Application of High Hydrostatic Pressure (Long Holding Time vs. Two Consecutive Short Cycles) for the Preservation of Lamb Burgers Enriched with Lupinus albus Flour

Abstract

High hydrostatic pressure (HHP) can extend the shelf life and ensure safety of meat products such as lamb burgers. Lupinus albus variety Orden Dorado (a low alkaloid content variety) flour, rich in protein and phenolic compounds, offers the potential to enhance the preservation of meat products during storage. Lamb burgers were formulated with Lupinus albus flours (1%, w/w; weight/weight), either conventional or obtained by compression milling, and processed by HHP treatments (untreated, two consecutive cycles at 600 MPa for 1 s; or a single cycle at 600 MPa for 4 min), with the total processing time using the HHP unit being the same for both. Then, they were subsequently stored for 14 days under refrigerated conditions. Proximate composition, microbiological changes, color, and oxidation of burgers during storage were evaluated. Flour obtained by compression milling presented higher phenolic compound content, while its antioxidant activity is similar to that obtained by conventional methods. In lamb burgers, the incorporation of both lupine flours maintained the proximate composition and fatty acids profile. Lipid oxidation after 14 days was significantly lower in burgers with lupine flour obtained by compression milling, whereas protein oxidation responses depended on treatment–formulation interactions. HHP drastically reduced microbial counts, with sustained inactivation of coliforms and Escherichia coli (E. coli) although instrumental color was significantly altered in fresh burgers after processing. However, sensory scores of grilled burgers remained unaffected by either flour type or HHP treatment. Incorporation of Lupinus albus flour into lamb burgers processed by HHP preserved sensory quality while enhancing the microbial and lipid oxidation stability of burgers. Finally, the application of two short (1 s) cycles at 600 MPa was more beneficial than a single 4 min cycle, offering similar microbial inactivation with less impact on the quality changes in burgers. Finally, applying two short (1 s) HHP cycles at 600 MPa was more beneficial than a single 4 min cycle, as it achieved similar microbial inactivation while better preserving the color and oxidative stability of the burgers.

1. Introduction

Lamb meat is a valuable source of protein, unsaturated lipids, iron, zinc, vitamins A and B12, folic acid, and other essential nutrients important for a well-balanced human diet [1,2,3]. Its production from native breeds such as Merino sheep also contributes significantly to environmental protection, biodiversity conservation, and the support of rural development through sustainable extensive farming practices [4]. It is considered a traditional, high-quality food; however, its consumption has declined markedly in recent years, primarily due to its seasonal availability, its association with festive occasions, and, consequently, its higher price [5]. As a result, consumers tend to opt for more affordable meat alternatives, such as pork, chicken or processed meat [5].

Processed meat consumption has increased recently due to shifting eating habits and limited time available for meal preparation, leading consumers to prefer quick and convenient food options. Among these, burgers are widely consumed due to their low cost and ease of preparation [6,7]. However, meat and meat products are highly perishable foods, which provide a suitable environment for microbial growth [8,9].

High hydrostatic pressure (HHP) is a feasible alternative to increase the safety and enhance the shelf life of meat products [10]. HHP is a non-thermal food preservation technology that has been successfully commercialized to produce microbiologically safe food products [11]. Factors affecting HHP efficiency, including process parameters and implementation challenges, have been evaluated across a range of meat types, such as beef burgers, caiman, cured sausages, goose, turkey, dry-cured products, and chicken [12]. Additionally, HHP extends shelf life without requiring chemical additives, aligning with consumer demand for clean-label foods [13,14,15]. HHP also inactivates pathogenic bacteria such as Salmonella, Listeria, and E. coli and this technology can be combined with other hurdles for further extension of shelf life [16]. In this respect, for the inactivation of bacterial and fungal spores, multi-pulse HHP treatment effectiveness has been documented [17].

However, the treatment of fresh meat products with HHP may induce color changes due to myoglobin denaturation, resulting in a loss of redness. Timón et al. [12] reported that HHP causes noticeable color modifications in chicken meat, which may negatively affect consumer acceptance. In addition, refrigerated storage can further exacerbate these color alterations [13,16]. Moreover, some authors have indicated that pressure levels between 300 and 600 MPa are critical for inducing lipid oxidation in fresh pork, beef, and poultry meat, which can lead to rancidity, off-flavors, and a reduction in shelf life, ultimately compromising the quality and consumer acceptance of meat products [16,18]. To mitigate this effect, these technologies are increasingly combined with natural antioxidants, aiming to minimize oxidative processes and the associated sensory deterioration [19].

While the main sources of natural antioxidants are vegetable products, other underexplored sources, such as legumes, could also provide valuable antioxidant compounds. Legumes are the dry, clean, and separated seeds from the pods of plants belonging to the Leguminosae family, including varieties such as chickpeas, lentils, beans, dried peas, and lupins [20]. Legumes have been a staple in human diets for centuries, playing an important role in the Mediterranean diet. Nutritionally, they are of great importance due to their protein, dietary fiber, and other vital nutrient contents [21]. From an agronomic perspective, they stand out for their ability to fix atmospheric nitrogen in the soil, enhancing soil biodiversity and fertility. Their inclusion in crop rotations can also improve the efficiency of chemical fertilizer use [20,22]. Although the recognition of legumes is increasing, their potential as ingredients in food product development is not being completely exploited due to sensory aspects such as inherent flavor, taste and aroma sensations which are often perceived as negative features by consumers as well as the occurrence of intestinal discomfort when ingested [23]. Among legumes, lupins (Lupinus spp.) stand out for their high protein content and low glycemic index, as their carbohydrates are primarily dietary fiber. They also have a low-fat content and a high proportion of unsaturated fatty acids such as oleic, linoleic and linolenic acids [24]. However, the main challenge associated with lupin consumption in human and animal nutrition is its high alkaloid content, which, while providing plant resistance to pest and pathogens, imparts a bitter taste and can be toxic. To reduce the concentration of these compounds and make the seeds suitable for consumption, low-alkaloid varieties have been selectively bred [25].

Recent marked trends have increased the substitution of meat protein by vegetable protein from different sources with high protein content like lupines. The incorporation of lupins into meat products offers a significant advantage due to their potential antioxidant activity, attributed to their high content of phenolic compounds and tannins, which possess antioxidant properties, as well as their low cost. This could help extend the shelf life of meat products [26].

Legume flours can be obtained through different procedures which could affect their composition and final properties [27]. The objective of this study was to evaluate the nutritional, physicochemical, sensory, and microbiological properties of lamb burgers enriched with flour from Lupinus albus var. Orden Dorado, a local low-alkaloid variety, processed using two milling techniques. Additionally, the synergistic effect of lupin flour enrichment and HHP treatment on extending the burgers’ shelf life was investigated. Two time-equivalent processing conditions were applied using the HHP unit (two consecutive cycles of 600 MPa, 1 s and one long cycle of 600 MPa, 4 min) in order to evaluate the differential effect for burger preservation.

2. Materials and Methods

2.1. Material

2.1.1. Manufacturing Process of Flours from Lupinus albus

Seeds of Lupinus albus var. “Orden Dorado,” a sweet variety known for or distinguished by its low alkaloid content, were obtained from the germplasm bank of the Technological and Scientific Research Centre of Extremadura (CICYTEX, Mérida, Badajoz, Spain) and used for the manufacture of flours added to burgers. This variety, developed by CICYTEX [28] through classical genetic breeding, involved crossing a Polish sweet lupin variety (Lupinus albus L.) with a local bitter variety with alkaloid content monitored by the Drangendorff method [29]. They were grown on the agricultural farm “La Orden” (Guadajira, Badajoz, Spain). Germination occurred in December and flowering in March. Harvesting took place in June, using a Wintersteiger harvester (Wintersteiger AG, Ried, Austria) for the plots under study, with an average yield of 1500 kg/ha. The harvested grain had an average humidity, crude protein and crude fat values of 7.5%, 42.7%, and 6.5%, respectively.

Around 10 kg of lupinus harvested in 2024 was milled at different conditions to obtain the two flour types which were evaluated for the manufacture of lamb burgers. The first flour (Flour 1) was obtained by conventional milling using a hammer mill equipped with sieves with a pore size of 1 mm. The second flour (Flour 2) was produced by compression milling, using a cold expeller press for oilseeds (Farmet, LIS DUO, CZ, Czech Republic) with a 15 mm barrel diameter and an L/D ratio of 15:1. The processing conditions were established at 5–7% moisture and a fixed temperature of 15 °C.

2.1.2. Manufacturing Process of Lamb Burgers

A total of 22 kg of lamb meat (local market, Badajoz, Spain) was required for the development of the different assays. Burger production was carried out in three independent batches. The burgers were prepared according to a traditional recipe: minced lamb meat was mixed at a ratio of 40% flank to 60% leg meat, along with 13 g kg⁻¹ of salt, 1.5 g kg⁻¹ garlic, 1.5 g kg⁻¹ dehydrated onion, 1.5 g kg⁻¹ parsley, and 0.5 g kg⁻¹ black pepper. Each burger had a weight of around 100 g. This formulation was used as the control. Burgers with the two types of flour were prepared using the same base proportions of salt and spices, with the addition of flour. Two types of lupin flour were used: conventional lupin flour (Flour 1) and lupin flour obtained by compression milling (Flour 2). Both were added to the burgers at different concentrations relative to the total mass.

