARS not significantly different from the Control), and (iv) absence of significant sensory differences from Control in a Dunnett post hoc test (α = 0.05) mean DFC ≤ 1.75.

Stage 2—Vegetable Nitrate Application (C treatments): Sausages were produced from the same base ingredients (Table 1) with slight modification in adding 2% red beet powder (Harissa, Zagreb, Croatia; dry matter 91.8%, fat 1.5%, carbohydrates 54.3% of which sugars 42.1%, protein 13.2%, salt 2.1%, dietary fibre 20.7%, nitrates 0.353%) and 1% leek powder (Harissa, Zagreb, Croatia; dry matter 92.3%, fat 1.8%, carbohydrates 55.1% of which sugars 13.1%, protein 11.2%, salt 1.5%, dietary fibre 25.7%, nitrates 0.294%). The amounts of red beet and leek powder were calculated to provide 100 mg nitrate per kg of meat batter. Additionally, more water was added to compensate for the higher dry matter content of red beet and leek powders. Based on the Stage 1 conversion efficiency assessment, only incubation at 30 °C for 90 and 180 min was retained to bracket the operational window while avoiding redundancy. After incubation, the sausages were cooked using the same procedure as applied in the Control (A) treatment.

2.2. Physical Analysis

pH and water activity (aw) were measured after 1 and 60 days of storage. pH was determined using a portable pH meter (HI98191, Hanna Instruments, Smithfield, RI, USA) with a spear-type electrode (BlueLine 21pH, Schott AG, Meinz, Germany) after homogenisation with distilled water (1:1) and equilibration for 30 min. Water activity was measured using a HygroPalm HP23-AW-A analyser and HC2-AW probe (Rotronic AG, Bassersdorf, Switzerland) after 120 min of equilibration at room temperature.

Colour (CIE L*, a*, b*) was measured after 1, 30, and 60 days using a Minolta Chroma Meter CR-410 (Konica Minolta, Tokyo, Japan) with D65 illuminant and 50 mm aperture. Measurements were taken immediately after the cross-section was cut. Total colour difference CIE ΔE*_ab were calculated; the experimental groups were counted with the control group comparation [32].

2.3. Basic Chemical Composition

Sample preparation followed ISO 3100–1:1991 [33]. Moisture content was determined gravimetrically [34] in a drying oven (UF75 Plus, Memmert, Schwabach, Germany). Ash was measured after incineration at 550 °C [35] (LV9/11/P320, Nabertherm, Lilienthal, Germany). Protein was determined by the Kjeldahl method [36] using a digestion block (Unit 8 Basic, Foss, Hilleroed, Denmark) and distillation unit (VAPODEST 50 s, Gerhardt, Königswinter, Germany). Fat was determined by Soxhlet extraction [37] using an automated Soxtherm 2000 (Gerhardt, Königswinter, Germany). Carbohydrate content was calculated by subtraction of fat, protein, and ash content from the dry matter content. Sugar content was measured enzymatically (R-Biopharm, Darmstadt, Germany). Results were expressed in g/kg. Quality control used reference material TET003RM (Fapas, York, UK).

2.4. Oxidative Stability (TBARS)

TBARS were determined in duplicate on three samples per treatment after 1 and 60 days, according to the method by Botsoglou et al. [38]. Absorbance of the thiobarbituric acid adduct was measured at 532 nm (Helios y, Thermo Electron Corp., Waltham, MA, USA). The method was slightly modified for samples containing red beet and leek powder due to the intensive red colour caused by red pigments. For these samples, malondialdehyde (MDA) was extracted from the reaction mixture of trichloroacetic acid extract and thiobarbituric acid with n-butanol. This organic solvent, which is immiscible with water, was chosen due to the low extractability of betalains [39] and its suitability for MDA extraction [40]. The results were expressed as mg MDA/kg using 1,1,3,3-tetramethoxypropane as standard.

2.5. Nitrite and Nitrate Determination

Residual nitrite was determined according to HRN EN 12014-3:2007 [41]. Briefly, 5 g of the sample was extracted with hot water, the pH was adjusted to 8.0–8.5, and then heated at 100 °C. After cooling, the sample was clarified with Carrez I and II solutions and filtered. Nitrite in the clear extract was reacted with sulfanilamide and N-(1-naphthyl)-ethylenediamine dihydrochloride to form a red azo dye, and absorbance was measured at 540 nm (DR/4000U, Hach, Loveland, CO, USA). Results were expressed as sodium nitrite equivalents (mg/kg).

Residual nitrate was determined using a validated enzymatic UV method with a commercial kit (R-Biopharm-Roche, Darmstadt, Germany). Five grams of sample were extracted in boiling water, clarified with Carrez I and II, and the pH was adjusted to 8.0. The sample was then diluted to 100 mL, cooled to allow fat separation, and filtered. Nitrate concentration was measured spectrophotometrically at 340 nm (DR/4000U, Hach, Loveland, CO, USA). Results were expressed as sodium nitrate equivalents (mg/kg).

2.6. Fatty Acid Analysis

Fatty acid methyl esters (FAME) were prepared as described by Vulić et al. [42] following EN ISO 12966–2:2011 [43] and EN ISO 12966–4:2015 [44]. Separation was performed by gas chromatography (7890BA GC-FID, Agilent Technologies, Santa Clara, CA, USA) on a DB-23 capillary column (60 m × 0.25 mm, 0.25 μm film). One μL was injected in split–splitless mode (270 °C; split 1:50) with helium as carrier gas. The oven temperature was programmed from 130 °C to 230 °C in increments; the detector temperature was maintained at 280 °C. Fatty acids were identified by comparing their retention times with standards, and the results were expressed as % of the total fatty acids. Analyses were performed in duplicate on three samples per treatment. Quality control used CRM BCR 163 (IRMM, Geel, Belgium).

2.7. Multielemental Analysis

Elements were quantified according to Vihnanek Lazarus et al. [45]. Samples were freeze-dried for 72 h (HyperCOOL HC3055, LabTech, Namyangju-si, Republic of Korea; average water content 66%), and homogenised in a Mixer Mill MM 400 (Retsch, Haan, Germany). Aliquots (160 mg) were acid-digested in an UltraCLAVE IV (Milestone, Sorisole, Italy) with ultrapure water and purified nitric acid (Merck, Darmstadt, Germany). Macro-elements (Ca, K, Na, Mg) and trace elements (As, Cd, Co, Cu, Fe, Pb, Mn, Hg, Mo, Se, Zn) were quantified by ICP-MS (8900, Agilent Technologies, Santa Clara, CA, USA). Quality control was performed using certified reference materials (ERM^®-BB184 Bovine muscle; SRM 1577a Bovine liver; SRM 1570a Spinach leaves), with recoveries ranging from 91 to 112%. Results are expressed on a dry mass basis.

2.8. Sensory Analysis

Sensory analyses were performed in the Sensory Laboratory of the Faculty of Agriculture, University of Zagreb, equipped according to ISO 8589:2007 [46] (room conditions: relative humidity 50–55%, temperature 20–22 °C, illumination 4000 K and 500 lux at working table level). All evaluations were conducted with Compusense software using digital ballots. Assessors signed informed consent prior to participation. All sensory evaluations were conducted under blind and randomised conditions. Samples were coded with three-digit random numbers and served in randomised order to avoid positional or expectation bias.

Two methods were applied:

Stage 1—Difference-From-Control (DFC) Test: Fourteen trained assessors according to the ISO 8586:2023 [47] evaluated after 30 days of storage five control–test pairs per session using Compusense software Version 24.0.17 (Compusense, ON, Canada) according to Meilgaard et al. [48]. Differences were rated on a 0–5 scale (0 = no difference; 5 = very large).

Stage 2—Modified QDA: A modified quantitative descriptive analysis (QDA) was performed after 1 and 60 days of storage according to Meilgaard et al. [48]. For that purpose, nine trained assessors (four men, five women; age 38–55) with prior experience in meat product evaluation were prepared according to the ISO 8586:2023 [47] and further trained according to Meilgaard et al. [48]. The panel training program was designed to ensure that assessors achieved a high level of sensitivity, discrimination ability, and reproducibility in descriptive sensory evaluation. The overall training process comprised approximately 24 h, conducted in two phases. In the first phase (terminology development, 8 h), assessors collectively identified and defined relevant appearance, odour, flavour, and texture attributes using consensus vocabulary through structured discussions using commercial reference products that represented the expected sensory range. This phase focused on establishing precise attribute definitions, reference standards, and evaluation procedures. The second phase (quantitative calibration and practice, 16 h) was devoted to familiarizing assessors with the scaling method and to refining their scoring consistency. Using unstructured 0–100 intensity scales anchored with defined reference points, panellists practiced evaluating known intensity levels across selected attributes. Progressive calibration sessions, involving both wide and subtle product differences, were held to train discrimination and repeatability skills. Each session concluded with group feedback and discussion to reinforce the use of terminology and scaling alignment.

Panel performance was monitored through repeatability (measure of intra-assessor variation between replicates as absolute differences between repeated samples), accuracy (measure of assessor’s performance against the panel median), and precision (measure of assessor’s ability to discriminate among samples). In total, eight samples were evaluated in one session (one per treatment plus one replicate) and two sessions took place following a balanced block design. Attribute intensity was rated on a 0–100 scale. Samples were individually coded with three-digit numbers and served at room temperature in sensory booths, on white ceramic plates. A 5 min break was provided between samples and 30 min were allowed between sessions. Assessors used water, bread, and green apples as palate cleansers.

2.9. Statistical Analysis

All results are expressed as mean ± SD. For each batch, three sausage samples per treatment were analysed, yielding a total of six observations per treatment (n = 6). Data from both batches were statistically evaluated together. For sensory data, replication followed the panel design, with the number of assessors or repetitions indicated in the respective section. Analyses were performed in SAS Studio Release: 3.82 [49]. Prior to applying parametric tests, data were examined for normality (Shapiro–Wilk test) and homogeneity of variance (Levene’s test), confirming that the assumptions for ANOVA were met. Descriptive statistics were obtained with PROC MEANS. Physicochemical data (pH, aw, colour, nitrate/nitrite, TBARS) were analysed by PROC GLM with Tukey’s post hoc test (p = 0.05). Sensory Stage 1 DFC data were analysed by ANOVA with Dunnett’s post hoc test (α = 0.05) to detect differences from control. Stage 2 QDA data were analysed with PROC MIXED (treatment fixed; assessor random) and Tukey’s post hoc test (p = 0.05). Graphs were prepared in Microsoft Excel.

3. Results

3.1. Nitrite and Nitrate Dynamics

Residual nitrite, nitrate, and total concentrations in sausages from control (A), Stage 1 (B treatments), and Stage 2 (C treatments) during storage are presented in

Table 2. Residual nitrite, nitrate, and total concentrations (mg/kg) in sausages from Control (A), Stage 1—Nitrate Conversion Validation (B), and Stage 2—Vegetable Nitrate Application (C) during storage. Values are mean ± SD (n = 6).

