The Effect of Reducing Fat and Salt on the Quality and Shelf Life of Pork Sausages Containing Brown Seaweeds (Sea Spaghetti and Irish Wakame)

Abstract

Edible brown seaweeds, sea spaghetti (SS) and Irish wakame (IW), were incorporated at 2.5% into the formulation of reduced-fat (fat reduced from 25% to 20%, 15%, and 10%) and -salt (sodium chloride—NaCl) (salt reduced from 2% to 1.5%, 1%, and 0.5%) pork sausages. The physicochemical and sensory characteristics of the reformulated sausages were analysed. Subsequently, shelf-life evaluation (lipid oxidation and microbiological analyses) was performed on selected sausages stored under aerobic (AP), MAP70/30 (70% N₂:30% CO₂), MAP80/20 (80% O₂:20% CO₂), and vacuum (VP) conditions. Relative to the control, seaweed sausages containing 10% fat had higher (p < 0.05) protein and 1.5% salt seaweed sausages had higher (p < 0.05) ash content. The addition of seaweed did not affect the pH of reduced-fat and -salt sausages, and cook loss increased in reduced-fat sausages. Reduced-fat and -salt seaweed sausages were darker in colour than the experimental controls. Based on sensory results, the most accepted sausages using SS and IW were 10% fat, 0.5% salt (SS10f/0.5s), and 15% fat, 1% salt (IW15f/1s), respectively. With regard to the shelf life of selected seaweed sausages, MAP70/30 (70% N₂, 30% CO₂) and VP (vacuum packaging) were the most effective approaches for the lipid oxidation and TVC (total viable counts), respectively.

1. Introduction

Fresh pork sausages are a comminuted type of processed meat products; typically, they are made with minced or ground pork meat, (back) fat, and a range of ingredients (water, cereal (rusk), or protein substitutes) and additives (salt (sodium chloride—NaCl), phosphates, seasoning, spices, and preservatives). These ingredients are mixed (chopped) together and filled into edible collagen casings. From independent in-store product research, fresh pork sausages on the Irish market typically contain 24–28% fat and 1.7–2.4% salt. Pork back fat, utilised in sausages, contains saturated fat at up to 40% [1].

The excess consumption of processed meats, which are often high in fat and salt (NaCl), has been consistently linked to several serious health issues. These include an increased risk of elevated blood pressure, obesity, cardiovascular disease, and certain types of cancer [2]. Consequently, the Department of Health and the Food Reformulation Taskforce in Ireland aims to reduce the intake of calories, saturated fat, sugar, and salt in the Irish diet; there is a target to reach a 10% reduction in saturated fat and salt intake by 2025 [3]. Such directives have prompted research into fat- and salt-reduction strategies for processed pork products, including pork sausages.

Fat and salt, as ingredients in processed meat formulations, play an important role with respect to product quality and shelf-life. Fat stores flavouring compounds and plays a role in product texture [4] and consumer acceptability. Salt enhances product flavour, improved by reducing water activity [5], and increases water-holding capacity through the extraction of myofibrillar proteins, resulting in enhanced cohesiveness and adhesiveness properties [6,7]. In addition to water binding, salt addition also contributes to the fat-binding properties of meat products, resulting in reduced cooking losses and enhanced textural characteristics [8].

The unique compositional, nutritional, and technological properties of seaweeds may offer the potential for their use as replacer ingredients in meat products [9,10]. A limited number of previously reported studies have examined the use of brown seaweed species such as wakame (Undaria pinnatifida) and sea spaghetti (Himanthalia elongata) as fat and/or salt replacers in beef patties and frankfurters at inclusion levels ranging from 1 to 3.3% [11,12,13].

Brown seaweeds are rich in polysaccharides, such as fucoidan, laminarin, and alginates, and these compounds can increase viscosity and provide thickening properties, but they may also confer textural characteristics similar to fat in processed meats [14]. Brown seaweeds also contain a high level of minerals (e.g., potassium) and amino acids, such as aspartic and glutamic acids, which may positively influence saltiness and umami (monosodium glutamate (MSG)) flavour profiles in pork products [15]. For instance, replacing 2.5 g of NaCl with 2 g of MSG, a sodium salt derivative of glutamic acid, can reduce NaCl intake by 37% [16]. The use of commercially available Irish brown seaweed species (sea spaghetti (Himanthalia elongata) and Irish wakame (Alaria esculenta)) as partial fat and salt replacers in fresh pork sausages has not been examined and merits investigation. Since seaweeds have an intense flavour profile, a maximum acceptable inclusion level was previously assessed, using 1%, 2.5%, and 5% in pork sausages. The results stated that 2.5% was adequate for sea spaghetti and Irish wakame in fresh pork sausages [17].