The optimum level of lupin flour (1% w/w) was chosen based on preliminary results of a sensory analysis with several formulations. For that experiment, 4 kg of meat (local market, Badajoz, Spain) was utilized and five burgers per batch were prepared with 0, 1, 5 and 10% of flour (w/w) (Flour 1 and Flour 2), resulting in a total of eight formulations and 40 burgers. Once grilled, the burgers were evaluated, and those containing 1% (w/w) flour showed a similar level of acceptance to those without flour. This concentration was considered optimal, as it did not cause rejection from the panelists.

Another different experiment was carried out following the previous recipe and with the optimized level of the two types of lupin flour (1% w/w) incorporated into lamb burgers. Around 12 kg of meat (local market, Badajoz, Spain) was required. In this experiment, the effect of HHP and the changes during the refrigerated storage of the enriched lamb burgers with lupinus flour were evaluated. All burgers were individually vacuum-packed in plastic bags (Eurobag, polyamide polyethylene 20/100, Madrid, Spain) with an oxygen permeability of 50 cm³ m⁻², 24 h⁻¹, at 0% relative humidity, 120 µm thickness. Some burgers were treated with HHP according to the experimental design and then stored under refrigeration conditions (at 6 °C) for 14 days in darkness. Five burgers per batch were manufactured to optimize HHP conditions, with a total of 90 burgers made for the experiment: 5 (replicates per batch) × 3 formulations (control, Flour 1 and Flour 2) × 3 pressure treatments (untreated, 2 cycles at 600 MPa/1 s and 600 MPa/4 min) × 2 storage times (day 1 and day 14). Additionally, 15 burgers (5 burgers per formulation) were produced for initial characterization (protein and fat content, humidity and fatty acid profile). Thus, a total of 115 (90 + 15) were manufactured. Non-treated burgers at day 14 (n = 5 × 3 = 15) were discarded because of the high microbial load and they were removed from the experiment. The burgers at days 1 and 14 were subjected to microbiological analysis, instrumental color measurement, lipid oxidation, protein oxidation and phenolic compound content.

For the sensory analysis, a third assay was carried out. A total of 54 lamb burgers were manufactured following the same experimental design as for the physicochemical and microbiological study with two types of lupine flour (1% w/w) (control, Flour 1, and Flour 2) and three HHP conditions (untreated, 600 MPa/1 s for 2 cycles, and 600 MPa/4 min). Six burgers per formulation were produced, so a total of 54 burgers were prepared (n = 3 × 3 × 6), using 6 kg of lamb meat in total (local market, Badajoz, Spain). Only burgers at day 1 were evaluated in the sensory analysis to avoid possible microbiological problems. A trained panel of eight regular panelists participated in the evaluation over seven structured sensory sessions. Each session included a balanced and randomized subset of samples.

2.1.3. Treatment of High Hydrostatic Pressure and Refrigerated Storage

Two HHP treatments were applied. Processing conditions were selected based on commercial-scale practices, where a maximum pressure of 600 MPa is commonly used. Short holding times were chosen, as pressure intensity generally has a greater effect than treatment duration. In some cases, two short consecutive pressure cycles are applied to enhance microbial inactivation, since the first cycle induces sublethal damage that increases microorganisms’ sensitivity to the second cycle [17]. Accordingly, the following treatments were evaluated: two cycles at 600 MPa for 1 s (HHP1) and a single cycle at 600 MPa for 4 min (HHP2), with untreated burgers used as the control. The HHP2 treatment had an equivalent total processing time to the two consecutive cycles applied in HHP1.

Pressurization was carried out using a semi-industrial high hydrostatic pressure unit (55 L capacity; Hiperbaric Wave 6000/55, Burgos, Spain) located in Badajoz. Vacuum-packed burgers were processed with an initial water temperature of 15 °C inside the pressure vessel. The pressure come-up time was 3.5 min, and decompression was instantaneous. The storage temperature of 6 °C was selected to simulate realistic refrigerated storage conditions commonly found in retail and domestic environments, where temperature fluctuations above the ideal 4 °C frequently occur.

Both treated and untreated burgers were stored in darkness at 6 ± 1 °C for 14 days. Analyses were conducted on day 1 (the day after production and processing) and after 14 days of refrigerated storage at 6 °C in darkness.

2.2. Methods

2.2.1. Proximate Analysis of Flours from Lupinus albus

Moisture, fat, and protein (g 100 g⁻¹) were determined by drying the samples at 104 °C until constant weight, gravimetrically after extraction with chloroform:methanol (2:1), and by the Kjeldahl method, respectively [30], with minor adaptations for flour matrices. The pH was analyzed with a pH meter Crison pH 25+ (Crison, Barcelona, Spain). The total content of fiber was determined using the modified Southgate method [31]. Total content of phenolic compounds was measured by the he Folin–Ciocalteu reagent-based colorimetric assay [32] and the antioxidant capacity was determined by the ABTS•+ method [33].

2.2.2. Proximate Analysis of Lamb Burgers and Fatty Acid Profile

The same standardized analytical methods described above were applied to burger samples with appropriate adjustments for meat matrices. The moisture, fat, and protein (%) of burgers were determined by drying the samples at 104 °C until constant weight, gravimetrically, after extraction with chloroform/methanol (2:1) and using the Kjeldahl method, respectively [30]. The pH was analyzed with a pH meter Crison pH 25+ (Crison, Barcelona, Spain), and the water activity (a_w) measurement was carried out at 25 °C using a Novasina Labmaster—a_w meter (Novasina AG, Lachen, Switzerland), which provides temperature-controlled measurements.

The fatty acid profile was determined by obtaining the fatty acid methyl esters (FAMEs) using KOH in methanol and analyzing them using an Agilent Technologies 6890 GC (Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector and an Agilent DB-23 (60 m × 0.25 mm ID × 0.25 μm) column. The system was operated in split mode (1:100). Split liners with glass wool and 11 mm septa were used. The injector and detector temperatures were maintained at 260 °C and 280 °C, respectively. The oven temperature program started at 185 °C (held for 2 min), then increased at 5 °C min⁻¹ to 220 °C and held for 15 min. Helium was used as a carrier gas at a constant flow of 1.2 mL min⁻¹. FAME MIX 37 Component (Supelco, LRAD1445, Bellefonte, PA, USA) identification was based on the retention times. Quantification was performed by determining the response factors after injecting with FAME. Results were expressed as required as a percentage.

2.2.3. Microbiological Analyses

Ten grams of burgers were taken aseptically and homogenized with 90 mL of peptone water (Merck, 1.07043, San José, CA, USA) using a laboratory blender (Stomacher^® 400 Circulator, West Sussex, UK) for 1 min. Serial decimal dilutions were prepared in sterile peptone water and 1 mL of each dilution was spread onto appropriate culture media. All microorganisms, except Staphylococcus aureus, were inoculated using the pour plate technique, while S. aureus was inoculated using the spread plate technique. Mesophilic aerobic microorganisms were enumerated using Plate Count Agar (Merck, 1.07881, Darmstadt, Germany), with incubation at 30 °C for 72 h. Psychrophilic microorganisms were counted on the same medium following incubation at 7 °C for 10 days. Molds and yeasts were incubated on Glucose Chloramphenicol Agar (CGA) (Scharlau Microbiology, Barcelona, Spain) at 25 °C for 5 days. Staphylococcus aureus was identified on Baird Parker Agar (Merck, 1.05406, Darmstadt, Germany) following incubation at 37 °C for 24–48 h. Anaerobic sulfite-reducing bacteria (and when needed, specifically Clostridium perfringens) were subjected to 80 °C initial suspension for 10 min to eliminate vegetative forms and leave spores, and seeded in TSC agar (Merck, 1.11972, Darmstadt, Germany) and incubated at 37 °C for 24 h in anaerobic jars using the GasPak™, Sparks, MD, USA, anaerobic system, which generates an oxygen-free environment suitable for the selective growth of anaerobic bacteria. Escherichia coli and total coliforms were detected on Chromocult Agar (Merck, 1.10426, Darmstadt, Germany) after incubation at 37 °C for 24–48 h. Results were expressed as Log₁₀ CFU (colony forming units) per gram. The detection limit for all methods was 10 CFU g⁻¹, except for S. aureus, whose detection limit was 100 CFU g⁻¹.