Trait	Day	Control 1	Stage 1—Nitrate Conversion Validation 2				Stage 2—Vegetable Nitrate Application 3
		A	B-30-90	B-30-180	B-40-90	B-40-180	C-30-90	C-30-180
Nitrites	1	71.30 ± 5.93 a	17.90 ± 1.58 e	28.70 ± 1.48 d	38.14 ± 3.34 c	48.62 ± 4.06 b	17.06 ± 0.85 e	28.18 ± 2.35 d
	15	65.02 ± 5.15 a	17.08 ± 1.47 e	25.95 ± 1.86 d	31.98 ± 2.60 c	45.46 ± 2.14 b	18.06 ± 0.85 e	26.58 ± 1.31 d
	30	52.32 ± 2.01 a	13.57 ± 0.50 e	18.91 ± 0.94 c	20.98 ± 1.56 c	29.74 ± 2.34 b	16.08 ± 1.14 d	31.05 ± 2.13 b
	60	33.23 ± 1.54 a	10.44 ± 0.51 e	11.51 ± 0.44 de	13.83 ± 1.14 d	9.00 ± 1.05 e	15.99 ± 1.07 c	20.92 ± 1.71 b
Nitrates	1	6.63 ± 0.33 f	73.96 ± 2.79 ab	60.11 ± 2.79 c	41.67 ± 4.06 d	29.07 ± 1.85 e	79.86 ± 7.77 a	66.24 ± 5.27 bc
	15	34.82 ± 2.51 d	84.13 ± 3.64 a	64.60 ± 4.81 b	62.54 ± 4.82 b	47.74 ± 2.36 c	80.03 ± 3.50 a	56.05 ± 4.88 bc
	30	47.77 ± 2.40 ef	71.10 ± 3.42 ab	60.93 ± 4.62 cd	66.24 ± 4.76 bc	42.16 ± 2.47 f	75.76 ± 3.17 a	53.79 ± 4.84 de
	60	45.14 ± 3.31 c	54.16 ± 3.34 ab	52.55 ± 3.59 ab	49.89 ± 3.07 bc	50.95 ± 3.40 bc	57.28 ± 2.86 a	46.99 ± 3.73 c
Total	1	77.93 ± 5.84 b	91.86 ± 3.19 a	88.81 ± 4.22 ab	79.80 ± 5.61 b	77.68 ± 5.91 b	96.92 ± 7.58 a	94.42 ± 7.62 a
	15	99.84 ± 2.65 a	101.21 ± 4.22 a	90.55 ± 2.96 c	94.52 ± 2.25 bc	93.18 ± 4.49 bc	98.10 ± 2.73 ab	82.63 ± 5.45 d
	30	100.08 ± 4.41 a	84.67 ± 2.92 c	79.85 ± 4.72 c	87.21 ± 6.31 bc	71.89 ± 3.68 d	91.85 ± 2.75 b	84.83 ± 2.70 c
	60	78.37 ± 3.48 a	64.60 ± 3.06 cd	64.05 ± 3.94 cd	63.72 ± 2.86 cd	59.95 ± 4.40 d	73.27 ± 2.92 ab	67.90 ± 2.02 bc

^abcdef Different superscript letters within the same row indicate significant differences (p < 0.05). ¹ Control treatment with nitrites added. ² B-30-90: added nitrates incubated at 30 °C for 90 min; B-30-180: added nitrates incubated at 30 °C for 180 min; B-40-90: added nitrates incubated at 40 °C for 90 min; B-40-180: added nitrates incubated at 40 °C for 180 min. ³ C-30-90: added red beet and leek powder incubated at 30 °C for 90 min; C-30-180: added red beet and leek powder incubated at 30 °C for 180 min.

. Significant differences (p < 0.05) were observed among most treatments within the same sampling day. In the Control (A) group, nitrite concentrations decreased progressively during storage, from 71 mg/kg on day 1 to 33 mg/kg on day 60. In Stage 1 (B) treatments, nitrite levels were significantly lower (p < 0.05) than in the Control group (A) and also declined over time. The lowest concentrations were consistently recorded in B-30-90 and B-30-180, whereas incubation at 40 °C resulted in higher initial values. In Stage 2 (C) sausages, nitrite levels on day 1 were comparable to B-30-90 and B-30-180, but C-30-180 retained higher concentrations by day 60 (20.92 mg/kg).

Residual nitrate concentrations showed an opposite pattern. The control (A) samples contained minimal nitrate on day 1, while B treatments started at higher levels that declined with increasing incubation temperature and duration. C treatments exhibited the highest nitrate concentrations, with C-30-9 maintaining values above most B treatments throughout storage.

When nitrite and nitrate contents were combined, total concentrations were initially highest in the C treatments, followed by B-30-90 and B-30-180, while the Control (A) showed comparatively lower values. During storage, total concentrations in the Control (A) remained relatively stable, whereas those in the experimental treatments decreased more substantially, with the lowest level determined in B-40-180 on day 60.

3.2. pH, Water Activity, and Colour Parameters

Residual pH, water activity (aw), and instrumental colour values are presented in

Table 3. pH, water activity (aw), colour parameters (CIE L*, a*, b*), and total colour difference (ΔE) in sausages from Control (A), Stage 1—Nitrate Conversion Validation (B), and Stage 2—Vegetable Nitrate Application (C) during storage. Values are mean ± SD (n = 6).

Trait	Day	Control 1	Stage 1—Nitrate Conversion Validation 2				Stage 2—Vegetable Nitrate Application 3
		A	B-30-90	B-30-180	B-40-90	B-40-180	C-30-90	C-30-180
pH value	1	6.35 ± 0.02	6.38 ± 0.02	6.37 ± 0.03	6.35 ± 0.00	6.37 ± 0.04	6.37 ± 0.04	6.34 ± 0.02
	60	6.34 ± 0.09	6.35 ± 0.06	6.39 ± 0.04	6.36 ± 0.01	6.42 ± 0.02	6.34 ± 0.04	6.35 ± 0.02
aw value	1	0.968 ± 0.008	0.974 ± 0.001	0.968 ± 0.002	0.971 ± 0.001	0.970 ± 0.002	0.972 ± 0.003	0.966 ± 0.004
	60	0.954 ± 0.003	0.956 ± 0.001	0.956 ± 0.002	0.962 ± 0.002	0.960 ± 0.001	0.960 ± 0.003	0.963 ± 0.003
CIE L*	1	61.78 ± 0.57 a	62.99 ± 1.01 a	62.13 ± 2.14 a	61.72 ± 0.63 a	61.67 ± 0.49 a	47.65 ± 1.12 b	45.81 ± 0.66 b
	30	62.50 ± 1.34 a	61.71 ± 0.65 a	61.49 ± 0.74 a	63.58 ± 2.05 a	61.06 ± 0.48 a	46.37 ± 0.42 b	45.57 ± 0.54 b
	60	62.94 ± 0.57 a	62.76 ± 0.97 a	62.00 ± 0.43 a	62.94 ± 1.34 a	62.06 ± 1.37 a	45.25 ± 0.65 b	45.40 ± 1.09 b
CIE a*	1	15.44 ± 0.09 b	14.33 ± 0.45 b	14.80 ± 1.14 b	15.92 ± 0.07 b	15.68 ± 0.44 b	22.24 ± 0.88 a	22.72 ± 0.66 a
	30	15.87 ± 0.91 b	14.93 ± 0.35 b	15.61 ± 0.16 b	15.15 ± 0.96 b	15.66 ± 0.73 b	23.25 ± 0.25 a	24.87 ± 0.50 a
	60	15.49 ± 0.24 c	14.33 ±0.48 c	15.38 ± 0.10 c	15.20 ± 0.88 c	15.28 ± 0.68 c	23.25 ± 0.65 b	25.38 ± 0.63 a
CIE b*	1	7.00 ± 0.04 b	6.41 ± 0.18 b	6.68 ± 0.05 b	6.93 ± 0.14 b	6.65 ± 0.19 b	13.89 ± 0.89 a	12.76 ± 0.61 a
	30	7.46 ± 0.23 b	6.59 ± 0.07 d	6.70 ± 0.14 cd	7.11 ± 0.26 bc	6.71 ± 0.20 cd	11.77 ± 0.37 a	11.75 ± 037 a
	60	7.26 ± 0.15 b	6.49 ± 0.19 c	6.79 ± 0.31 b	7.04 ± 0.21 b	6.66 ± 0.31 b	10.89 ± 0.34 a	11.34 ± 0.33 a
CIE ΔE*ab	1	-	1.75	0.80	0.48	0.44	17.13	18.47
	30	-	1.50	1.29	1.35	1.63	18.25	19.65
	60	-	1.41	1.06	0.36	1.09	19.66	20.55

^abcd Different superscript letters within the same row indicate significant differences (p < 0.05). ¹ Control treatment with nitrites added. ² B-30-90: added nitrates incubated at 30 °C for 90 min; B-30-180: added nitrates incubated at 30 °C for 180 min; B-40-90: added nitrates incubated at 40 °C for 90 min; B-40-180: added nitrates incubated at 40 °C for 180 min. ³ C-30-90: added red beet and leek powder incubated at 30 °C for 90 min; C-30-180: added red beet and leek powder incubated at 30 °C for 180 min.

. pH values in the Control (A) group remained stable throughout storage with no significant fluctuations. Stage 1 (B) treatments exhibited similarly stable pH values across all incubation conditions and storage days. Stage 2 (C) treatments showed pH comparable to both the Control (A) and Stage 1 (B) treatments, with no significant changes during storage. No significant differences in pH were detected among treatments within each sampling day (p > 0.05).

Water activity (aw) in Control (A) samples decreased slightly over the 60-day storage period, with a reduction of 0.014 units between day 1 to day 60. Stage 1 (B) and Stage 2 (C) treatments followed a similar trend, showing minor reductions during storage. No significant differences (p > 0.05) in aw were found between treatments at any sampling day.

Instrumental colour measurements indicated clear treatment effects. Lightness (CIE L*) values remained stable in the Control (A) and Stage 2 (B) treatments throughout storage, whereas Stage 2 (C) samples exhibited significantly lower (p < 0.05) lightness. Redness (CIE a*) and yellowness (CIE b*) values did not differ significantly between the Control (A) and Stage 1 (B) treatments but were significantly higher (p < 0.05) in Stage 2 (C) treatments at all sampling points.

Overall colour differences (CIE ΔE*_ab) remained low in the Stage 1 (B) treatments, indicating minimal variation during storage. In contrast, Stage 2 (C) treatments displayed consistently higher ΔE values, clearly differentiating these samples from the Control (A) groups.