The objective of this study was to investigate the effectiveness of Irish brown seaweed species (sea spaghetti (Himanthalia elongata) and Irish wakame (Alaria esculenta)) as partial fat (from 25% to 20%, 15%, and 10%) and salt (from 2% to 1.5%, 1%, and 0.5%) replacers in fresh pork sausages at an inclusion level of 2.5%. The physicochemical, sensory, and shelf-life stability (lipid oxidation and microbiological analysis) of selected pork sausages stored in various packaging formats were also examined.

2. Materials and Methods

2.1. Chemicals and Reagents

Kjeltabs, sodium chloride (NaCl), hydrogen peroxide (H₂O₂), boric acid (H₃BO₃), hydrochloric acid (HCl), sulphuric acid (H₂SO₄), Trichloroacetic acid (TCA), and 2-thiobarbituric acid (TBA) were supplied by Sigma Aldrich Ireland Ltd., Vale Road, Arklow, Wicklow, Ireland. Silver nitrate and potassium chromate (K₂CrO₄) were purchased from Fisher Scientific, Loughborough, UK. Potassium dichromate (K₂Cr2O₇) was supplied by BDH Limited Supplies, Poole, UK. All solvents were of analytical grade.

2.2. Raw Materials

Brown seaweed species (Himanthalia elongata (Sea spaghetti (SS)) and Alaria esculenta (Irish wakame (IW))) were purchased from Wild Irish Seaweed, Co., Clare, Ireland. Seaweeds were hand-harvested off the coast of Co. Clare, Ireland, and were 100% naturally grown, sustainably harvested, and organically certified. According to the supplier’s specifications, all seaweeds were air-dried, dehumidified, and milled prior to purchase. The average particle size of the brown and red seaweeds was approximately 2.5 mm [18]. The nutritional composition (protein, fat, moisture, ash, salt (NaCl), pH, minerals, amino acids, dietary fibre), bioactive, thermal, and technological properties of these brown seaweeds have been quantified and assessed in a previous study [19].

Pork oyster (longissimus thoracis et lumborum) and pork backfat were supplied by Ballyburden Meat Processors (Cork, Ireland). Food-grade salt was obtained from British Salt Ltd. (Cheshire, UK). A custom-formulated reduced-salt sausage seasoning (0.39% salt) containing rusk (wheat flour (calcium carbonate, iron, niacin, thiamin), salt), sugar, dextrose, stabiliser (E450, E452), flavour enhancer (E621), preservative (E223), antioxidant (E301), natural flavourings, anticaking agent (E551), rapeseed oil, and hydrolysed vegetable protein (soya, maise) was obtained from Redbrook Ingredient Services (Mulhuddart, Dublin 15, Ireland). Rusk (wheat flour (wheat flour, calcium carbonate, iron, niacin, thiamin), salt, raising agent E503) and collagen casing were purchased from Viscofan UK Ltd. (Sevenoaks, Kent, UK).

2.3. Sausage Manufacture and Treatments

2.3.1. Sausage Manufacture

Fresh pork oyster and backfat were processed and delivered under hygienic conditions and were minced through a 4 mm perforated plate (Talsabell SA., Valencia, Spain). Pork oyster, half of the water, salt, and the seasoning was mixed for 60 s at high-speed using a bowl chopper (Seydleman Bowl chopper, Burgstallstraße, Germany). The backfat and various seaweeds were added and mixed at high speed for 30 s. The remaining water was added, and the batter was mixed for an additional 30 s at high speed. Finally, the rusk was added and mixed at low speed for 30 s. Sausage batter was stuffed using a piston-type sausage filler (Mainca UK Ltd., Berkshire, UK) into 21 mm diameter collagen casings and hand-linked into individual pork sausages (~10 cm in length).