2.2.4. Instrumental Color of Burgers

Instrumental color determinations were performed with a Minolta CM-5 spectrophotometer (Minolta Camera, Osaka, Japan), with an illuminant/angle of D65/10⁰ and a measuring area of 30 mm. The color coordinates of lightness (L*), redness (a* red/green axis), and yellowness (b* yellow/blue axis) in the CIE Lab color space were analyzed. In addition, hue angle was calculated (h° = tan⁻¹ (b*/a*)) as well as the saturation index or Chroma (C*) (C = (a*² + b*²)^0.5). Two measurements were recorded (one for each side of the burger), and the mean of the two readings was obtained.

2.2.5. Lipid and Protein Oxidation of Burgers

Lipid oxidation was assessed by thiobarbituric acid reactive substances (TBA-RS) according to Sørensen and Jørgensen’s method [34]. TBA-RS values were calculated from the standard (1,1,1,3-tetraethoxypropane, TEP) curve, and the results were expressed as mg of malondialdehyde per kg of sample (mg MDA kg⁻¹). Protein oxidation was assessed by measuring the carbonyl groups formed during incubation with 2,4-dinitrophenylhydrazine (DNPH) in 2 N HCl following the method described by Oliver et al. [35]. The absorbance measurement of protein concentration was detected at λ-280 nm spectrophotometry using a spectrophotometer (Evolution 201 UV–Visible, Thermo Scientific, Waltham, MA, USA) with bovine serum albumin (BSA) as standard. Protein oxidation was expressed as nmol carbonyls mg protein⁻¹.

2.2.6. Total Phenolic Compounds (TPC)

To carry out the extraction, 5 g of lupine flour was added to 45 mL of solution (water 80%, 19.9% methanol, 0.1% citric acid). The mixture was agitated for 1 h at 60 °C and centrifuged at 1000 rpm for 15 min at 4 °C. Subsequently, samples were centrifuged at 1000 rpm for 15 min at 4 °C and the supernatant was evaporated at 37 °C (10 min) using a rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) under a vacuum of 50 mbar. The solvent-free extract was then made up to a final volume of 50 mL with ultra-pure water. The results were expressed in mg gallic acid equivalent (GAE) on 100 g of wet sample by the utilization of a calibration curve with gallic acid.

2.2.7. Sensory Analysis Methodology

The aim of the sensory analysis was to assess the effect of the addition of lupine flour and the effect of HHP on the sensory quality of grilled burgers. For the sensory analysis, an independent assay was conducted (detailed in Section 2.1.2). The cooking conditions were optimized in advance, and the internal temperature of each burger was controlled (72 °C). Each burger was grilled on a contact grill (Lacor, Bergara, Gipuzkoa, Spain, 43 × 35 × 19 cm; 1400 W) at maximum temperature (220 °C) for two minutes. After cooking, each burger was cut into four pieces and wrapped in aluminum foil. A random identification number was assigned to each batch. Mineral water and bread were provided to each panelist. Each panelist tasted one piece from each batch on each day of analysis. A trained panel of eight individuals aged between 20 and 60 years participated in the sensory analysis. All panelists were expert tasters from the center, routinely involved in sensory evaluation of meat and meat products. The analyses were performed in individual booths under white light. Sensory sessions were conducted in a controlled environment at approximately 22 °C, with relative humidity around 50–60% and adequate ventilation, following standard conditions for expert sensory panels. Each session included a balanced and randomized subset of samples. A total of seven sessions were carried out on separate days, during which samples from four randomly selected batches were evaluated in each session. The following parameters were evaluated using an unstructured scale from 0 to 10 points (from “I dislike it very much” to “I like it very much”): general appearance, odor, taste, texture, and overall acceptability. Additionally, the presence or absence of undesirable flavors was assessed (absence/presence). Only burgers from day 1 were evaluated in the sensory analysis to ensure panelist safety.

2.3. Statistical Analysis

Three samples (n = 3) of Flour 1 and Flour 2 were analyzed for the initial characterization of the lupine flour used in the preparation of lamb burgers. To evaluate differences between the two types of flour, a Student’s t-test was applied (p-value).

In the burger study, five burgers (n = 5) per treatment were analyzed. A one-way ANOVA was performed to evaluate differences in the composition among the three formulations (p-formulation), followed by Tukey’s HSD test when significant differences were found (Table 2).

To evaluate the effects of treatment (NO HHP vs. HHP1 vs. HHP2) and formulation (Lamb vs. Flour 1 vs. Flour 2) and their interaction at day 1, a two-way ANOVA was applied (Table 3). Similarly, after 14 days of refrigerated storage, a two-way ANOVA was conducted to assess the effects of treatment (HHP1 vs. HHP2), formulation (Lamb vs. Flour 1 vs. Flour 2), and their interaction (Table 3). When significant differences were detected, Tukey’s HSD test was performed for multiple mean comparisons.

For the study of the effect of HHP and storage (Tables 4–6) on burgers with lupinus flour, a one-way ANOVA was performed twice: (i) to analyze the effect of formulation within the different HHP treatments (p-formulation) followed by Tukey’s HSD test when significant differences were found; (ii) another ANOVA to analyze changes after storage (day 1 vs. day 14) (p-storage) in burgers with the same formulation and with the same HHP conditions.

Statistical analyses were performed using SPSS software, v21.0 (SPSS Inc., Chicago, IL, USA). Data were analyzed at three levels of significance (ns: p ≥ 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). Results are expressed as mean ± standard deviation.

3. Results and Discussion

3.1. Characterization of Lupine and Obtained by Compression Milling Lupine Flours

The proximate composition (g 100 g⁻¹) and the bioactivity of the two lupine flours used in the formulation of lamb burgers, designated as Flour 1 and Flour 2, are shown in

Table 1. Initial characterization of the lupine flour used in the preparation of lamb burgers.

	Flour 1 (Mean ± SD)	Flour 2 (Mean ± SD)	p-Value
Proximate composition
pH	5.6 ± 0.1	5.7 ± 0.0	ns
Moisture (%)	7.8 ± 0.1	5.9 ± 0.2	***
Protein (%)	42.7 ± 0.7	42.5 ± 0.8	ns
Fiber (%)	39.0 ± 0.7	31.9 ± 0.7	***
Fat (%)	5.7 ± 0.2	7.1 ± 0.4	***
Fatty acids profile (%)
∑SFA	13.1 ± 0.1	13.2 ± 0.1	ns
∑MUFA	58.8 ± 0.1	58.4 ± 0.0	**
∑PUFA	27.3 ± 0.0	27.6 ± 0.0	**
Bioactivity
Phenolic compounds (mg 100 g−1)	36.8 ± 0.7	74.0 ± 3.8	***
Antioxidant activity (mM Trolox g−1)	517.9 ± 17.1	539.3 ± 22.6	ns

Student’s t-Test. ns: non-significant differences; ** Significance at p ≤ 0.01; *** Significance at p ≤ 0.001.

. The pH values of the lupine flours showed no statistically significant differences between Flour 1 (5.6 ± 0.1) and Flour 2 (5.7 ± 0.0). Moisture content was significantly lower in Flour 2 compared to Flour 1. Both flours exhibited high protein levels and no significant differences were observed between them. This is consistent with lupine’s well-documented high protein content, which ranges between 30% and 42%, with variations attributed to the specific lupin variety [36]. Moreover, lupin proteins offer several additional benefits, including their improved ease of handling from a technological standpoint and having a higher denaturation temperature compared to animal proteins [37]. Meanwhile, Flour 1 contained significantly more fiber than Flour 2, the inclusion of which in meat products could be beneficial for human health, improving digestive function and contributing to the regulation of lipid metabolism [38]. Fat content was significantly higher in Flour 2. In this context, although lupine is classified as a legume and not traditionally considered an oilseed crop, its seeds contain a substantial amount of oil, ranging from 5% to 20% crude oil based on the total seed weight [39] which is in line with our results. Regarding fatty acid composition monounsaturated fatty acids (MUFAs) showed the highest percentage (58.4–58.8%; p < 0.01), with oleic acid (C18:1) being the predominant fatty acid (Table S1) (p < 0.001) followed by polyunsaturated fatty acids (PUFA) (27.3–27.6%; p < 0.01) and by saturated fatty acids (SFA) (13.1–13.2%). The predominant fatty acids amongst the SFA were palmitic acids (C16:0), whereas the most abundant PUFA was linoleic acid (C18:2 n-6). Although these differences are subtle, the higher PUFA content in Flour 2 could impart greater nutritional value due to the well-known benefits of PUFA on cardiovascular health [40]. However, PUFAs are structurally more susceptible to oxidation, and their higher oxidation rate negatively impacts meat quality by promoting sensory deterioration and the formation of harmful compounds [41]. No significant differences were observed in total SFA. One of the most notable differences was observed in the concentration of phenolic compounds, which was more than doubled in Flour 2 (74.0 mg 100 g⁻¹) compared to the Flour 1 (36.8 mg 100 g⁻¹). However, despite this substantial increase, the antioxidant activity did not significantly differ between flours, suggesting that either the phenolic profile was qualitatively different or that the matrix effects limited the expression of antioxidant potential. Previous studies have highlighted that not all phenolics contribute equally to antioxidant performance, and their effectiveness may depend on structure, solubility, and interactions with other components [42]. In line with our results, previous studies by Lampart-Szczapa et al. [43] evaluated different lupin seed products and found no correlation between antioxidant properties and phenolic compounds content. Other antioxidants like alkaloids, tocopherols, or bioactive peptides might be involved in this bioactivity. Several studies have demonstrated that the degree of milling is positively correlated with nutrient bioaccessibility [44,45]. In general, the effect of milling depends on the parameters of the process, but, generally, increasing temperatures or pressures increase the bioavailability of bioactive compounds, including phenolic compounds [46]. This more intense process could promote extensive cell rupture during the operation, which enhances nutritional bioaccessibility. Changes in the specific phenolic profile of lupine flour during the flour production process were not evaluated in the present study, which limits the interpretation of the observed results.