3.3. Oxidative Stability (TBARS)

TBARS values (mg MDA/kg) are presented in

Table 4. Thiobarbituric acid reactive substances (TBARS, mg MDA/kg) in sausages from Control (A), Stage 1—Nitrate Conversion Validation (B), and Stage 2—Vegetable Nitrate Application (C) during storage. Values are mean ± SD (n = 6).

Trait	Day	Control 1	Stage 1—Nitrate Conversion Validation 2				Stage 2—Vegetable Nitrate Application 3
		A	B-30-90	B-30-180	B-40-90	B-40-180	C-30-90	C-30-180
TBARS	1	0.03 ± 0.005 b	0.08 ± 0.003 b	0.07 ± 0.014 b	0.08 ± 0.003 b	0.09 ± 0.002 b	0.353 ± 0.088 a	0.396 ± 0.012 a
	60	0.08 ± 0.002 c	0.08 ± 0.003 c	0.08 ± 0.006 c	0.07 ± 0.006 c	0.09 ± 0.023 c	0.354 ± 0.015 b	0.417 ± 0.043 a

^abc Different superscript letters within the same row indicate significant differences (p < 0.05). ¹ Control treatment with nitrites added. ² B-30-90: added nitrates incubated at 30 °C for 90 min; B-30-180: added nitrates incubated at 30 °C for 180 min; B-40-90: added nitrates incubated at 40 °C for 90 min; B-40-180: added nitrates incubated at 40 °C for 180 min. ³ C-30-90: added red beet and leek powder incubated at 30 °C for 90 min; C-30-180: added red beet and leek powder incubated at 30 °C for 180 min.

. In the Control (A) group, values remained low and stable throughout storage, with only slight increases by the end of the period. Stage 1 (B) treatments followed a similar trend, with minimal variations across incubation conditions and storage days. No significant differences (p > 0.05) were detected between the Control (A) and Stage 1 (B) treatments or among Stage 1 (B) treatments at any sampling day.

In contrast, Stage 2 (C) treatments, displayed significantly (p < 0.05) higher TBARS values compared with both the Control (A) and Stage 1 (B) groups. These elevated values were present at the beginning of storage and remained consistently higher throughout the experimental period, with only minor additional increases over time.

3.4. Basic Chemical Composition

Basic chemical composition values of sausages are presented in

Table 5. Basic chemical composition (g/kg) of sausages from Control (A), Stage 1—Nitrate Conversion Validation (B), and Stage 2—Vegetable Nitrate Application (C). Values are mean ± SD (n = 6).

Trait	Day	Control 1	Stage 1—Nitrate Conversion Validation 2				Stage 2—Vegetable Nitrate Application 3
		A	B-30-90	B-30-180	B-40-90	B-40-180	C-30-90	C-30-180
Dry matter	1	316.76 ± 7.29	317.89 ± 5.77	315.31 ± 3.68	315.90 ± 5.32	316.43 ± 4.17	317.11 ± 2.84	318.17 ± 5.34
	60	317.12 ± 9.37	316.07 ± 9.11	316.12 ± 6.66	317.89 ± 3.79	314.85 ± 4.79	318.04 ± 0.89	317.93 ± 3.71
Protein	1	169.38 ± 4.65	169.47 ± 3.22	170.18 ± 1.88	168.92 ± 5.58	169.08 ± 1.41	167.11 ± 3.98	165.85 ± 5.95
	60	170.81 ± 1.60	168.76 ± 3.05	169.22 ± 4.05	168.86 ± 2.71	168.49 ± 3.43	166.02 ± 3.00	166.47 ± 5.73
Fat	1	114.97 ± 3.06 ab	116.30 ± 5.51 a	113.02 ± 1.88 b	114.98 ± 2.92 ab	115.36 ± 3.46 ab	107.73 ± 3.95 c	110.43 ± 3.39 bc
	60	113.86 ± 5.14 ab	115.38 ± 4.73 a	114.78 ± 4.09 ab	117.02 ± 4.79 a	114.03 ± 1.46 ab	108.75 ± 2.62 b	108.91 ± 2.79 b
Ash	1	25.56 ± 0.67	25.32 ± 0.58	25.00 ± 0.46	24.62 ± 0.34	25.27 ± 0.72	25.30 ± 0.33	24.80 ± 1.46
	60	25.66 ± 1.29	24.99 ± 1.39	24.59 ± 1.94	25.06 ± 0.69	25.15 ± 0.49	25.95 ± 1.21	25.36 ± 0.84
Carbohydrate	1	6.85 ± 0.89 b	6.80 ± 0.57 b	7.11 ± 0.63 b	7.38 ± 0.58 b	6.72 ± 0.64 b	16.97 ± 1.32 a	17.09 ± 1.29 a
	60	6.79 ± 0.62 b	6.94 ± 0.65 b	7.53 ± 0.72 b	6.95 ± 0.71 b	7.18 ± 0.68 b	17.32 ± 1.25 a	17.19 ± 1.39 a
Sugar	1	4.22 ± 0.69 b	4.11 ± 0.35 b	4.47 ± 0.51 b	4.75 ± 0.42 b	3.99 ± 0.44 b	14.18 ± 0.75 a	14.16 ± 0.74 a
	60	4.15 ± 0.53 b	4.26 ± 0.57 b	4.89 ± 0.41 b	4.23 ± 0.39 b	4.22 ± 0.31 b	14.03 ± 1.15 a	14.11 ± 1.01 a

^abc Different superscript letters within the same row indicate significant differences (p < 0.05). ¹ Control treatment with nitrites added. ² B-30-90: added nitrates incubated at 30 °C for 90 min; B-30-180: added nitrates incubated at 30 °C for 180 min; B-40-90: added nitrates incubated at 40 °C for 90 min; B-40-180: added nitrates incubated at 40 °C for 180 min. ³ C-30-90: added red beet and leek powder incubated at 30 °C for 90 min; C-30-180: added red beet and leek powder incubated at 30° C for 180 min.

. Dry matter content in the Control (A) group increased slightly during storage. Stage 1 (B) treatments showed comparable values across all sampling days, and a similar trend was observed in Stage 2 (C) treatments. No significant differences (p > 0.05) in dry matter were found between treatments at any time.

Protein content remained stable throughout storage in all treatments, with no significant differences observed among Control (A), Stage 1 (B), and Stage 2 (C) groups.

Fat content in the Control (A) sausages decreased slightly by the end of storage. Stage 1 (B) treatments showed comparable results, with no significant differences compared to the Control (A). Stage 2 (C) treatments, however, exhibited a significantly lower (p < 0.05) fat content than both the Control (A) and Stage 1 (B) treatments, most likely due to the lower fat content of the incorporated vegetable powders.

Ash content remained consistent across treatments and storage periods, with no significant variations detected. Carbohydrate and sugar content showed the clearest treatment-related differences. The Control (A) and Stage 1 (B) treatments contained comparable amounts, whereas Stage 2 (C) treatments displayed significantly higher (p < 0.05) levels, which remained consistent over time. As shown in Table 5, most of the carbohydrate fraction consisted of sugars.

3.5. Fatty Acid Composition and Indices

Fatty acid composition and ratios are presented in

Table 6. Fatty acid composition (%) and ration in sausages from Control (A), Stage 1—Nitrate Conversion Validation (B), and Stage 2—Vegetable Nitrate Application (C) after 1 and 60 days of storage. Values are mean ± SD (n = 6).

Trait	Day	Control 1	Stage 1—Nitrate Conversion Validation 2				Stage 2—Vegetable Nitrate Application 3
		A	B-30-90	B-30-180	B-40-90	B-40-180	C-30-90	C-30-180
SFA	1	40.82 ± 0.67 a	36.54 ± 0.65 d	36.02 ± 0.72 de	38.50 ± 0.31 bc	37.60 ± 0.62 cd	39.12 ± 0.84 ab	38.03 ± 0.91 bc
	60	40.89 ± 0.40 a	37.95 ± 0.68 bc	37.25 ± 0.65 c	39.33 ± 0.54 ab	38.00 ± 0.27 bc	38.74 ± 0.86 b	37.37 ± 0.64 c
MUFA	1	45.11 ± 0.50	45.37 ± 1.76	45.43 ± 1.07	45.20 ± 0.81	45.40 ± 1.05	44.86 ± 0.41	44.90 ± 0.86
	60	45.15 ± 0.21	44.66 ± 0.88	44.63 ± 0.51	44.74 ± 0.35	44.52 ± 0.68	45.12 ± 0.74	44.33 ± 0.99
PUFA	1	12.56 ± 0.73 e	15.77 ± 0.57 ab	16.32 ± 0.53 a	14.63 ± 0.25 cd	14.71 ± 0.47 bcd	13.47 ± 0.48 de	15.34 ± 0.54 abc
	60	12.41 ± 0.11 c	15.25 ± 0.58 ab	15.64 ± 0.19 a	14.65 ± 0.52 b	15.24 ± 1.08 ab	14.03 ± 0.66 b	15.50 ± 0.30 a
n-3 PUFA	1	0.35 ± 0.01 e	0.98 ± 0.02 b	1.09 ± 0.02 a	0.83 ± 0.01 d	0.94 ± 0.02 bc	0.77 ± 0.03 d	0.90 ± 0.02 c
	60	0.38 ± 0.02 e	0.93 ± 0.04 abc	0.97 ± 0.04 ab	0.85 ± 0.05 cd	0.81 ± 0.03 d	0.92 ± 0.03 bc	1.04 ± 0.05 a
n-6 PUFA	1	12.21 ± 0.42 c	14.79 ± 0.48 a	15.23 ± 0.53 a	13.80 ± 0.44 b	13.77 ± 0.47 b	12.70 ± 0.50 bc	14.44 ± 0.56 ab
	60	12.03 ± 0.12 d	14.33 ± 0.37 ab	14.67 ± 0.18 a	13.80 ± 0.47 bc	14.42 ± 1.08 a	13.12 ± 0.64 c	14.46 ± 0.33 a
SFA/PUFA	1	3.25 ± 0.16 a	2.32 ± 0.05 d	2.21 ± 0.08 d	2.63 ± 0.07 bc	2.56 ± 0.11 bc	2.91 ± 0.17 ab	2.48 ± 0.12 cd
	60	3.29 ± 0.02 a	2.49 ± 0.11 bc	2.38 ± 0.07 c	2.68 ± 0.10 b	2.50 ± 0.20 bc	2.76 ± 0.10 b	2.41 ± 0.04 c
n-6/n-3	1	34.88 ± 1.39 a	15.09 ± 0.72 bc	13.97 ± 0.51 c	16.63 ± 0.61 b	14.65 ± 0.79 c	16.49 ± 1.19 b	16.04 ± 1.14 b
	60	31.66 ± 1.72 a	15.41 ± 0.55 c	15.12 ± 0.64 cd	16.24 ± 0.61 bc	17.80 ± 1.30 b	14.26 ± 0.41 d	13.90 ± 0.66 d

^abcde Different superscript letters within the same row indicate significant differences (p < 0.05). ¹ Control treatment with nitrites added. ² B-30-90: added nitrates incubated at 30 °C for 90 min; B-30-180: added nitrates incubated at 30 °C for 180 min; B-40-90: added nitrates incubated at 40 °C for 90 min; B-40-180: added nitrates incubated at 40 °C for 180 min. ³ C-30-90: added red beet and leek powder incubated at 30 °C for 90 min; C-30-180: added red beet and leek powder incubated at 30 °C for 180 min.