2.3.2. Sausage Treatments

Fresh pork sausages (4.3 kg batches) (Control (C)) were formulated containing 41.1% pork oyster muscle, 25% pork back fat (f), 18.5% water (50:50, water: ice), 11.4% rusk, 2.0% seasoning, and 2% salt (sodium chloride (s)) (0.39% salt from the sausage seasoning, 0.14% from the rusk, and 1.5% salt added as an ingredient into the formulation) (

Table 1. Sausage treatments (%).

Treatment	Pork Oyster	Pork Backfat	Water	Rusk	Seasoning	Salt	Seaweed
C25f/2s *	41.1	25	18.5	11.4	2	2.0	0
20f/1.5s	44.1	20	18.5	11.4	2	1.5	2.5
20f/1.0s	44.6	20	18.5	11.4	2	1.0	2.5
20f/0.5s	45.1	20	18.5	11.4	2	0.5	2.5
15f/1.5s	49.1	15	18.5	11.4	2	1.5	2.5
15f/1.0s	49.6	15	18.5	11.4	2	1.0	2.5
15f/0.5s	50.1	15	18.5	11.4	2	0.5	2.5
10f/1.5s	54.1	10	18.5	11.4	2	1.5	2.5
10f/1.0s	54.6	10	18.5	11.4	2	1.0	2.5
10f/0.5s	55.1	10	18.5	11.4	2	0.5	2.5

* Control, C = control; f = fat; s = salt. %—g/100 g.

). The control formulation, designated C25f/2s—25% fat and 2% salt—was based on the average composition of pork sausages typically available on the Irish market.

Additional batches of pork sausages (n = 9) were manufactured with a sequential reduction in fat (f) (20%, 15%, and 10%) and salt (s) (1.5%, 1%, and 0.5%) levels (at each fat level) resulting in 20f/1.5s, 20f/1.0s, 20f/0.5s, 15f/1.5s, 15f/1.0s, 15f/0.5s, 10f/1.5s, 10f/1.0s, and 10f/0.5s treatments. Seaweed species (SS or IW) were added to the additional pork sausage batches at a level of 2.5%, previously identified as the maximum acceptable inclusion level in pork sausages [17].

2.4. Physicochemical Properties

2.4.1. Proximate Composition

The protein (Kjeldahl) and ash (muffle furnace at 550 °C) contents of sausage samples were analysed following AOAC Methods 954.01 and 942.05, respectively [20]. The moisture and fat content of sausages were determined using a SMART Trac system (CEM GmbH, Kamp-Lintfort, Germany). The findings were given as g/100 g of the sample.

2.4.2. Salt Analysis

The salt content of sausage samples was determined by titration using silver nitrate [21]. The silver nitrate (AgNO₃) solution was standardised against 20 mL of 0.1% sodium chloride (NaCl) to confirm the molarity of AgNO₃ (0.1 N).

Sausage samples were ashed at 550 °C in a muffle furnace, as described in Section 2.4.1. Ash samples were subsequently washed into conical flasks with 20 mL distilled water to ensure all ashed samples were adequately transferred. The indicator (potassium chromate and potassium dichromate) (2 mL) was added and standardised silver nitrate was used to titrate the solution from a clear yellow colour to an opaque light orange. Blank titrations were performed using 20 mL distilled water. The salt content was calculated using the following equation:

% salt = Titre for sample (mL) − Titre for blank (mL)/Mass of sample (g) × Molarity of AgNO₃ × 5.844

(1)

2.4.3. pH Determination

An Ultra Turax T25 homogenizer (Janke and Kunkel, IKA-Labortechnik, GmbH and Co., Staufen, Germany) was used to homogenise 5 g of fresh sausage samples for 1 min at 24,000 rpm in 45 millilitres of distilled water. A pH metre (Seven Easy portable, Mettler-Toledo GmbH, Schweizenbach, Switzerland) was used to measure the pH at 20 °C (room temperature). The pH metre was calibrated with buffer solutions 4.0 and 7.0.