3.2. Proximate Composition, Fatty Acids Profile, pH and Water Activity of Lamb Burgers with Lupine Flour

Table 2. Proximate composition (%), fatty acids profile (%), pH and a_w of lamb burgers with different lupine flours.

	Lamb	Flour 1 (Burger)	Flour 2 (Burger)	p-Formulation
pH	5.8 a ± 0.0	5.8 a ± 0.0	5.7 b ± 0.0	*
aw	0.869 ± 0.001	0.868 ± 0.002	0.868 ± 0.002	ns
Moisture (%)	65.5 ± 1.3	65.2 ± 0.6	65.5 ± 0.7	ns
Protein (%)	20.7 ± 0.5	20.7 ± 0.3	20.3 ± 0.5	ns
Fat (%)	11.6 ± 1.4	11.1 ± 1.1	11.2 ± 1.8	ns
Fatty acids profile (%)
∑SFA	43.2 ± 0.5	42.9 ± 0.2	42.9 ± 0.7	ns
∑MUFA	52.3 ± 0.4	52.5 ± 0.2	52.4 ± 0.5	ns
∑PUFA	4.5 ± 0.1	4.6 ± 0.2	4.6 ± 0.2	ns

Different letters in the same row indicate differences due to formulation by the Tukey test. ns: non-significant differences; * Significance at p ≤ 0.05.

presents the proximate composition, fatty acid profile, pH, and water activity (a_w) of lamb burgers formulated with different lupine flours. The pH values showed a slight but statistically significant decrease (p ≤ 0.001) in the burgers with Flour 2 (5.7 ± 0.0) compared to the lamb control and Flour 1 formulation (5.8 ± 0.0). Both flours presented similar pH values (5.6 ± 0.1) so they may not affect the pH of burgers differently; differences could be attributed to the high repeatability of the measurements. Water activity remained high (0.868–0.869 ± 0.001–0.002) across all treatments, which is characteristic of fresh meat products and indicates a high susceptibility to microbial growth unless properly stored [47]. No significant differences were observed in moisture, protein, or fat content between the formulations, which could be explained by the low levels of addition of flour (1%).

The incorporation of conventional lupine flour (Flour 1) or compression milling lupine flour (Flour 2) did not significantly affect the main fatty acid groups (SFA, MUFA, PUFA) compared to the control formulation. Despite the high content of MUFA and PUFA in the added lupine flour, the levels of these fatty acids in the burgers remained unchanged. SFA remained around 43%, MUFA around 52–53%, and PUFA around 4–5% in all treatments, with no significant differences observed between formulations. The lack of changes in the fatty acid profile of burgers could be attributed to the low inclusion level (1%) and the relatively low-fat content of the flours (6–7%). The fatty acid composition of meat plays a critical role in determining key quality traits, including nutritional value, flavor, and textural properties. PUFAs are essential for human health, playing crucial roles in reducing cardiovascular risk, modulating inflammation, and supporting brain function. On the other hand, a higher content of polyunsaturated fatty acids also increases its susceptibility to oxidation, leading to rancidity and off-flavors [48]. Therefore, balancing the PUFA content is crucial to maximize health benefits while maintaining meat quality. The stability of proximate composition observed in our study aligns with findings in beef burgers added at higher level, where 5–15% lupine flour did not adversely affect technological parameters or macronutrient balance [49].

3.3. Color Changes in Lamb Burgers with Lupine Flour Treated by High Hydrostatic Pressure

Table 3. Two-way ANOVA and interactions between treatment and formulation at day 1 and after 14 days of storage.

Initial 1		Treatment	Formulation	Treatment * Formulation
Color	CIE L*	***	ns	ns
	CIE a*	***	ns	ns
	CIE b*	***	**	*
	Chroma	***	*	*
	Hue	***	ns	ns
Oxidation	Lipid oxidation	***	***	ns
	Protein oxidation	*	ns	ns
Microbiology	Mesophilic	***	ns	ns
	Psychrophilic	***	ns	ns
	Molds and yeasts	***	ns	ns
	S. aureus	**	ns	ns
	C. perfringens	ns	ns	ns
	Total coliforms	***	ns	ns
	E. coli	***	ns	ns
Sensory analysis	General appearance	ns	ns	ns
	Odor intensity	ns	ns	ns
	Taste intensity	ns	ns	ns
	Texture	ns	ns	ns
	Unpleasant taste	ns	ns	ns
	General evaluation	ns	ns	ns
14 days 2		HHP1 vs. HHP2	Formulation	Treatment * Formulation
Color	CIE L*	***	ns	*
	CIE a*	***	**	*
	CIE b*	***	**	**
	Chroma	***	*	***
	Hue	***	**	ns
Oxidation	Lipid oxidation	***	***	**
	Protein oxidation	*	ns	***
Microbiology	Mesophilic	ns	*	ns
	Psychrophilic	ns	ns	ns
	Molds and yeasts	ns	ns	**
	S. aureus	ns	ns	*
	C. perfringens	ns	ns	ns
	Total coliforms	ns	ns	ns
	E. coli	ns	ns	ns

ns non-significant differences; * significance at p ≤ 0.05; ** significance at p ≤ 0.01; *** significance at p ≤ 0.001. ¹ Two-way ANOVA and interactions of the treatment (NO HHP vs. HHP1 vs. HHP2) and formulation (Lamb vs. Flour 1 vs. Flour 2) at day 1. ² Two-way ANOVA and interactions of the treatment (HHP1 vs. HHP2) and formulation (Lamb vs. Flour 1 vs. Flour 2) after 14 days of storage.

shows the two-way ANOVA results evaluating the effects of treatment and formulation, as well as their interaction, on instrumental color parameters (CIE L*, a*, b*, Chroma, and Hue) of lamb burgers at day 1. HHP significantly influenced all color parameters of lamb burgers at day 1 (p ≤ 0.001), confirming its strong impact on visual appearance of fresh burgers. Formulation effects (lupine flour addition) were significant only for CIE b* and Chroma, while a significant treatment × formulation interaction was also detected for these same parameters.

Instrumental colors of lamb burgers formulated with different lupine flours, evaluated after HHP treatment and during refrigerated storage, are shown in

Table 4. Instrumental color of lamb burgers with different lupine flours after HHP and during the refrigerated storage (day 1 and day 14).

Storage (Days)	NO HHP				HHP1				HHP2
	Lamb	Flour 1	Flour 2	p-Formulation	Lamb	Flour 1	Flour 2	p-Formulation	Lamb	Flour 1	Flour 2	p-Formulation
L*
1	46.7 ± 0.7	47.3 ± 0.6	46.9 ± 1.4	ns	55.4 ± 0.8	55.4 ± 0.5	55.1 ± 0.7	ns	58.1 ± 0.2	58.0 ± 0.2	58.0 ± 0.6	ns
14	-	-	-	-	55.9 ± 0.5	55.4 ± 0.4	55.5 ± 0.5	ns	57.0 b ± 1.0	58.2 a ± 0.6	57.6 ab ± 0.2	*
p-storage					ns	ns	ns		*	ns	ns
a*
1	8.3 ± 0.5	7.3 ± 1.0	7.4 ± 0.6	ns	3.2 ± 0.2	3.2 ± 0.2	3.2 ± 0.1	ns	2.7 ± 0.3	2.6 ± 0.2	2.6 ± 0.1	ns
14	-	-	-	-	3.1 b ± 0.4	4.0 a ± 0.7	3.1 b ± 0.3	*	2.3 ± 0.3	2.6 ± 0.3	2.7 ± 0.3	ns
p-storage					ns	*	ns		*	ns	ns
b*
1	13.2 ± 0.4	12.7 ± 0.4	13.1 ± 0.5	ns	11.9 b ± 0.5	12.3 ab ± 0.2	12.7 a ± 0.6	*	11.8 ± 0.5	11.9 ± 0.4	12.3 ± 0.1	ns
14	-	-	-	-	13.1 ± 0.2	13.0 ± 0.3	13.1 a ± 0.4	ns	12.7 a ± 0.2	11.8 b ± 0.1	12.7 a ± 0.2	***
p-storage					***	**	ns		**	ns	*
Chroma *
1	15.6 ± 0.5	14.7 ± 0.7	15.1 ± 0.7	ns	12.3 b ± 0.5	12.7 ab ± 0.2	13.1 a ± 0.5	*	12.1 ± 0.5	12.2 ± 0.4	12.6 ± 0.1	ns
14	-	-	-	-	13.5 ± 0.2	13.7 ± 0.4	13.4 ± 0.4	ns	12.9 a ± 0.2	12.1 b ± 0.2	12.9 a ± 0.3	***
p-storage					***	***	ns		**	ns	*
Hue *
1	57.9 ± 1.6	60.2 ± 3.0	60.4 ± 1.2	ns	75.0 ± 1.2	75.5 ± 0.8	75.9 ± 0.9	ns	77.2 ± 1.0	77.8 ± 1.1	78.2 ± 0.4	ns
14	-	-	-	-	76.7 a ± 1.5	73.1 b ± 2.8	76.5 a ± 1.3	*	79.9 a ± 1.2	77.7 bA ± 1.4	78.1 ab ± 1.0	*
p-storage					ns	ns	ns		**	ns	ns