, with individual fatty acids detailed in

Table A1. Fatty acid composition of cooked sausages from Control (A), Stage 1—Nitrate Conversion Validation (B), and Stage 2—Vegetable Nitrate Application (C) at day 1 and 60. Values are mean ± SD (n = 6).

Fatty Acid	Day	Control 1	Stage 1—Nitrate Conversion Validation 2				Stage 2—Vegetable Nitrate Application 3
		A	B-30-90	B-30-180	B-40-90	B-40-180	C-30-90	C-30-180
C10:0	1	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
	60	0.00 ± 0.00	0.13 ± 0.01	0.13 ± 0.01	0.12 ± 0.01	0.13 ± 0.01	0.14 ± 0.01	0.14 ± 0.01
C12:0	1	0.11 ± 0.01	0.10 ± 0.01	0.10 ± 0.01	0.09 ± 0.01	0.09 ± 0.01	0.10 ± 0.01	0.11 ± 0.01
	60	0.11 a ± 0.01	0.09 ab ± 0.01	0.09 ab ± 0.01	0.08 b ± 0.01	0.08 b ± 0.01	0.09 ab ± 0.01	0.10 ab ± 0.01
C14:0	1	1.50 a ± 0.03	1.34 b ± 0.04	1.29 b ± 0.04	1.36 b ± 0.04	1.26 c ± 0.07	1.37 b ± 0.02	1.47 a ± 0.03
	60	1.50 a ± 0.14	1.31 ab ± 0.08	1.32 ab ± 0.05	1.28 bc ± 0.04	1.25 bc ± 0.02	1.36 ab ± 0.08	1.32 ab ± 0.06
C15:0	1	0.06 ± 0.01	0.06 ± 0.01	0.06 ± 0.01	0.07 ± 0.01	0.06 ± 0.01	0.06 ± 0.01	0.06 ± 0.01
	60	0.06 ± 0.01	0.06 ± 0.01	0.06 ± 0.01	0.07 ± 0.01	0.07 ± 0.01	0.06 ± 0.01	0.06 ± 0.01
C15:1	1	0.11 b ± 0.01	0.00 c ± 0.00	0.00 c ± 0.00	0.00 c ± 0.00	0.00 c ± 0.0	0.09 b ± 0.01	0.19 a ± 0.01
	60	0.11 a ± 0.01	0.00 b ± 0.00	0.00 b ± 0.00	0.00 b ± 0.00	0.00 b ± 0.00	0.00 b ± 0.00	0.00 b± 0.00
C16:0	1	24.22 a ± 0.89	22.55 b ± 0.32	22.67 b ± 0.75	23.64 ab ± 0.34	23.80 a ± 0.73	24.72 a ± 0.93	22.46 b ± 0.56
	60	24.68 a ± 0.87	23.46 ab ± 0.41	22.76 b ± 0.96	23.98 a ± 0.18	22.66 b ± 0.25	23.69 ab ± 0.52	22.61 b ± 0.64
C16:1	1	2.98 ± 0.10	2.95 ± 0.09	2.96 ± 0.07	2.88 ± 0.15	2.75 ± 0.05	2.94 ± 0.12	2.75 ± 0.08
	60	3.02 ± 0.29	2.99 ± 0.12	2.88 ± 0.12	2.82 ± 0.11	2.70 ± 0.18	2.86 ± 0.15	2.75 ± 0.09
C17:0	1	0.47 d ± 0.01	0.48 d ± 0.01	0.45 d ± 0.02	0.66 a ± 0.01	0.59 b ± 0.01	0.53 c ± 0.01	0.52 c ± 0.02
	60	0.47 b ± 0.05	0.46 b ± 0.03	0.46 b ± 0.02	0.69 a ± 0.04	0.63 a ± 0.03	0.53 ab ± 0.02	0.48 b ± 0.02
C17:1	1	0.27 c ± 0.01	0.31 c ± 0.01	0.30 c ± 0.01	0.45 a ± 0.03	0.39 b ± 0.02	0.32 c ± 0.01	0.29 c ± 0.01
	60	0.28 a ± 0.01	0.25 a ± 0.01	0.00 c ± 0.00	0.25 a ± 0.01	0.25 a ± 0.02	0.18 b ± 0.01	0.00 c ± 0.00
C18:0	1	13.51 a ± 0.51	11.84 b ± 0.59	11.26 b ± 0.33	12.48 ab ± 0.40	11.64 b ± 0.29	12.18 b ± 0.25	13.23 a ± 0.31
	60	13.16 ± 0.54	12.17 ± 0.24	12.12 ± 0.46	12.92 ± 0.27	12.99 ± 0.48	12.67 ± 0.28	12.35 ± 0.42
C18:1	1	41.11 ± 0.44	41.47 ± 1.67	41.55 ± 1.01	41.17 ± 0.66	41.60 ± 1.00	40.85 ± 0.39	41.07 ± 0.94
	60	41.18 ± 0.51	40.76 ± 0.95	41.13 ± 0.58	40.97 ± 0.45	40.86 ± 0.57	41.41 ± 0.85	40.96 ± 1.04
C18:2 n6	1	11.75 b ± 0.41	14.04 a ± 0.58	14.32 a ± 0.51	13.07 a ± 0.25	13.10 a ± 0.75	11.99 b ± 0.51	13.91 a ± 0.55
	60	11.62 c ± 0.13	13.49 a ± 0.35	13.92 a ± 0.19	13.07 ab ± 0.47	13.68 a ± 1.11	12.35 bc ± 0.64	13.70 a ± 0.32
C18:3 n3	1	0.35 e ± 0.01	0.83 b ± 0.02	0.93 a ± 0.02	0.71 c ± 0.01	0.79 b ± 0.02	0.65 d ± 0.02	0.72 c ± 0.02
	60	0.38 d ± 0.02	0.78 b ± 0.03	0.83 a ± 0.04	0.72 bc ± 0.04	0.68 c ± 0.02	0.71 c ± 0.02	0.89 a ± 0.04
C20:0	1	0.18 ± 0.01	0.18 ± 0.01	0.19 ± 0.02	0.21 ± 0.02	0.17 ± 0.01	0.16 ± 0.01	0.18 ± 0.01
	60	0.19 ± 0.01	0.20 ± 0.01	0.20 ± 0.01	0.20 ± 0.01	0.20 ± 0.01	0.21 ± 0.01	0.20 ± 0.01
C20:1	1	0.65 ± 0.03	0.64 ± 0.02	0.63 ± 0.02	0.70 ± 0.05	0.65 ± 0.02	0.66 ± 0.02	0.64 ± 0.02
	60	0.64 ± 0.03	0.66 ± 0.03	0.62 ± 0.02	0.71 ± 0.03	0.71 ± 0.02	0.67 ± 0.02	0.62 ± 0.02
C20:2 n6	1	0.35 d ± 0.01	0.54 ab ± 0.02	0.58 a ± 0.03	0.44 c ± 0.01	0.50 b ± 0.02	0.46 bc ± 0.01	0.42 c ± 0.02
	60	0.32 c ± 0.01	0.53 a ± 0.02	0.54 a ± 0.02	0.46 b ± 0.01	0.47 b ± 0.02	0.49 ab ± 0.01	0.54 a ± 0.01
C20:3 n6	1	0.00 b ± 0.00	0.00 b ± 0.00	0.11 a ± 0.01	0.08 a ± 0.01	0.00 b ± 0.00	0.08 a ± 0.01	0.00 b ± 0.00
	60	0.00 b ± 0.00	0.10 a ± 0.01	0.00 b ± 0.00	0.08 a ± 0.01	0.08 a ± 0.01	0.09 a ± 0.01	0.00 b ± 0.00
C20:3 n3	1	0.00 c ± 0.00	0.15 a ± 0.01	0.16 a ± 0.01	0.13 b ± 0.01	0.15 a ± 0.01	0.12 b ± 0.01	0.18 a ± 0.01
	60	0.00 b ± 0.00	0.15 a ± 0.01	0.14 a ± 0.01	0.13 a ± 0.01	0.14 a ± 0.01	0.13 a ± 0.01	0.15 a ± 0.01
C20:4 n6	1	0.10 c ± 0.01	0.22 ab ± 0.01	0.24 a ± 0.01	0.20 b ± 0.01	0.20 b ± 0.01	0.17 b ± 0.01	0.11 c ± 0.01
	60	0.10 b ± 0.01	0.21 a ± 0.01	0.22 a ± 0.01	0.19 a ± 0.02	0.20 a ± 0.02	0.19 a ± 0.02	0.22 a ± 0.01
C21:0	1	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
	60	0.00 b ± 0.00	0.12 a ± 0.01	0.00 b ± 0.00	0.00 b ± 0.00	0.00 b ± 0.00	0.11 a ± 0.01	0.00 b ± 0.00
C22:5 n3	1	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
	60	0.00 b ± 0.00	0.00 b ± 0.00	0.00 b ± 0.00	0.00 b ± 0.00	0.00 b ± 0.00	0.08 a ± 0.01	0.00 b ± 0.00
C23:0	1	0.78 a ± 0.03	0.00 b ± 0.00	0.00 b ± 0.00	0.00 b ± 0.00	0.00 b ± 0.00	0.00 b ± 0.00	0.00 b ± 0.00
	60	0.72 a ± 0.04	0.07 b ± 0.01	0.00 c ± 0.00	0.00 c ± 0.00	0.00 c ± 0.00	0.00 c ± 0.00	0.00 c ± 0.00

^abcde Different superscript letters within the same row and the same sampling day (1 or 60) indicate significant differences (p < 0.05). ¹ Control treatment with nitrites added. ² B-30-90: added nitrates incubated at 30 °C for 90 min; B-30-180: added nitrates incubated at 30 °C for 180 min; B-40-90: added nitrates incubated at 40 °C for 90 min; B-40-180: added nitrates incubated at 40 °C for 180 min. ³ C-30-90: added red beet and leek powder incubated at 30 °C for 90 min; C-30-180: added red beet and leek powder incubated at 30 °C for 180 min.