2.4.4. Instrumental Colour Analysis

The colour of the fresh sausage samples was measured according to the CIE L*a*b* colour system [22] on the day of production. The samples were left to adjust to room temperature for 30 min before measurement. A Minolta chromameter (CR400, Minolta Camera Co. Ltd., Osaka, Japan) with an 11 mm diameter aperture and D65 illuminant was used to measure the surface colour. A white tile was used to calibrate the chromameter (L* = 97.79, a* = −0.11, b* = 2.69).

2.4.5. Texture Profile Analysis

The texture profile analysis (TPA) of sausage samples was carried out using a texture analyser TA-XF10S1.5i (Stable Micro System, Surrey, UK) based on a method described by Bourne [23]. The cooked sausage samples were allowed to equilibrate at room temperature for 30 min before measuring. The samples were cut into cylindrical slices (10 mm thickness), and four pieces were measured per sausage sample to ensure accuracy and repeatability. Two cycles of compression were applied to the samples with a 30 kg load cell. All samples were compressed to 40% of their original height using a 35 mm cylindrical probe (SMP/35 compression plate) at a cross-head speed of 1.5 mm/s. The determining factors include the following: hardness (N—Newton), the maximum force required for the first compression; springiness (mm—millimetre), distance the sample recovers after initial compression; cohesiveness (dimensionless), the ratio of positive force area during the second compression; chewiness (N*mm—Newton*millimetre), the product of gumminess and springiness.

2.4.6. Water-Holding Capacity

The water-holding capacity (WHC) was measured using the method described by Lianji and Chen [24]. Fresh sausage samples (10 g) were weighed into glass jars and heated in a water bath for 10 min at 90 °C. The samples were taken out of the heating process, wrapped in cheesecloth, and put into 30 mL centrifuge tubes with cotton wool lining the bottom of each tube. The samples were centrifuged (Avanti J-E centrifuge, Beckman Coulter, Brea, CA, USA) at a speed of 13,300× g for 10 min at 4 °C. After centrifugation, the cheesecloth was removed, and samples were reweighed. WHC was calculated using the following calculation:

% Water-holding capacity = 1 − (Sample weigh before heating − Sample weight after heating)\(Sample weight before heating × Moisture of the sample) × 100

(2)

2.4.7. Cook Loss

Sausage weights were recorded before and after cooking. Sausages were cooked in a preheated Zanussi convection oven (Zanussi Professional, Dublin, Ireland) at 180 °C for 22 min until an internal temperature of 72 °C was reached. Cook loss was calculated using the following equation:

% Cook loss = Sample weight before cooking − Sample weight after cooking\Sample weight before cooking × 100

(3)

2.5. Sensory Analysis (Sensory Acceptance Testing)

Sensory acceptance testing (SAT), also known as affective testing or hedonic testing, is a sensory technique used to assess product acceptability and liking/preference or its unique sensory properties [25].

Sausages were cooked as described in Section 2.4.7, cooled, and cut into cylindrical pieces (~2 cm). SAT was carried out using untrained assessors (n = 25), who were familiar with the taste of pork sausages [26]; SAT was conducted in sensory booths conforming to international standards at room temperature [27]. Sausage samples (20 samples − 10 treatments × 2 replicates) were presented to assessors in a randomised presentation order and were placed on plates with randomly assigned three-digit codes. SAT was carried out over two sessions to reduce panellist fatigue and water for palate cleansing between samples was provided. Assessors were asked to indicate their liking scores in relation to the following hedonic terms; appearance, aroma, flavour, texture, and overall acceptability on a 10 cm hedonic scale (1 = extremely dislike and 10 = extremely like). Assessors gave informed consent before commencing in the sensory study to ensure they participated voluntarily and granted permission of data usage. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the University College Cork Social Research and Ethics Committee (Log 2021-188).

2.6. Packaging and Shelf-Life of Pork Sausages

Optimum packaging conditions for the control (C25f/2s) and two selected sausages (SS10f/0.5s and IW15f/1s) were investigated using four packaging treatments. Sausage samples were placed in low-oxygen-permeable (<1 cm³/m²/24 h at STP) polystyrene/ethyl vinyl alcohol/polyethylene trays and over-wrapped with oxygen-permeable film (cling film) for aerobic packaging (AP) conditions. Trays containing the sausage treatments were flushed with 80% O₂: 20% CO₂ (MAP80/20) or 70% N₂: 30% CO₂ (MAP70/30) using modified atmosphere packaging (MAP) technology. This was performed using a vacuum-sealing unit (VS 100, Gustav Müller and Co. KG, Bad Homburg, Germany) fitted with a gas mixer (Witt-Gasetechnik GmbH and Co. KG, Witten, Germany) [28].