T0: initial T1: 14 days. Flour 1: lamb burgers with 1% (w/w) of conventional lupine flour. Flour 2: lamb burgers with 1% (w/w) of lupine obtained by compression milling. HHP1: 600 MPa 1 s (2 cycles). HHP2: 600 MPa 4 min. Means values followed by different letters indicate the existence of significant differences between formulation by the Tukey test (p ≤ 0.05) Student’s t-test. ns: non-significant differences; * Significance at p ≤ 0.05; ** significance at p ≤ 0.01; *** significance at p ≤ 0.001.

. On day 1, within each group, no significant differences in lightness CIE L* were observed between formulations, suggesting that neither Flour 1 nor Flour 2 influenced the initial lightness. The lowest L* values were observed in the non-high-pressure-treated group, with values ranging from 46.7 in lamb burgers to 47.3 in Flour 1 formulation. Application of HHP markedly increased lightness in both treatments (HHP1 and HHP2). Other authors have reported an increase in L* values in beef M. biceps femoris samples after HHP treatment (350–600 MPa) [50]. According to Cheftel & Culioli, [51], this “whitening/brightening” effect induced by HHP processing could be attributed to globin denaturation, displacement or release heme iron, and oxidation of the ferrous atom. Regarding CIE a* parameter values were not affected by the flour inclusion. Values were highest in NO HHP samples, showing lower redness in HHP samples. HHP2 (1 cycle 600 MPa/4 min) showed lower values than HHP1 (2 cycles 600 MPa/1 s). The changes in redness of meat after HHP is mainly due to myoglobin modification.

Meat color is largely determined by both the concentration of myoglobin and its chemical form. Upon exposure to air, freshly cut meat undergoes oxygen binding to myoglobin, producing oxymyoglobin, which imparts the bright red appearance commonly linked to freshness. The reduction in red color intensity after HHP is primarily attributed to changes in protein configuration and protein denaturation triggered by HHP, a phenomenon extensively documented in meat products. These effects are mainly due to the conversion of oxymyoglobin to metmyoglobin and the denaturation of globin chains, both of which lead to a paler or brownish appearance [52].

For yellowness (CIE b*), non-HHP samples showed similar values between formulations at day 1. However, in HHP1, Flour 2 presented significantly higher b* values than the Lamb and Flour 1, while in HHP2 no significant differences were found initially. This would explain the significant interaction between treatment and formulation (Table 3). Chroma followed a similar pattern, with HHP1 Flour 2 showing higher values compared with the other formulations, whereas no significant differences were observed in HHP2 at day 1. Hue values were not affected by formulation, although they were higher in HHP-treated samples than in NO HHP samples. Overall, HHP application induced changes in all color parameters, while formulation effects were limited to b* and Hue.

Two-way ANOVA of color parameters after 14 days of storage comparing treatments (HHP1 vs. HHP2) and formulations (Lamb vs. Flour 1 vs. Flour 2) are shown in Table 3. After 14 days of storage, HHP application of two short cycles or one long treatment (HHP1 vs. HHP2) significantly affected all color parameters (p ≤ 0.001). Formulation effects (flour addition) were significant for CIE a*, b*, Chroma, and Hue, indicating that the addition of flour influenced redness, yellowness, saturation, and hue values. Significant treatment × formulation interactions were observed for CIE L*, a*, b*, and Chroma. Individual changes were detailed at day 14 (Table 4); lightness showed no significant differences among formulations in HHP1, with values ranging from 55.4 to 55.9, whereas in HHP2, Flour 1 exhibited significantly higher L* (58.2) compared with Lamb (57.0) and Flour 2 (57.6) (p ≤ 0.5). Redness (a*) differed among formulations only in HHP1, with Flour 1 showing higher values (4.0) than Lamb and Flour 2 (3.1), while no significant differences were observed in HHP2 formulations. Yellowness showed no formulation effect in HHP1, while in HHP2 Flour 1 exhibited significantly lower values than Lamb and Flour 2. A similar trend was observed for Chroma, where HHP1 did not differ across formulations; however, in HHP2, Flour 1 was significantly lower than the other two formulations. Regarding Hue, significant formulation effects were found in both treatments: in HHP1, Flour 1 had lower values than Lamb and Flour 2, while in HHP2, Flour 1 was also lower compared with Lamb and Flour 2.

Storage time (changes day 1 vs. day 14, Table 4) had no significant effect on lightness values, except in lamb burgers treated with HHP2. In HHP1 burgers, a* values significantly increased after 14 days in samples containing Flour 1, whereas in HHP2 a significant decrease was observed in the control samples. Yellowness (b*) values increased in burgers with Flour 1 and HH1 and decreased in lamb with HHP2. Chroma followed a similar trend, showing a significant increase in the control and Flour 1 samples under HHP1, and in the control and Flour 2 samples under HHP2, suggesting a reduction in color intensity. The Hue angle significantly increased in the control samples under HHP2.

The decline in redness (CIE a*) during refrigerated storage is mainly attributed to the oxidation of oxymyoglobin or deoxymyoglobin into brownish metmyoglobin, as well as myoglobin denaturation [52]. Lightness also typically increases over storage. These shifts can influence purchase decisions; however, color changes in HHP-treated burgers are generally less pronounced [12]. The visual appearance of food plays a fundamental role in consumers’ perception of quality and strongly influences purchasing decisions. In addition, the absence of significant effects from flour addition may also be beneficial for its inclusion in lamb burger formulations.

However, HHP treatment induced noticeable changes in burger appearance. The color alterations observed after HHP are consistent with previous studies on beef and poultry burgers treated at 400–600 MPa, where myoglobin denaturation and conversion to metmyoglobin resulted in whitening and discoloration [50,52]. Nonetheless, other studies have reported that these HHP-induced visual changes generally disappear after cooking [53].

Regarding differences between HHP1 vs. HHP2, they were evidenced in the two way-ANOVA at day 14 (Table 3) but they could also be appreciated at day 1 (Table 4). In general, lightness was lower in HHP1 than in HHP2, indicating that whitening induced effect was more marked in HHP2. CIE a* values in HHP2 were generally lower than in HHP1 (2 cycles), indicating a decrease in redness at the longer one cycle of HHP. Similarly, the decrease in yellowness after HHP was less intense in HHP1 (two cycles) than in HHP2 (long cycle). Therefore, the discoloration often induced in meat products after HHP treatment was less intense when the two short cycles were applied. In this regard, Timon et al. [12] reported greater color changes at higher pressure levels (600 vs. 400 MPa) and found that two cycles caused more color alteration than a single cycle under the same conditions in chicken burgers. The current results indicate that the application of short HHP cycles opens the door to mitigate the noticeable color changes induced after HHP.

3.4. Oxidative Status of Lamb Burgers with Lupine Flour and Treated by High Hydrostatic Pressure

At day 1, HHP treatment had a significant effect on lipid and protein oxidation of burgers (Table 3). Formulation also significantly influenced lipid oxidation, whereas no effect was observed on protein oxidation. No significant treatment × formulation interactions were detected for lipid and protein oxidation.

The initial lipid oxidation levels ranged from 0.1 to 0.3 mg MDA kg⁻¹ across the three burger formulations (with 2 lupines flour) and HHP treatments (

Table 5. Lipid (mg MDA kg⁻¹) and protein oxidation (nmols carbonyls mg protein⁻¹) lamb burgers with different lupine flours after HHP and during the refrigerated storage (day 1 and day 14).