. Significant treatment effects (p < 0.05) were observed for several parameters. On day 1, saturated fatty acids (SFAs) were highest in the Control (A) treatment, lower in Stage 1 (B) treatments, particularly in the B-30-180, and intermediate in Stage 2 (C) treatments. This pattern persisted on day 60, ted, with Control (A) sausages maintaining the highest SFA values and B-30-180 and C-30-180 showing the lowest.

Monounsaturated fatty acids (MUFAs) content did not differ significantly (p > 0.05) among treatments at any sampling day. In contrast, polyunsaturated fatty acids (PUFAs) showed significant differences (p < 0.05). Control (A) sausages consistently exhibited the lowest PUFA values, Stage 1 (B) treatments showed significantly (p < 0.05) higher levels, and Stage 2 (C) treatments displayed intermediate to high values. Within Stage 2 (C), C-30-180 contained significantly more (p < 0.05) PUFAs than C-30-90 at both sampling points. Table A1 indicates that these differences were mainly attributable to variations in C16:0, C18:0, C18:2n-6 and C18:3n-3 fatty acids.

n-3 PUFA content was significantly higher (p < 0.05) in Stage 1 (B) and Stage 2 (C) treatments compared do Control (A), with the highest values observed in B-30-180 and B-30-90 on day 1 and in C-30-180 and B-30-180 on day 60. Similarly, n-6 PUFA levels were significantly higher (p < 0.05) in Stage 1 (B) and Stage 2 (C) treatments compared to Control (A) on both sampling days. Within Stage 1 (B), incubation at 40 °C tended to increase SFAs and reduce PUFAs (including n-3 and n-6), with several differences reaching statistical significance (p < 0.05).

Lipid quality ratios followed these compositional trends. The SFA/PUFA ratio was significantly higher (p < 0.05) in Control (A) compared with all other treatments, except C-30-90 on day 1. Within Stage 1 (B), incubation at 30 °C resulted in a significantly lower (p < 0.05) SFA/PUFA ratio than incubation at 40 °C. The n-6/n-3 ratio was also significantly higher (p < 0.05) in Control (A) than in all other treatments on both sampling days.

3.6. Elemental Composition

Elemental composition data are presented in

Table 7. Elemental composition of sausages from Control (A), Stage 1—Nitrate Conversion Validation (B), and Stage 2—Vegetable Nitrate Application (C). Values are mean ± SD (n = 6).

Trait	Control 1	Stage 1—Nitrate Conversion Validation 2				Stage 2—Vegetable Nitrate Application 3
	A	B-30-90	B-30-180	B-40-90	B-40-180	C-30-90	C-30-180
Na (mg/kg)	25,351.88 ± 2034.11 a	23,627.77 ± 1461.10 ab	21,698.74 ± 966.61 cd	19,915.68 ± 1418.07 d	21,299.03 ± 1241.96 cd	21,384.73 ± 1048.28 cd	22,728.35 ± 2398.22 bc
K (mg/kg)	7525.18 ± 543.63 bc	7455.97 ± 392.12 bc	7213.73 ± 281.43 c	6136.37 ± 455.17 d	6959.79 ± 377.69 c	7900.78 ± 382.80 ab	8491.79 ± 881.01 a
Mg (mg/kg)	711.25 ± 41.95 bc	663.12 ± 35.55 cd	626.51 ± 27.36 d	553.56 ± 34.39 e	611.84 ± 26.68 d	758.53 ± 38.83 ab	793.86 ± 82.58 a
Ca (mg/kg)	230.22 ± 6.54 b	256.56 ± 23.07 b	229.04 ± 8.74 b	210.83 ± 12.02 b	226.78 ± 17.3 b	601.75 ± 39.05 a	607.65 ± 61.39 a
Zn (mg/kg)	71.23 ± 4.58	76.01 ± 6.73	74.01 ± 3.3	71.19 ± 6.88	70.36 ± 3.42	69.62 ± 5.68	73.13 ± 8.58
Fe (mg/kg)	24.24 ± 1.64 bc	26.17 ± 3.79 b	23.50 ± 1.29 bc	21.53 ± 1.18 c	23.02 ± 1.12 c	31.34 ± 1.68 a	33.68 ± 3.97 a
Cu (mg/kg)	2.33 ± 0.20 b	2.36 ± 0.19 b	2.21 ± 0.07 b	2.13 ± 0.10 b	2.12 ± 0.05 b	2.83 ± 0.52 a	2.70 ± 0.31 a
Mn (mg/kg)	1.95 ± 0.07 bc	2.19 ± 0.31 b	1.71 ± 0.06 c	1.75 ± 0.04 c	1.77 ± 0.11 c	4.82 ± 0.35 a	4.72 ± 0.45 a
Se (μg/kg)	314.35 ± 17.95 bcd	339.68 ± 14.12 a	334.82 ± 14.13 ab	325.96 ± 16.05 abc	336.40 ± 8.96 ab	302.86 ± 20.4 d	312.21 ± 31.39 c
Mo (μg/kg)	30.71 ± 2.38 bc	35.91 ± 10.70 b	32.06 ± 1.51 bc	29.86 ± 2.34 c	30.81 ± 1.08 bc	52.79 ± 3.13 a	55.03 ± 4.83 a
Co (μg/kg)	17.37 ± 6.84 a	3.64 ± 0.63 c	3.01 ± 0.21 c	4.17 ± 1.97 c	2.76 ± 0.19 c	10.94 ± 0.73 b	10.91 ± 1.28 b
Pb (μg/kg)	4.44 ± 1.00 c	6.55 ± 1.00 b	3.81 ± 0.42 c	4.43 ± 0.74 c	4.03 ± 0.21 c	12.24 ± 1.21 a	13.10 ± 1.15 a
As (μg/kg)	2.61 ± 0.42 b	2.75 ± 0.30 b	2.57 ± 0.33 b	2.22 ± 0.36 b	2.60 ± 0.28 b	5.88 ± 0.50 a	6.76 ± 0.76 a
Cd (μg/kg)	1.51 ± 0.19 b	2.04 ± 0.26 b	1.71 ± 0.24 b	1.44 ± 0.16 b	1.39 ± 0.10 b	16.14 ± 1.26 a	15.66 ± 1.24 a

^abcde Different superscript letters within the same row indicate significant differences (p < 0.05). ¹ Control treatment with nitrites added. ² B-30-90: added nitrates incubated at 30 °C for 90 min; B-30-180: added nitrates incubated at 30 °C for 180 min; B-40-90: added nitrates incubated at 40 °C for 90 min; B-40-180: added nitrates incubated at 40 °C for 180 min. ³ C-30-90: added red beet and leek powder incubated at 30 °C for 90 min; C-30-180: added red beet and leek powder incubated at 30 °C for 180 min.

, grouped as macroelements (Na, K, Mg, and Ca) and trace elements (Zn, Fe, Cu, Mn, Se, Mo, Co, Pb, As, and Cd).

For macroelements, sodium concentration was highest in the Control (A) treatment and significantly lower (p < 0.05) in all experimental treatments, except for B-30-90, which showed comparable values. Stage 2 (C) sausages contained significantly higher (p < 0.05) concentrations of potassium, magnesium, and calcium compared to the Control (A).

Regarding trace elements, no significant differences in zinc content were observed among treatments (p > 0.05). Iron, copper, and manganese levels were significantly higher (p < 0.05) in Stage 2 (C) sausages compared with the Control (A) and Stage 1 (B) treatments. Selenium content was significantly lower (p < 0.05) in Stage 2 (C) sausages compared with the Control (A) and Stage 1 (B). Molybdenum content was significantly higher (p < 0.05) in Stage 2 (C) than in other treatments. Cobalt concentrations were highest in Control (A), lower in Stage 1 (B), and intermediate but significantly higher (p < 0.05) in Stage 2 (C) compared with Stage 1 (B).

Lead, arsenic, and cadmium were significantly higher (p < 0.05) in Stage 2 (C) sausages compared with the Control (A) and Stage 1 (B) sausages. Lead content in the B-30-90 treatment was significantly higher (p < 0.05) than in the Control (A) and other Stage 1 (B) treatments.

3.7. Sensory Analysis

Two sensory methods were applied: difference-from-control (DFC) for Stage 1 and quantitative descriptive analysis (QDA) for Stage 2.

Stage 1—Difference-from-control (DFC) results are presented in

Table 8. Difference-from-control (DFC) test scores of sausages from control (A) and Stage 1—Nitrate Conversion Validation (B) at day 30. Values are mean ± SD (n = 14).

	Control 1	Stage 1—Nitrate Conversion Validation 2
	A	B-30-90	B-30-180	B-40-90	B-40-180
DFC score 3	1.17 ± 0.71	1.61 ± 1.04	1.17 ± 1.25	2.00 ± 1.28	2.28 ± 1.36
p-value	-	0.60	1.00	0.11	0.02

¹ Control treatment with nitrites added. ² B-30-90: added nitrates incubated at 30 °C for 90 min; B-30-180: added nitrates incubated at 30 °C for 180 min; B-40-90: added nitrates incubated at 40 °C for 90 min; B-40-180: added nitrates incubated at 40 °C for 180 min. ³ Mean difference from control (DFC) on a 0–5 scale, where 0 = no difference and 5 = very large difference.

. On day 30, the Control (A) treatment showed a low mean DFC score, confirming the validity of the methodology and assessors’ consistency. Within Stage 1 treatments, B-30-180 showed the lowest mean DFC score, closely matching the Control (A). B-30-90 treatment did not differ significantly from Control (A), while B-40-90 and B-40-180 showed higher mean DFC scores, with B-40-180 significantly different (p < 0.05) from the Control (A).

Stage 2—Quantitative descriptive analysis (QDA) results are presented in

Table 9. Quantitative descriptive analysis (QDA) of sausages from Control (A), and Stage 2—Vegetable Nitrate Application (C) at day 1 and 60. Values are mean ± SD (n = 9).