Sausage samples were also placed in vacuum pouches (polyamide/polyethylene 90, O₂: 50 cm³/m²/24 h STP, N₂: 10 cm³/m²/24 h STP, CO₂: M150 cm³/m²/24 h STP) and vacuum-packaged (VP) using a vacuum machine (Henkelmann, Polar 80, Henkelmann, Titaniumlaan 10, 5221 CK’s-Hertogenbosch, The Netherlands). All packaging treatments (AP, MAP80/20, MAP70/30, and VP) were stored for up to 27 days at 4 °C. Shelf-life measurements included lipid oxidation and microbiological analysis and were carried out on days 1, 6, 13, 20, and 27 to establish the time (day) at which the sausage samples exceed the acceptable limits.

2.6.1. Lipid Oxidation

The lipid oxidation measurements were conducted using the methodology outlined by Siu and Draper [29] on aerobically packaged (AP), MAP80/20, MAP70/30 and vacuum-packaged (VP) sausages on days 1, 6, 13, 20, and 27 stored at 4 °C.

Sausage samples (5 g) were weighed into beakers and homogenised for 2 min in 25 mL distilled water using an Ultra Turrax T25 homogeniser (Janke and Kunkel, IKA-Labortechnik, GmbH and Co., Staufen, Germany). Trichloroacetic acid (10%) was added (25 mL) and the mixture was shaken vigorously and filtered through Whatman No. 1 filter paper. The clear filtrate (4 mL) was added to 1 mL of 0.06 M 2-thiobarbituric acid (TBA). Assay blanks contained all reagents except the filtrate (2 mL distilled water, 2 mL 10% TCA, and 1 mL of 0.06 M TBA reagent). Samples were placed in a water bath and held at 80 °C for 90 min. After heating, samples were cooled at room temperature and vortex-mixed. The absorbance of the filtrate was measured using a spectrophotometer at 532 nm against the assay blank. The malondialdehyde content of the samples was calculated using an extinction coefficient of 1.56 × 105 M⁻¹ cm⁻¹.

Results were presented as TBARS (Thiobarbituric acid reactive substances) in mg malondialdehyde (MDA)/kg sausage.

2.6.2. Microbiological Analysis

Microbiological analyses (total viable counts (TVC), Escherichia coli (E. coli) and Salmonella) were determined as described by O’ Neill et al. [30].

Raw sausage samples (10 g) were weighed aseptically into a stomacher bag and a primary 10-fold dilution was carried out by adding 90 mL of sterile maximum recovery diluent (MRD) (Oxoid, Basingstoke, UK), stomaching (Steward Stomacher 400 Lab Blender, London, UK) the samples for 3 min, and then serially diluting the homogenates by 10-fold using MRD. In order to count TVC, 1 mL of each suitable dilution in the middle of compact dry-total count plates (20 cm²) (Nissui Pharmaceutical, Co. Ltd., Tokyo, Japan) and incubated for 48 h at 37 °C. E. coli was detected using Compact Dry EC plates (Nissui Pharmaceutical, Japan). Measures of 1 mL of each suitable dilution were inoculated in the middle of the plates and incubated at 37 °C for 24 h.

Sausage samples were examined for the presence or absence of Salmonella in Compact dry SL plates at the start and finish of storage (Nissui Pharmaceutical, Co. Ltd., Tokyo, Japan). The pre-enrichment procedure involved weighing 25 g of the sample into a filter stomacher bag, adding 225 mL of buffered peptone water (Oxoid), homogenising the mixture with a stomacher for one minute, and then incubating the mixture for 24 h at 37 °C. After removing the bag from the incubator, 0.1 mL of the enriched specimen was carefully dropped onto the sheet, approximately 1 cm from the plate’s edge, and 1 mL of sterile water was added adjacent to the specimen. The plates were incubated for 48 h at 42 °C.

Analyses were carried out on AP, MAP80/20, MAP70/30, and VP sausages on days 1, 6, 13, 20, and 27 stored at 4 °C. Except for Salmonella, the results were given as log₁₀ colony-forming units per gram of sausage (CFU/g).