Storage (Days)	NO HHP				HHP1				HHP2
	Lamb	Flour 1	Flour 2	p-Formulation	Lamb	Flour 1	Flour 2	p-Formulation	Lamb	Flour 1	Flour 2	p-Formulation
Lipid oxidation 1
1	0.2 b ± 0.0	0.3 a ± 0.0	0.1 b ± 0.0	***	0.2 a ± 0.0	0.3 a ± 0.0	0.1 b ± 0.0	***	0.3 ± 0.1	0.3 ± 0.0	0.3 ± 0.0	ns
14	-	-	-		0.5 a ± 0.1	0.4 a ± 0.0	0.1 b ± 0.0	***	0.9 a ± 0.1	0.6 b ± 0.1	0.3 c ± 0.1	***
p-storage					**	***	ns		***	***	ns
Protein oxidation 2
1	3.1 ± 0.6	3.0 ± 0.2	2.5 ± 0.3	ns	3.8 ± 1.1	3.8 ± 0.3	4.1 ± 1.8	ns	3.6 ± 0.4	3.5 ± 0.3	3.2 ± 0.3	ns
14	-	-	-		3.7 b ± 0.9	4.2 b ± 0.1	5.9 a ± 0.9	**	5.6 a ± 0.7	5.2 ab ± 0.5	4.6 b ± 0.5	*
p-storage					ns	**	ns		***	***	***

T0: initial T1: 14 days. Flour 1: lamb burgers with 1% (w/w) of conventional lupine flour. Flour 2: lamb burgers with 1% (w/w) of lupine obtained by compression milling HHP1: 600 MPa 1 s (2 cycles). HHP2: 600 MPa 4 min. Means values followed by different letters indicate the existence of significant differences between formulation by the Tukey test (p ≤ 0.05) ns: non-significant differences; * Significance at p ≤ 0.05; ** significance at p ≤ 0.01; *** significance at p ≤ 0.001. ¹ mg MDA kg^{−1 2} nmol carbonyls mg protein⁻¹.

). At day 1, lipid oxidation levels were generally low across all samples, as expected with freshly prepared meat systems. However, statistically significant differences were observed between formulations within the same treatment group for both NO HHP and HHP1. In both, burgers with Flour 2 showed the lowest oxidation values (p < 0.001). In the NO HHP group, burgers with Flour 1 showed the highest oxidation levels; this trend was not found the other HHP burgers. The MDA values of samples subjected to HHP2 treatment were the highest, around 0.3 mg, with no significant differences observed among formulations. These findings are consistent with previous studies, indicating that HHP reduces the oxidative stability of fresh meat [54]. In agreement with the present results, Ma et al. [54] observed increased TBARS values in M. longissimus dorsi beef samples subjected to pressures ≥400 MPa at 20 °C and 40 °C. On the other hand, protein oxidation did not show significant differences among formulations at day 1 with carbonyl values ranging from 2.5 to 4.1 nmol mg protein⁻¹. Values of HHP-treated burgers were higher than non-treated, in line with lipid oxidation.

At day 14, according to the two-way ANOVA (Table 3) both HHP treatment (HHP 1 vs. HHP2) and formulation had significant effects on lipid oxidation, while protein oxidation was influenced only by HHP treatment. A significant treatment × formulation interaction was observed for lipid and protein oxidation. Under HHP1, burgers formulated with Flour 2 exhibited the lowest MDA value (0.1 mg kg⁻¹), significantly lower than both the control (0.5 mg kg⁻¹) and the Flour 1 (0.4 mg kg⁻¹) (p ≤ 0.001) (Table 5). Under HHP2, a similar trend was observed; burgers formulated with Flour 2 exhibited the lowest MDA value (0.3 mg kg⁻¹), significantly lower than both the Flour 1 (0.6 mg kg⁻¹) and the highest value was found in the control (0.9 mg kg⁻¹) (p ≤ 0.001). On the other hand, MDA values were higher in HHP2 than in HHP1 confirming that a longer pressurization time intensified lipid oxidation. Nevertheless, burgers containing Flour 1 (at HHP2) Flour 2 (at HHP1 and HHP2) still showed the greatest oxidative stability (0.3 mg kg⁻¹; p ≤ 0.001).

At day 14, the highest levels of protein oxidation were detected in the samples containing Flour 2 treated with HHP1 (5.9 nmols mg protein⁻¹; p < 0.01), contrarily to lipid oxidation results. In contrast, same flour samples subjected to HHP2 exhibited the lowest carbonyl contents in line with lipid oxidation. This differential effect at HHP1 and HHP2 is in agreement with the interaction between HHP and formulation (Table 3).

During the refrigerated storage (changes day 1 vs. day 14, Table 5), TBA-RS significantly increased in all burgers except in those with Flour 2. Protein oxidation significantly increased in all HHP-treated burgers except in Lamb and Flour 2 burgers at HHP1.

As far as we are concerned, the application of lupine flour to meat products is underexplored. The available literature mainly focuses on the characterization of the nutritional composition and beneficial properties of lupine seeds [55,56]. The lower oxidation observed in burgers containing lupin flour, compared to control samples, might be attributed to the presence of natural antioxidants such as phenolic compounds, alkaloids and tocopherols found in lupin seeds. In our study, the compression milling process of Flour 2 may have further enhanced the bioavailability of these antioxidants, resulting in a slight protective effect. Several studies have reported that pressure levels ranging from 300 to 600 MPa are critical thresholds for initiating lipid oxidation in fresh lamb pork, beef, and poultry [18,52]. This oxidation process can result in rancidity, the development of off-flavors, and a reduction in shelf life; however, the inclusion of lupine flour may delay this process due to the incorporation of antioxidant compounds into the meat matrix, which may be effective in mitigating oxidation. Similarly, D’Arrigo et al., [13], demonstrated that the addition of broccoli and cauliflower by-product ingredients recognized for their high polyphenolic content enhanced the oxidative stability of samples subjected to HHP.

Regarding the effect of HHP on protein oxidation, some authors [57] reported that treatments at 600 MPa for 6 min in pork altered the degradation pattern of myofibrillar proteins due to increased cathepsin activity, which could lead to enhanced protein oxidation; however, other studies [58] have indicated that legume-derived antioxidants like phenolic compounds can mitigate protein oxidation during storage or processing, which could partially explain the behavior observed in our study. Regarding the differential and unexpected effect of Flour 2 addition in HHP1 and HHP2, it should be added that protein oxidation has been less studied and remains less understood, mainly due to its complex chemistry, limited analytical methods, and the traditional focus on lipid oxidation and microbial spoilage as the primary causes of quality loss [58].

During refrigerated storage, lipid oxidation increased significantly, confirming that storage promoted the formation of secondary oxidation products. Protein oxidation also rose significantly during storage reflecting ongoing oxidative damage to proteins. However, burgers formulated with Flour 1 and especially with Flour 2 showed an enhanced lipid oxidative stability. This might be attributed to the antioxidants from lupines flour which could persist in the HHP-treated burgers during the storage. The effects of HHP (HHP1 vs. HHP2) and the type of lupine flour (Flour 1 vs. Flour 2) showed significant interactions for lipid and protein oxidation at day 14 (Table 3) which reflect the complexity of the reactions evaluated. The interactions between HHP and lupine flour were also significant for all color parameters. These results indicate that the effects of HHP (two cycles vs. one long cycle) on lamb burger quality are modulated by the presence and type of lupine flour, highlighting a clear interaction between processing intensity and ingredient functionality.

The reduced lipid oxidation in burgers containing compression-milled lupine flour (Flour 2) after HHP treatment is likely due to the higher availability of phenolic compounds acting as radical scavengers, whose effectiveness may be enhanced by pressure-induced modifications of meat proteins and microstructure. A similar effect would be applied to color changes at day 14 which were modulated by type of flour and the processing conditions applied.

3.5. Microbial Counts in Lamb Burgers with Lupine Flour Treated by High Hydrostatic Pressure

The results of the two-way ANOVA assessing the effects of treatment, formulation, and their interaction on the different microorganisms at day 1 (Table 3) showed that HHP treatment exerted a highly significant effect (p ≤ 0.001) on mesophilic, psychrophilic, molds and yeasts, total coliforms, and E. coli, while S. aureus was significantly affected at p ≤ 0.01. In contrast, no significant differences were observed for C. perfringens. Neither formulation nor the treatment × formulation interaction had a significant impact on any of the evaluated microorganisms. These findings indicate that HHP treatment was the main determinant of microbial reduction at day 1, regardless of formulation.

Table 6. Microbiological counts (log colony forming units; CFU g⁻¹) of lamb burgers with different lupine flours after HHP and during the refrigerated storage (day 1 and day 14).