Attribute	Control 1		Stage 2—Vegetable Nitrate Application 2
	A		C-30-90		C-30-180
	Day 1	Day 60	Day 1	Day 60	Day 1	Day 60
Slice red colour	20.00 ± 8.36 b	13.38 ± 7.09 b	71.17 ± 14.52 a	62.88 ± 18.88 a	71.83 ± 11.48 a	67.75 ± 18.16 a
Slice brown colour	4.50 ± 2.73 b	5.63 ± 3.37 b	48.50 ± 16.73 a	30.25 ± 13.33 a	46.33 ± 17.67 a	34.75 ± 13.07 a
Slice coherence	68.67 ± 14.76 a	63.63 ± 22.20	35.33 ± 13.01 b	55.95 ± 19.39	30.50 ± 13.49 b	53.25 ± 16.95
Slice appearance typicality	75.00 ± 8.29 a	87.50 ± 8.45 a	49.33 ± 9.77 b	58.75 ± 16.27 b	47.50 ± 15.54 b	59.75 ± 14.64 b
Heat-treated meat odour	61.33 ± 20.16	69.63 ± 18.04	60.67 ± 15.4	60.50 ± 13.8	59.53 ± 15.9	59.50 ± 16.54
Red beet odour	1.33 ± 1.97 b	4.25 ± 5.69 b	25.17 ± 13.16 a	35.13 ± 16.09 a	24.34 ± 17.14 a	30.25 ± 16.35 a
Leek odour	2.33 ± 3.39 b	13.25 ± 13.06 b	35.17 ± 16.97 a	50.63 ± 22.37 a	41.69 ± 17.83 a	51.50 ± 22.73 a
Off-odours	10.21 ± 10.1	8.25 ± 3.54	21.54 ± 15.9	14.25 ± 11.35	19.67 ± 11.64	9.50 ± 8.45
Odour typicality	69.17 ± 23.4	83.58 ± 11.35 a	52.92 ± 17.56	51.38 ± 16.71 b	53.50 ± 13.2	68.63 ± 17.58 ab
Firmness	67.00 ± 12.25 a	60.13 ± 11.84 a	31.33 ± 12.27 b	40.25 ± 14.98 b	37.17 ± 16.5 b	38.38 ± 12.75 b
Crumbliness	34.17 ± 13.29 b	50.63 ± 21.53	63.50 ± 27.13 b	63.50 ± 16.77	73.17 ± 15.34 b	66.75 ± 8.58
Juiciness	87.51 ± 7.54 a	67.50 ± 17.29	73.50 ± 7.23 b	65.63 ± 19.73	76.67 ± 9.46 ab	63.47 ± 15.97
Fat mouthfeel	23.17 ± 11.11	37.63 ± 17.18	28.5 ± 13.84	38.88 ± 17.63	27.67 ± 11.14	37.13 ± 12.61
Salty	48.67 ± 11.78	56.02 ± 9.46	39.67 ± 17.88	39.13 ± 14.79	40.33 ± 19.94	41.63 ± 15.69
Sweet	22.67 ± 7.2	11.53 ± 9.43 b	33.50 ± 12.21	31.13 ± 12.62 b	26.67 ± 10.02	26.52 ± 12.77 ab
Sour	7.50 ± 6.69	6.63 ± 6.39	6.67 ± 7.20	11.13 ± 9.27	10.32 ± 8.26	9.10 ± 6.58
Bitter	4.17 ± 4.25	2.50 ± 3.07	7.83 ± 5.13	2.75 ± 3.15	7.00 ± 6.8	2.63 ± 2.67
Spice aroma	49.32 ± 17.72	65.75 ± 15.54	48.33 ± 15.71	55.75 ± 21.39	47.50 ± 19.03	62.54 ± 17.5
Red beet aroma	3.00 ± 4.65 b	3.13 ± 5.25 b	26.67 ± 12.33 a	36.50 ± 17.23 a	22.33 ± 12.77 a	32.38 ± 14.84 a
Leek aroma	2.67 ± 4.18 b	12.13 ± 10.62 b	50.17 ± 19.45 a	52.63 ± 17.86 a	51.83 ± 19.99 a	53.13 ± 18.85 b
Heat-treated meat aroma	66.17 ± 8.40	64.25 ± 19.71	53.33 ± 16.49	56.25 ± 13.47	55.85 ± 16.37	60.75 ± 15.44
Off-flavours	8.17 ± 5.71	2.13 ± 3.64	16.17 ± 12.89	13.63 ± 13.77	20.83 ± 11.69	9.38 ± 12.12
Aftertaste	63.83 ± 15.92	66.13 ± 16.57	58.67 ± 19.77	59.25 ± 18.38	55.83 ± 18.63	60.84 ± 15.85
Aroma harmony	83.83 ± 13.26 a	77.25 ± 17.57 a	61.67 ± 12.97 b	58.13 ± 14.73 ab	62.33 ± 15.45 b	53.38 ± 14.16 b
Aroma typicality	84.17 ± 11.03 a	78.38 ± 19.91 a	53.83 ± 19.45 b	51.75 ± 17.82 b	54.83 ± 11.86 b	53.38 ± 16.35 b

^ab Different superscript letters within the same row and the same sampling day (1 or 60) indicate significant differences (p < 0.05). ¹ Control treatment with nitrites added. ² C-30-90: added red beet and leek powder incubated at 30 °C for 90 min; C-30-180: added red beet and leek powder incubated at 30 °C for 180 min.

. Control (A) sausages were consistently rated significantly higher (p < 0.05) for slice coherence, appearance typicality, odour typicality, firmness, juiciness, aroma harmony, and aroma typicality. Stage 2 (C) sausages were rated higher for slice red and brown colour, red beet and leek odour and aroma, crumbliness, and sweet taste. These differences were consistent across incubation times. Sampling time influenced several attributes: slice red colour, off-odours, bitter taste, off-flavours, and aroma harmony scores decreased during storage, whereas appearance typicality, red beet and leek odour and aroma, fat mouthfeel, and spice aroma increased across all treatments.

4. Discussion

The two-stage design of this study allowed systematic validation of microbial nitrate-to-nitrite conversion before extending the process to vegetable nitrate sources.

4.1. Stage 1—Nitrate Conversion Validation (A vs. B Treatments)

The results of Stage 1 demonstrated that microbial nitrate curing with Staphylococcus carnosus can result in technological outcomes comparable to those of direct nitrite curing, provided that incubation conditions are carefully optimized. In this experiment, two incubation temperatures (30 and 40 °C) and two incubation times (90 and 180 min) were applied to validate the efficiency of nitrite formation and the effect on nutritional, physical, and sensory properties of sausages. Nitrite levels in the Control (A) sausages (from 71.30 mg/kg on day 1 to 33.23 mg/kg on day 60) were within the range reported by in previous studies [11,50,51], although higher than values reported by Jantapirak et al. [52], Sheng et al. [53] (2025), and Stamenić et al. [54]. The comparatively higher residual nitrite levels observed in our study may be attributed to the absence of a vacuum during nitrite addition and sausage preparation. The application of vacuum processing enhances the efficiency of nitrite reduction by lowering the partial pressure of oxygen and thereby shifting the redox potential toward a more reducing state. Under such anaerobic conditions, the chemical and enzymatic conversion of nitrite to nitric oxide is favoured [16,17]. Furthermore, oxygen removal limits the oxidative reconversion of nitrite to nitrate and reduces the degradation of nitric oxide, resulting in improved curing efficiency [1,6]. As shown in Table 2, nitrate-to nitrite conversion was effective, confirming efficient microbial conversion. These findings are in line with earlier reports demonstrating that coagulase-negative staphylococci, including Staphylococcus carnosus, reduce nitrate effectively under mild incubation conditions [17].

Nitrite levels in nitrate-incubated sausages from Stage 1 (B) treatments were lower (17.90–48.62 mg/kg on day 1) than in Control (A) sausages (71.30 mg/kg on day 1), where sodium nitrite was directly added. This difference indicates that the method of nitrite introduction influences its final concentration. In Stage 1 (B) treatments, nitrite was generated gradually by S. carnosus and a portion of it was immediately consumed in reactions forming nitric oxide and subsequent curing pigments. In contrast, direct nitrite addition in the Control (A) provided an immediate and higher initial concentration, leading to higher measurable residual nitrite after processing. Similar findings were reported by Sindelar et al. [16] and Terns et al. [17], who observed that microbial nitrate reduction systems tend to yield lower residual nitrite. On the first sampling day, residual nitrite concentrations were reduced by between 3.98-fold (B-30-90) and 1.47-fold (B-40-180) compared with the Control (A). A similar trend was observed by Krause et al. [55], who observed consistently higher residual nitrite in nitrite-added control group across the entire 42-day storage period. From a toxicological perspective, residual nitrite is undesirable due to its role in the formation of potentially carcinogenic N-nitroso compounds (nitrosamines) [1]. Therefore, lower residual nitrite values can be considered favourable. Nevertheless, adequate nitrite concentrations remain essential for safety, particularly to provide a stronger and longer-lasting barrier against pathogens, as well as for colour stabilization and antioxidant protection [1,2].

In contrast to nitrite, nitrate levels were significantly higher in Stage 1 (B) sausages (from 73.96 mg/kg in B-30-90 to 29.07 mg/kg in B-40-180) than in Control (A) group (6.63 mg/kg) on day 1, indicating that the total curing potential (nitrite + nitrate) was maintained, although conversion to nitrite was slower and less complete than with direct nitrite addition. This aligns with the review by Premi et al. [56], which highlighted that, when combined with coagulase-negative staphylococci, nitrate undergoes gradual microbial reduction, leaving relatively high residual concentrations during the early stages of processing. Moreover, the accumulation of nitrite from microbial reduction is constrained by the redox state of the system and microbial competition [56]. Therefore, direct nitrite addition ensures immediate and predictable curing effects, while microbial nitrate curing depends on microbial activity, which is more variable and less efficient.

The higher nitrate levels observed in Stage 1 (B) sausages reflect incomplete conversion during incubation, as expected in systems relying on microbial nitrate reduction. Although this resulted in lower residual nitrite, no adverse effects on product quality were detected. Colour development (Table 3), oxidative stability (Table 4), and sensory properties (Table 8) remained comparable to the Control (A), particularly in samples incubated at 30 °C. Research specifically examining the curing efficiency of chemically added nitrates in form of NaNO₃ combined with S. carnosus, as applied in Stage 1, is limited. However, studies using vegetable-derived nitrate sources have shown that microbial curing does not compromise product safety, provided that bacterial cultures, primarily S. carnosus, remain active and incubation conditions allow sufficient nitrite formation to inhibit spoilage bacteria [10,30,57]. Overall, these results confirm that microbial nitrate curing maintained both technological functionality and safety despite lower nitrite concentrations. During storage, nitrite declined steadily across all treatments, consistent with the well-documented depletion of curing agents during shelf life [1,3,15,27,51]. Meanwhile, nitrate levels increased from day 1 to day 15, likely due to nitrite oxidation to nitrate [1], as also confirmed by Sheng et al. [53]. Residual nitrite and nitrate are important for colour preservation in cured meat products because they can interconvert with each other or form nitric oxide, thus serving as a “reservoir” that stabilizes cured meat colour during storage [2]. This effect is supported by the results in Table 3, where no significant changes in the CIE L* and CIE a* colour parameters of the Control (A) (CIE L* 61.78–62.94; CIE a* 15.44–15.87) and Stage 1 (B) sausages (CIE L* 61.06–63.58; CIE a* 14.33–15.92) was observed during storage. Incubation at 30 °C promoted gradual and sustained nitrite formation, whereas 40 °C accelerated formation, indicating stable curing at both temperatures as previously determined for S. carnosus by Casaburi et al. [21], and Terns et al. [17]. However, nitrite content decreased more rapidly from day 30 to day 60 in treatments incubated at 40 °C. As reported by Domínguez et al. [58], lipid oxidation exhibits temperature-dependent kinetics, where elevated temperature accelerates hydroperoxide decomposition and radical propagation. These reactive compounds can subsequently interact with nitrite-derived nitric oxide or nitrous acid. Consequently, nitrite is likely consumed more rapidly at higher temperatures through coupled oxidative and nitrosative pathways, resulting in an accelerated decline of residual nitrite content, despite the initially higher conversion efficiency.

pH and water activity remained stable and unaffected by treatment on day 1 (pH 6.35–6.38; aw 0.968–0.974) and day 60 (pH 6.34–6.42; aw 0.954–0.962), confirming that incubation did not compromise product stability. These results are in line with earlier studies showing that nitrate-reducing cultures function effectively without altering the acid–base balance of cooked sausages [16]. Similarly, colour attributes of Control (A) and Stage 1 (B) sausages were comparable and within the ranges reported in other studies [11,28,50]. Since as little as 10–15 ppm residual nitrite is sufficient for colour stability [59], the observed levels were adequate to ensure the formation of stable cured pigments such as nitrosylmyoglobin and its heat-induced derivative, nitrosylhemochrome [1,2].