Storage (Days)	NO HHP				HHP1				HHP2
	Lamb	Flour 1	Flour 2	p-Formulation	Lamb	Flour 1	Flour 2	p-Formulation	Lamb	Flour 1	Flour 2	p-Formulation
Mesophilic
1	7.4 ± 0.1	7.6 ± 0.3	7.5 ± 0.2	ns	4.1 ± 0.3	4.3 ± 0.3	4.1 ± 0.5	ns	3.7 ± 0.3	4.5 ± 1.4	3.9 ± 0.3	ns
14	-	-	-		4.7 ± 0.8	4.6 ± 0.9	5.0 ± 0.9	ns	4.7 b ± 0.5	4.8 b ± 0.6	6.6 a ± 1.6	*
p-storage	-	-	-		ns	ns	ns		**	ns	**
Psychrophilic
1	7.6 ± 0.1	7.7 ± 0.3	7.8 ± 0.2	ns	1.6 a ± 0.4	1.5 ab ± 0.4	1.0 b ± 0.1	*	1.1 ± 0.3	1.1 ± 0.3	1.0 ± 0.2	ns
14	-	-	-		3.5 ± 1.3	4.1 ± 1.8	4.6 ± 0.9	ns	4.5 ± 0.6	4.5 ± 1.2	2.6 ± 2.4	ns
p-storage	-	-	-		*	*	***		***	***	ns
Molds and yeast
0	3.2 ± 0.4	3.3 ± 0.1	3.1 ± 0.5	ns	1.1 ± 0.4	0.9 ± 0.1	1.0 ± 0.1	ns	0.9 ± 0.1	1.1 ± 0.3	1.0 ± 0.2	ns
14	-	-	-		2.0 a ± 0.3	1.4 b ± 0.2	1.0 b ± 0.3	***	1.3 ± 0.4	1.5 ± 0.4	1.4 ± 0.5	ns
p-storage	-	-	-		**	***	ns		ns	ns	ns
S. aureus
1	2.6 ± 0.5	2.2 ± 0.3	2.2 ± 0.2	ns	<2	<2	<2	ns	<2	2.0 ± 0.1	2.0 ± 0.1	ns
14	-	-	-		2.0 ± 0.1	2.0 ± 0.1	2.1 ± 0.2	ns	<2	2.5 ± 0.6	2.0 ± 0.1	ns
p-storage	-	-	-		ns	ns	ns		ns	ns	ns
C. perfringens
1	<1	<1	<1	ns	<1	<1	<1	ns	<1	<1	<1	ns
14	-	-	-		<1	<1	<1	ns	<1	<1	<1	ns
p-storage	-	-	-		ns	ns	ns		ns	ns	ns
Total coliforms
1	2.8 ± 0.4	3.2 ± 0.1	2.9 ± 0.3	ns	<1	<1	<1	ns	<1	<1	<1	ns
14	-	-	-		<1	<1	<1	ns	<1	<1	<1	ns
p-storage	-	-	-		ns	ns	ns		ns	ns	ns
E. coli
1	1.4 ± 0.3	1.2 ± 0.4	1.3 ± 0.2	ns	<1	<1	<1	ns	<1	<1	<1	ns
14	-	-	-		<1	<1	<1	ns	<1	<1	<1	ns
p-storage	-	-	-		ns	ns	ns		ns	ns	ns

T0: initial T1: 14 days. Flour 1: lamb burgers with 1% (w/w) of conventional lupine flour. Flour 2: lamb burgers with 1% (w/w) of lupine obtained by compression milling. HHP1: 600 MPa 1 s (2 cycles). HHP2: 600 MPa 4 min. Means values followed by different letters indicate the existence of significant differences between formulation by the Tukey test (p ≤ 0.05) ns: non-significant differences, * significance at p ≤ 0.05; ** significance at p ≤ 0.01; *** significance at p ≤ 0.001.

displays the microbial counts in lamb burgers for each treatment and formulation (CFU g⁻¹) at 1 and 14 days of storage. At day 1, mesophilic counts were high in all formulations not subjected to HHP treatment, with values above 7 log CFU g⁻¹ regardless of the flour type used (no flour, Flour 1 or Flour 2), indicating an initially elevated microbial load, likely attributable to pre-processing handling and to the intrinsic characteristics of lamb meat. In contrast, burgers subjected to HHP1 and HHP2 exhibited a drastic reduction in mesophilic counts at day 1, dropping to values between 3.7 and 4.5 log CFU g⁻¹. These reductions reflect the well-documented bactericidal effect of HHP, attributed to cell membrane disruption and protein denaturalization [59].

For psychrophilic counts a similar pattern was observed. NO HHP samples exhibited the highest counts (7.6–7.8 log CFU g⁻¹), whereas both HHP1 and HHP2 treatments significantly reduced the counts to approximately 1 log CFU g⁻¹. Psychrophilic microorganisms are well-adapted to grow at low temperatures, which is often associated with a higher proportion of unsaturated and branched-chain fatty acids in their membranes, which help maintain membrane fluidity and low-temperature functionality [60]. Some authors have pointed out that the cell wall and membranes of microorganisms, as well as membrane fluidity, may be factors influencing an organism’s pressure sensitivity [61], which could explain the higher susceptibility of these microorganisms to pressure; however, further research is still needed in this regard.

Mold and yeast counts were also significantly reduced by HHP, with reduction of around 2.2 log CFU g⁻¹. Staphylococcus aureus counts in untreated burgers ranged between 2.2 and 2.6 log CFU g⁻¹ across formulations, whereas after HHP treatment, the counts fell below the detection limit of the method (<2 log CFU g⁻¹). Clostridium perfringens remained below detection limits (<1 log CFU g⁻¹) in all samples. At day 1, NO HHP samples presented total coliforms levels of 2.8–3.2 log CFU g⁻¹ and E. coli of approximately 1.2–1.4 log CFU g⁻¹. Total coliforms and E. coli were both significantly reduced by HHP. In contrast, both HHP1 and HHP2 treatments reduced these counts to below the detection limits (<1 log CFU g⁻¹). The drastic reduction in coliforms, as well as in mesophilics and psychrophilics, at 600 MPa is consistent with studies showing 2–5 log cycle reductions and complete inactivation of Gram-negative bacteria under similar conditions [59,62]

At 14 days of storage, the two-way ANOVA (Table 3) showed that there were no significant main effects of treatment (HHP1 vs. HHP2) on mesophiles, psychrophiles, molds and yeasts, S. aureus, C. perfringens, total coliforms, or E. coli. Therefore, from a microbiological point of view, the application of two short HHP cycles had a similar effect as an equivalent (on time) long one. However, formulation had a significant effect on mesophiles (p ≤ 0.05), while treatment-by-formulation interactions were significant for molds and yeasts and S. aureus.

The microbiological counts after 14 days of storage are shown in Table 6. Mesophilic counts increased in HHP-treated samples compared with day 1, reaching values of 4.6–5.0 log CFU g⁻¹ in HHP1 and up to 6.6 log CFU g⁻¹ in one HHP2 formulation. This increase could be hypothesized to be related to microbial recovery or repair of sublethal injury during storage. As previously suggested by other authors, when microorganisms are exposed to chemical or physical processes that do not cause complete inactivation, they may enter a sublethally injured but reversible state [63].

Psychrophilic counts followed a similar trend. At day 14, an increase in psychrophilic levels was observed in the HHP-treated groups, with values of 3.5–4.6 log CFU g⁻¹ in HHP1 samples and 2.6–4.5 log CFU g⁻¹ in HHP2. These findings indicate that although HHP is effective in reducing psychrophilic populations initially, surviving sublethally injured cells may recover and proliferate under refrigerated conditions, particularly in nutrient-rich environments such as meat emulsions. The partial recovery of psychrotrophic populations during storage has also been observed in L. monocytogenes surviving sublethal HHP treatments, confirming the potential for microbial regrowth under refrigeration [64].

At day 14, molds and yeasts showed differences between formulations in HHP1, with values ranging from 1.0 to 2.0 log CFU g⁻¹ and with lower values in burgers with flours, whereas no significant differences were observed among formulations in HHP2. Staphylococcus aureus remained at very low levels after 14 days, with counts close to 2 log CFU g⁻¹ in HHP1 and HHP2 samples and with no significant differences between formulations. These low levels suggest that HHP prevented significant growth of this pathogen during storage. Clostridium perfringens remained undetectable across most treatments indicating no relevant risk associated with this microorganism. Finally, both total coliforms and E. coli stayed below the detection limit throughout storage in all HHP-treated samples with no recovery observed at day 14. These findings align with earlier work by Silhavy et al., [62], which highlighted the sensitivity of Gram-negative bacteria to HHP. The complete inactivation and sustained absence of coliforms and E. coli suggest that HHP is particularly effective against Gram-negative facultative anaerobes in meat matrices.

Regarding the effect of storage time (changes day 1 vs. day 14), mesophilic counts remained unchanged during storage at HHP1; however, in HHP2, lamb burgers and those with Flour 2 significantly increased. After refrigerated storage, psychrotrophic counts increased in all HHP-treated samples, indicating partial recovery. Only burgers treated at HHP2 with Flour 2 showed low similar counts at day 14 as at day 1. Refrigerated storage led to a significant increase in molds and yeasts in HHP1-treated samples, except in those with Flour 2, which remained constant, indicating partial recovery, whereas HHP2 samples remained stable over 14 days. This suggests that longer HHP is more effective in controlling fungal growth during storage, and this would be the only advantage of long cycles compared to short cycles from a microbiological point of view. Refrigerated storage had no significant effect on S. aureus, C. perfringens, total coliforms, or E. coli counts in HHP-treated lamb burgers, indicating that these microorganisms remained stable over 14 days. These results highlight the efficacy of HHP in ensuring microbiological stability, with storage time having minimal impact on key pathogenic and spoilage microorganisms [65]. The incorporation of the flour did not exert a significant impact on the overall microbiological profile; in any case, it generally did not have a detrimental effect (only observed for mesophilics in HHP2 burgers with flour 2) and even contributed to improved control of molds and psychrotrophic counts under certain HHP conditions. This outcome may be partly attributed to the antimicrobial properties of phenolic compounds, although their contribution remained marginal when compared with the pronounced reductions achieved by HHP.