Oxidative stability of Stage 1 (B) sausages was also maintained, as TBARS values (0.07–0.09 mg MDA/kg) were comparable to those of Control (A) sausages (0.03–0.08 mg MDA/kg) throughout storage (Table 4), confirming that nitrite generated from nitrate provided sufficient antioxidant protection. Nitrite and its derivatives (NO, NO₂, N₂O₃) act as radical scavengers and stabilize heme iron, thereby limiting lipid oxidation [1,3]. Similar TBARS values (0.065 mg MDA/kg) have been reported in nitrite-cured (150 ppm sodium nitrite) pork products [11]. These results agree with reports that the combination of nitrate and nitrite lowers TBARS values compared to nitrite alone [60]. However, findings in fermented sausages have been less consistent, with several reports showing higher TBARS and peroxide values in nitrate-cured compared to nitrite-cured products [61,62]. Such discrepancies could be influenced by factors including microbial activity, incubation temperature, and product composition [17,21,56], which affect the rate and extent of nitrate-to-nitrite conversion and the subsequent formation of nitric oxide. In the present study, controlled incubation with S. carnosus ensured sufficient nitrate reduction and nitrite availability, resulting in oxidative stability comparable to direct nitrite curing despite differences in nitrite source and concentration obtained. The basic chemical composition presented in Table 5 was unaffected by incubation or microbial activity. Levels of protein, fat, ash, carbohydrates, and sugars remained within expected ranges, supporting the view that starter cultures influence curing chemistry without causing major alterations in proximate composition [4].

Fatty acid composition revealed a modest nutritional benefit of microbial nitrate curing, as Stage 1 (B) sausages showed lower SFA proportions (36.02–38.50%) and higher PUFA content (14.63–16.32%) compared with Control (A) (SFA 40.82%; PUFA 12.56%). Since SFA cannot be directly converted into PUFA during meat processing, these shifts likely reflect relative compositional changes rather than true inter-conversion. A possible explanation is that microbial activity of Staphylococcus carnosus influenced PUFA release and retention within the sausage matrix. Many starter cultures used in meat fermentation, especially coagulase-negative staphylococci (CNS) such as S. carnosus, are known to possess lipase and esterase activities [21,56,63,64]. Lipases primarily act on long-chain triglycerides, releasing free fatty acids, while esterases hydrolyse short-chain esters and phospholipids, both of which are typically rich in PUFA. The incubation conditions applied in this study (30 or 40 °C for 90 or 180 min) likely influenced the activity of these enzymes, as moderate heating can enhance lipolytic reaction rates by improving substrate availability and enzyme–substrate interaction within the meat matrix. In addition, endogenous tissue lipases may have contributed to this process. As demonstrated by Hierro et al. [64], meat lipases remain active during processing and can account for up to 60% of total lipolysis in dry fermented sausages, indicating that both microbial and endogenous enzymes play complementary roles. Additionally, some strains of Staphylococcus carnosus also produce catalase [21], which decomposes hydrogen peroxide and reduces pro-oxidant stress. By mitigating oxidative reactions, these microbes may indirectly protect PUFA from peroxidation, thereby preserving higher proportions during storage.

Elemental profiles were generally comparable across treatments, with no systematic differences in major elements. However, cobalt concentrations were notably lower in nitrate-incubated Stage 1 (B) sausages (2.76–4.17 μg/kg) compared with Control (A) (17.37 μg/kg). For such a result we do not have an apparent explanation.

Sensory evaluation confirmed 30 °C as the optimal incubation temperature for nitrite curing. Sausages incubated at 30 °C, especially with extended incubation, were perceived as highly similar to nitrite controls (DFC score < 1.75), while those incubated at 40 °C were consistently rated as more different (DFC score > 2.00). This aligns with previous studies showing that nitrite production from natural sources is maximized at 30 °C [56]. Furthermore, Dasiewicz et al. [65] reported that utilizing “cold” fermentation instead of “warm” fermentation, improves nutritional and sensory characteristics and extends the shelf life of salami-type sausages. These results reinforce the present findings that excessive incubation intensity can negatively shift flavour balance.

Based on the selection criteria defined in the Materials and Methods, nitrate-to-nitrite conversion in Stage 1 (B) sausages was effective at both incubation temperatures and durations. Higher temperature and longer duration showed conversion efficiency closer to Control sausages (A). Colour equivalence between Stage 1 (B) sausages and Control (A) was maintained (ΔE < 2), while oxidative stability was not compromised, as TBARS values remained statistically equivalent. Finally, sausages incubated at 30 °C showed smaller sensory deviations from Control (A) with mean DFC < 1.75, whereas those incubated at 40 °C constantly exceeded mean DFC > 2.00. Overall, these findings demonstrate that microbial nitrate curing with Staphylococcus carnosus at 30 °C provides chemical, nutritional, and sensory outcomes equivalent to conventional nitrite curing. This establishes Stage 1 as a robust baseline for subsequent testing of vegetable-derived curing agents.

4.2. Stage 2—Vegetable Nitrate Application

The second stage of this study transferred validated incubation conditions from Stage 1 to produce sausages cured with vegetable nitrate sources (red beet and leek powders). Stage 1 confirmed that nitrate reduction by Staphylococcus carnosus at 30 °C for 90 and 180 min produced outcomes comparable to direct nitrite curing, thereby establishing a baseline. This was crucial to separate the effects of vegetable ingredients from incubation conditions in Stage 2.

Residual nitrite levels in C-30-90 (17.06 mg/kg on day 1) and C-30-180 (28.18 mg/kg) sausages were comparable to nitrate-incubated counterparts (17.90 mg/kg in B-30-90 and 28.70 mg/kg in B-30-180), but consistently lower than nitrite Control (A) sausages (71.30 mg/kg). Residual nitrate remained elevated, reflecting slower release or limited bioavailability from the plant matrix. Similar findings have been reported for celery, spinach, radish, and red beet powders, where nitrite generation efficiency depends strongly on both vegetable type and incubation time [18,19,23,26,28,66]. Indeed, incubation time and temperature are consistently reported as critical factors for achieving efficient conversion, often exerting a greater influence than vegetable nitrate concentration [17,66]. The lower nitrite levels observed in Stage 2 (C) treatments thus reflect a combined effect of microbial nitrate reduction, as observed in Stage 1, and intrinsic limitations of the vegetable sources themselves.

Physicochemical properties such as pH and water activity remained stable across treatments on day 1 (pH 6.34–6.37; aw 0.966–0.972) and day 60 (pH 6.34–6.35; aw 0.960–0.963), indicating no safety compromises. However, treatments cured with vegetable powders (C-30-90 and C-30-180) demonstrated markedly higher redness (CIE a* 22.24 and 22.72, respectively) and yellowness (CIE b* 13.89 and 12.76, respectively), accompanied by reduced lightness (CIE L* 47.65 and 45.81, respectively), relative to both the Control (A) (CIE a* 15.44, CIE b* 7.00, CIE L* 61.78) and Stage 1 (B) (CIE a* 14.33–15.94, CIE b* 6.41–6.93, CIE L* 61.67–62.99) sausages. Similar colour shifts have been reported by other authors [23,26,28] and are most likely attributable to betalain pigments in red beet [67,68], which persist throughout storage. Consequently, CIE ΔE*_ab values compared with Control (A) exceeded 17, far higher than values reported in previous studies (e.g., CIE ΔE*_ab = 2.6 [25]; or CIE ΔE*_ab = 6.22−7.40 [23]). Comparable findings were noted when red beet extracts were fermented to produce pre-converted nitrite; they imparted strong redness but showed limitations in oxidative protection unless combined with accelerators such as ascorbic acid [14,23]. These outcomes highlight that vegetable curing inherently creates visually distinct products, as confirmed by large colour differences (CIE ΔE*_ab values). The discrepancy with earlier reports can be explained by the direct addition of red beet powder in this study, rather than the pre-converted red beet juice employed in previous works. In pre-converted systems, microbial or enzymatic reduction of nitrate to nitrite is completed before application, enabling uniform nitric oxide formation and more typical cured colour development [10,16]. In contrast, the direct incorporation of red beet powder introduces a high content of betalain pigments [67,68], which impart an intense purplish-red hue independent of nitrosylmyoglobin formation [28,60]. Furthermore, the presence of natural antioxidants and sugars in red beet can alter pigment stability and Maillard browning during heating, contributing to enhanced redness and CIE ΔE*_ab values relative to conventional curing systems [20]. Furthermore, vegetable powder addition in Stage 2 (C) was adjusted to match nitrite levels in Control (A) and nitrate levels in Stage 1 (B) sausages, requiring the incorporation of 2% red beet and 1% leek powder. Under these conditions, beet pigments dominated colour development, masking the typical cured appearance. Schopfer et al. [28] got similar conclusions, noting that comparable amounts of beet extract produced highly detectable colour change compared to chemical curing during incubation, thereby limiting its ability to reproduce the typical appearance of nitrite-cured products.

TBARS values in Stage 2 (C) sausages on day 60 (0.354–0.417) were four to five times higher than in Control (A) (0.08) or Stage 1 nitrate-incubated (B) sausages (0.07–0.09), despite comparable total curing potential (Table 2). Similar findings were previously reported by Jeong et al. [11] who found that nitrite-added control samples had significantly lower (p < 0.05) TBARS values (0.065 mg MDA/kg) than alternatively cured pork products containing 0.4% Chinese cabbage, radish, or spinach powder (0.083–0.094 mg MDA/kg). However, in their experiment, the increase was only 1.3–1.4 fold, notably smaller than in our study. Sindelar et al. [16] demonstrated that nitrite-added hams had lower TBARS values compared with those cured using either 0.2% or 0.35% celery juice powder, which is consistent with our findings. The elevated TBARS values in alternatively cured products were unexpected, given that vegetables typically contain natural antioxidants that should supress lipid oxidation. However, it should be noted that despite the very low solubility of red beet betalains in n-butanol [39], it is possible that some were co-extracted with MDA during extraction, resulting in increased absorbance values and a higher MDA content in sausages. In addition, the red beet and leek powders used in this study were particularly rich in sugars (42.1% and 13.1%, respectively), increasing the total sugar content of Stage 2 (C) sausages (Table 5). Elevated sugar levels can enhance Maillard and caramelization reactions during processing and storage, generating reactive intermediates that act as pro-oxidants and thereby accelerate lipid oxidation, despite the presence of antioxidant compounds [69]. TBARS values are relevant for predicting off-odour development. According to Tarladgis et al. [70], the threshold for detecting off-odours in cooked pork is 0.5–1.0 mg MDA/kg. In this study, TBARS levels in Stage 2 (C) sausages (0.353–0.417) remained below this threshold, suggesting that rancid odours were unlikely to be evident. Nevertheless, the substantially higher oxidation in Stage 2 (C) treatments compared with Control (A) and Stage 1 (B) sausages indicates a potential risk for quality deterioration, especially during extended storage.

Basic composition (Table 5) confirmed the diluting effect of plant powders, resulting in lower fat (107.73–110.43 g/kg) and higher sugar (13.98–14.36 g/kg) contents in C-30-90 and C-30-180 sausages compared to the Control (A) and Stage 1 (B) treatments. A similar trend has also been observed in formulations incorporating celery, radish, or kimchi powders [18,65,71]. Nutritional lipid indices and ratios improved (Table 6), with reductions in SFA and increases in PUFA and n-3 fatty acids, suggesting that microbial nitrate curing systems may promote better PUFA preservation under certain conditions. Notably, a comparable shift was already evident in Stage 1 (B) sausages cured with nitrate, indicating that these effects are more likely attributable to microbial activity during incubation than to the direct contribution of red beet and leek powders.

Elemental analysis revealed substantial modifications in the profile of Stage 2 (C) sausages compared to Control (A), with both nutritional advantages and safety considerations. On the positive side, the addition of red beet and leek powders increased the concentration of nutritionally valuable elements such as potassium, magnesium, calcium, iron, and manganese, which is in agreement with the intrinsic composition of these vegetables [15,72]. These elements enhance dietary quality and can be considered a beneficial nutritional trait of naturally cured sausages. On the contrary, elevated levels of lead, arsenic, and cadmium (12.24–13.10 μg/kg, 5.88–6.76 μg/kg, and 15.66–16.14 μg/kg, respectively) were found in Stage 2 (C) sausages compared with the Control (A) group (4.44, 2.61, and 1.51 μg/kg, respectively). This trend is consistent with the well-documented ability of root and leafy vegetables to accumulate these elements from soil [73]. According to Commission Regulation (EC) No. 1881/2006 [74] and subsequent amendments, the maximum permissible levels for lead and cadmium in muscle meats are 0.10 mg/kg and 0.05 mg/kg, respectively, while no regulatory limit is currently set for arsenic. Importantly, although lead, arsenic, and cadmium concentrations in Stage 2 (C) sausages were elevated compared with the Control (A), all values remained far below the EU regulatory thresholds, indicating no direct compliance or safety concerns. Taken together, Stage 2 (C) sausages demonstrated both advantages and limitations compared with conventional nitrite curing. Their enriched essential element content adds nutritional value, whereas the elevated presence of lead, arsenic, and cadmium underscores the importance of careful ingredient selection and sourcing. As emphasized by Rivera et al. [7], microbial nitrate curing systems are particularly sensitive to the compositional variability of plant ingredients, making sourcing practices a critical factor in ensuring both safety and product quality.

Quantitative descriptive analysis confirmed that vegetable nitrate curing defines a distinct sensory profile. Stage 2 (C) sausages were characterized by an intensified red and brown colour, pronounced red beet and leek odour and aroma, enhanced sweetness, and greater crumbliness. By contrast, the Control (A) sausages were rated higher in slice coherence, typicality, firmness, juiciness, and aroma harmony. Similar sensory shifts have been reported in sausages cured with celery, radish, spinach, or fruit and vegetable extracts, where flavour and texture deviations were evident despite technological feasibility [17,25,75]. Previous studies indicate that red beet can maintain acceptable sensory quality in meat products. In fermented beef sausages, red beet powder preserved colour, flavour, and overall quality for up to 56 days [26]. In meat emulsions, fermented beet extract combined with ascorbic acid maintained colour and flavour stability and achieved sensory acceptance comparable to synthetic nitrite [8]. In low-salt frankfurters, beet extract improved appearance, colour, and juiciness, with only minor flavour changes during storage [24]. Research on leek demonstrates similar trends. For instance, Zaki and Khallaf [31] reported that camel sausages formulated with leek achieved acceptable appearance, texture, and flavour, in some cases surpassing controls. Luka et al. [76] found that leek powder improved flavour, juiciness, and tenderness in suya, with moderate inclusion yielding the highest overall acceptability. Tsoukalas et al. [30] showed that fermented sausages produced with freeze-dried leek powder and reduced nitrite obtained sensory scores for appearance, flavour, and overall acceptability comparable to nitrite controls, though leek odour became apparent at higher inclusion levels. Likewise, Eisinaite et al. [57] demonstrated that sausages cured with vegetable powders, including leek, retained acceptable sensory profiles but exhibited distinctive vegetable-associated flavour notes that reduced typicality compared with conventional products.

Compared with these earlier studies, the results presented in Table 9 demonstrate more pronounced differences between Stage 2 (C) treatments and the nitrite Control (A). This contrast may reflect differences in vegetable forms (powder versus fermented extract), incubation and processing conditions influencing nitrate reduction and pigment development, or the product matrix, which can alter colour stability and flavour release. Extended storage conditions in our study may also have intensified oxidative changes and sensory divergence, resulting in clearer treatment-related differences. During storage, lipids and proteins undergo complex oxidation reactions that generate large quantities of free radicals, which can accelerate the oxidation of nitrosylmyoglobin and lead to reduced redness in cooked sausages [77]. Over prolonged periods, the release of iron from heme proteins further catalyses these reactions, promoting lipid peroxidation and the formation of secondary oxidation products that contribute to rancidity and flavour deterioration [58]. Also, lipid oxidation can be indirectly stimulated by protein oxidation through interactions with lipid radicals and oxidative intermediates [58]. However, the phenolic compounds present in vegetable powders may scavenge free radicals and delay nitrosylmyoglobin oxidation, partially stabilising the red colour [77] as observed in present study. While such differentiation may limit direct equivalence to conventional products, it supports the development of distinct “natural” product categories appealing to specific consumer groups [7,22]. The lower typicality scores observed in Stage 2 (Table 9) further suggest that assessors perceived these sausages as belonging to a different sensory category, underscoring their differentiation potential in the marketplace, but also a challenge for consumer acceptance when traditional flavour is expected.

4.3. Critical Considerations of the Approach

By first validating nitrate reduction and incubation conditions in Stage 1, the study design ensured that deviations observed in Stage 2 could be attributed specifically to the addition of red beet and leek powders, rather than microbial activity or incubation regime. This two-phase strategy aligns with recent recommendations emphasizing the importance of distinguishing conversion efficiency from plant-matrix effects in microbial nitrate curing studies [4,17,65]. Nevertheless, incubation requires additional time, strict control of temperature and hygiene, and reliable microbial performance, making the process more complex than direct nitrite addition. The temporary holding step at mild temperatures may also increase microbiological risks if not rigorously managed. These limitations reflect broader concerns regarding the scalability of starter culture-based nitrate curing, which, though effective under experimental conditions, poses challenges for industrial application due to added complexity and cost [3,4]. In this context, direct use of plant-derived nitrate sources may be more feasible for fermented products than for heat-treated sausages.

5. Conclusions

This study used a two-stage experimental design to evaluate strategies for reducing synthetic nitrite use in cooked sausages while preserving essential curing functions. Stage 1 established a controlled baseline by validating microbial nitrate reduction using Staphylococcus carnosus. Incubation at 30 °C for 90 and 180 min with 100 mg/kg sodium nitrate resulted in partial conversion to nitrite, yielding residual nitrite concentrations equivalent to approximately 35–40% of those found in directly nitrite-treated controls. Despite this lower conversion efficiency, sausages exhibited an equivalent colour profile (CIE ΔE* < 2), comparable oxidative stability (TBARS: 0.07–0.09 mg MDA/kg vs. 0.08 mg MDA/kg in controls), and no statistically significant sensory differences from the control group (mean DFC ≤ 1.75). These findings confirm that microbial nitrate curing can reproduce the key technological functions of nitrite. However, not all incubation conditions were equally effective: treatments incubated at 40 °C for 180 min generated higher initial nitrite concentrations but failed to maintain sensory neutrality during storage (mean DFC > 2.00). This outcome underscores the necessity of optimising incubation temperature and duration to balance conversion efficiency with product stability.

Stage 2 applied the validated microbial conversion protocol to sausages cured with red beet and leek powders as natural nitrate sources. Although these powders supplied nitrate levels equivalent to those in Stage 1 (100 mg/kg), they induced pronounced compositional and sensory changes. Sausages displayed intensified redness (CIE a* values), increased sugar content, and fourfold higher lipid oxidation (TBARS: 0.35–0.42 mg MDA/kg). Fatty acid profiles shifted towards higher unsaturation, and multielemental analysis revealed elevated concentrations of lead, arsenic, and cadmium, indicating potential safety concerns. Sensory evaluation further highlighted enhanced slice redness, dominant vegetable aroma, increased sweetness and crumbliness, and reduced firmness and juiciness compared with conventionally cured counterparts.

By separating the effects of microbial nitrate conversion in Stage 1 from those associated with vegetable composition in Stage 2, this study demonstrated both technological feasibility and product differentiation. Collectively, the results indicate that microbial nitrate curing provides a robust and reproducible platform for nitrite replacement with minimal sensory change, whereas vegetable-based nitrate curing introduces both desirable and undesirable compositional and sensory modifications requiring further optimisation. Future research should focus on refining vegetable nitrate formulations, implementing targeted antioxidant and stabilisation strategies, reducing heavy metal accumulation, and assessing consumer perception to ensure safe, consistent, and appealing naturally cured meat products.