From a microbiological perspective, the application of two short HHP cycles (600 MPa for 1 s) produced effects comparable to those of a single, longer cycle of equivalent total duration (600 MPa for 4 min). The beneficial effect of multi-pulse application has been previously demonstrated at microbial level [17]. Previous work has shown that applying two cycles at 600 MPa/1 s can be more effective in reducing microbial counts than a single cycle (600 MPa/1 s) although the two-cycle treatment also produced more color alterations in chicken burgers, leading to a light gray–blue appearance [12]. In our study, the longer treatment at 600 MPa (HHP2) was only more effective in reducing mold and yeast counts. However, that treatment also exerted a stronger influence on instrumental color changes and oxidation parameters, potentially compromising the final quality of the product. As far as we are concerned, this is the first time that the application of two cycles has been shown to provide a clear advantage over longer single-cycle treatments (equivalent on time), not only from a microbiological point of view but also from a meat quality perspective. Therefore, the application of HHP1 appears to confer greater advantages than HHP2 since the time of utilization of the HHP equipment at the commercial level would be similar.

3.6. Sensory Analysis

The organoleptic properties of meat products are predominantly governed by the compositional ingredients used in their formulation and by the technological processes applied. No significant differences were found in sensory attributes of the grilled burgers, including general appearance, odor intensity, taste intensity, texture, unpleasant taste, and overall evaluation—due to treatment, formulation, or their interaction (Table 3).

Table 7. Sensory evaluation of lamb burgers with lupine flour and high-pressure processing.

	NO HHP				HHP1				HHP2
	Lamb	Flour 1	Flour 2	p-Formulation	Lamb	Flour 1	Flour 2	p-Formulation	Lamb	Flour 1	Flour 2	p-Formulation
General appearance	7.5 ± 0.3	7.3 ± 0.6	7.1 ± 0.7	ns	7.2 ± 0.4	7.4 ± 0.3	7.3 ± 0.4	ns	7.2 ± 0.6	7.5 ± 0.1	7.5 ± 0.3	ns
Odor intensity	7.7 ± 0.3	7.8 ± 0.2	7.6 ± 0.2	ns	7.3 ± 0.5	7.7 ± 0.2	7.3 ± 0.5	ns	7.4 ± 0.3	7.9 ± 0.5	7.6 ± 0.6	ns
Taste intensity	7.5 ± 0.1	7.0 ± 0.5	7.4 ± 0.2	ns	7.5 ± 0.4	7.5 ± 0.5	7.2 ± 0.4	ns	7.2 ± 0.6	7.2 ± 0.3	7.7 ± 0.5	ns
Texture	6.8 ± 0.5	6.3 ± 0.6	6.6 ± 0.3	ns	6.3 ± 0.2	6.4 ± 0.4	6.1 ± 0.1	ns	6.0 ± 1.0	6.7 ± 0.2	6.0 ± 0.3	ns
Unpleasant taste	0.8 ± 0.2	0.9 ± 0.8	0.3 ± 0.2	ns	0.6 ± 0.1	0.8 ± 0.5	1.1 ± 0.8	ns	1.1 ± 0.2	1.4 ± 1.1	1.0 ± 0.3	ns
General evaluation	7.2 ± 0.5	6.7 ± 0.4	7.2 ± 0.4	ns	6.8 ± 0.4	7.0 ± 0.3	6.8 ± 0.4	ns	6.7 ± 0.7	7.4 ± 0.2	7.1 ± 0.3	ns

Flour 1: lamb burgers with 1% (w/w) of lupine flour. Flour 2: lamb burgers with 1% (w/w) of lupine obtained by compression milling. HHP1: 600 MPa 1 s (2 cycles). HHP2: 600 MPa 4 min. ns—non-significant.

provides a detailed description of the sensory attributes of lamb burgers formulated with Lupinus albus flours and subjected to HHP treatment. Scores for general appearance, odor intensity, taste intensity and overall acceptability remained consistently high across all samples (6.8–7.9), and no statistically significant differences were observed. Texture scores remained within acceptable consumer levels (≥6.0), with no significant differences among formulations. Unpleasant taste was minimal across all samples, indicating that the incorporation of the sweet lupin variety, whether in normal flour or flour obtained by compression milling, did not lead to the development of off-flavors or negatively impact the overall sensory acceptance at the concentration used.

The sensory evaluation demonstrated that the inclusion of Lupinus albus flour—Flour 1 or 2—did not negatively affect the sensory attributes of lamb burgers, regardless of HHP treatment. Reviews highlight that processed lupin fractions possess a neutral flavor and are white in color, attributes that support their use as functional ingredients in meat systems [66]. The sensory evaluation supports the high acceptability of the lupin flour and increases its potential for use in food formulations or in gluten-free products. The results of the sensory analysis confirm that the level of addition of flour chosen (1% w/w) was adequate to avoid the rejection of the consumers but provided beneficial antioxidant effects. Previous research also has demonstrated that lupin flour can be incorporated into meat products without compromising sensory quality. Alrahaife & Abu-Alruz [49] revealed that the incorporation of Lupinus albus flour at levels of 5%, 10%, and 15% in beef burgers did not negatively affect key sensory parameters, such as color, flavor, juiciness, or overall acceptability. In our study, those high levels received lower scores, when initially, the limit of admission of lupine flour was evaluated to set the levels added to the burgers.

Despite the important effect of HHP on instrumental color or in lipid and protein oxidation in the fresh burgers, panelists did not perceive differences in any of the parameters evaluated in the grilled burgers (No HHP vs. HHP). This observation is consistent with previous studies reporting that the inclusion of Brassica by-products and the application of HHP did not adversely affect the sensory characteristics of lamb burgers [13]. Comparable findings have been described in pork systems, where paste addition and HHP did not significantly modify the sensory attributes of grilled burgers [67]. Overall, these results reinforce the feasibility of incorporating lupin-based ingredients into meat matrices processed by HHP without compromising consumer acceptability when the purchase decision does not depend on the fresh appearance of the product.

4. Conclusions

The present study demonstrated the suitability of incorporating both conventional flour and flour obtained by compression milling Lupinus albus into lamb burger formulations, offering a viable strategy to enhance the oxidative stability of the product without compromising nutritional, technological or sensory attributes. Although compression milling increased the phenolic content of lupine flour, no significant improvement in antioxidant activity was observed, possibly due to matrix interactions. The inclusion of lupine flour, regardless of the type of milling, did not significantly alter the proximate composition of lamb burgers at the levels of inclusion studied; however, its combination with lupine flour—especially the compression milling variant—helped delay lipid oxidation and color changes during storage, which provides oxidative and color stability to lamb burgers during the storage. Sensory evaluation confirmed that the inclusion of both types of Lupinus albus flour did not negatively affect the sensory attributes of lamb burgers.

HHP effectively controlled microorganisms’ population in lamb burgers, with minimal microbial recovery during storage, while formulation had no significant impact, highlighting HHP as the main determinant of microbial stability and safety. Although HHP significantly reduced indicator microorganisms and ensured microbiological stability during refrigerated storage, the present results should not be interpreted as a formal validation of pathogen inactivation. According to FDA guidelines, a ≥5-log reduction in relevant foodborne pathogens (e.g., Listeria monocytogenes, Salmonella spp., and STEC E. coli) is required for process validation and industrial application; therefore, further studies including challenge tests with these pathogens are necessary to confirm compliance with this regulatory performance criterion.

HHP also induced expected color changes, increasing lightness and decreasing redness. The application of two short HHP cycles produced effects comparable to those of a single longer cycle of equivalent total duration from a microbiological perspective. However, color and oxidation changes induced by pressure were less pronounced when the two short-cycle treatment were applied compared to the longer (equivalent on time) treatment. This long treatments induced more pronounced changes on instrumental color and oxidation parameters, which could potentially compromise the final product quality. Therefore, the application of two short (1 s) cycles at 600 MPa appears to be more advantageous than a single 4 min cycle at 600 MPa, not only due to their comparable microbial efficacy but also because of the reduced impact on quality parameters. To our knowledge, this is the first report demonstrating these benefits.

In summary, the incorporation of lupine flour in combination with HHP represents a promising strategy to develop healthier, microbiologically safer, and oxidatively stable meat products without compromising quality. Although HHP showed strong antimicrobial efficacy, its interaction with natural ingredients such as lupine flour warrants further investigation. Future studies should address long-term storage stability, consumer acceptance, and potential synergistic effects with other preservation methods.