2.7. Statistical Analysis

Statistical analysis was carried out using the IBM SPSS for Windows (Version 28.0.1.1) (SPSS, Chicago, IL, USA) software package. All analyses were performed in duplicate and three independent experimental trials were carried out. The physiochemical data of seaweed sausages was analysed using a two-way ANOVA to investigate the effects of fixed factors (salt level, fat level, and fat × salt interactions) and random factor (batch). Tukey’s post hoc test was used to adjust for multiple comparisons between treatment means when significance was detected at p < 0.05.

A mixed-model ANOVA was used to analyse the sensory analysis data with treatment, salt level, fat level, and fat × salt interactions as fixed effects, with the batch, panellist, and session as the random effects. The interactions between the fixed and random effects were measured and Tukey’s post hoc test was used to adjust for multiple comparisons between treatment means.

A full repeated-measures ANOVA was performed in the packaging study (lipid oxidation and microbiological analysis) to investigate the impact of storage time and packaged sausage treatments on microbial growth and lipid oxidation. The ‘between-subjects’ factor was represented by the sausage treatment, while the effect of time was measured using the ‘within-subjects’ factor. Tukey’s post hoc test was used to adjust for multiple comparisons between treatment means when significance was detected at p < 0.05.

3. Results and Discussion

3.1. Physicochemical Properties of Sausages

3.1.1. Proximate Composition

The proximate composition of sausages containing sea spaghetti (SS) and Irish wakame (IW) is presented in

Table 2. Proximate composition of reduced-fat and -salt pork sausages containing brown seaweeds.

Treatment	Sea Spaghetti (SS)				Irish Wakame (IW)
	Protein (%)	Moisture (%)	Fat (%)	Ash (%)	Protein (%)	Moisture (%)	Fat (%)	Ash (%)
C25f/2s	12.65 ± 0.19 a	53.58 ± 0.19 a	20.31 ± 0.30 a	2.80 ± 0.02 a	12.61 ± 0.24 a	52.78 ± 0.43 a	19.50 ± 0.18 a	2.61 ± 0.01 a
20f/1.5s	13.83 ± 0.53 abc	54.78 ± 1.01 ab	16.43 ± 1.51 b	3.11 ± 0.01 b	12.63 ± 0.25 a	54.43 ± 0.67 ab	16.16 ± 0.35 b	3.00 ± 0.05 b
20f/1.0s	13.44 ± 0.40 ab	54.92 ± 0.18 ab	16.56 ± 0.25 b	2.45 ± 0.04 c	13.05 ± 0.14 ab	55.58 ± 0.32 b	15.83 ± 0.32 b	2.51 ± 0.02 a
20f/0.5s	13.21 ± 0.07 ab	56.26 ± 0.24 b	15.62 ± 0.23 b	2.02 ± 0.05 d	12.94 ± 0.17 ab	55.58 ± 0.24 b	16.28 ± 0.15 b	2.16 ± 0.04 c
15f/1.5s	14.05 ± 0.40 bc	55.64 ± 0.79 ab	14.42 ± 0.67 b	3.08 ± 0.04 b	13.35 ± 0.24 abc	58.04 ± 0.35 c	12.30 ± 0.28 c	3.00 ± 0.02 b
15f/1.0s	13.12 ± 0.29 ab	55.70 ± 0.56 ab	14.40 ± 0.85 b	2.50 ± 0.02 c	14.00 ± 0.35 abc	58.01 ± 0.21 c	12.82 ± 0.23 c	2.52 ± 0.04 a
15f/0.5s	13.95 ± 0.32 abc	57.12 ± 0.46 bc	14.54 ± 0.81 b	2.03 ± 0.03 d	14.04 ± 0.06 abc	57.72 ± 0.15 c	12.65 ± 0.22 c	2.03 ± 0.04 c
10f/1.5s	14.67 ± 0.27 bc	60.30 ± 0.38 d	9.31 ± 0.02 c	3.05 ± 0.02 b	14.19 ± 0.26 bc	59.11 ± 0.51 cd	10.13 ± 0.42 d	3.04 ± 0.03 b
10f/1.0s	15.35 ± 0.16 c	59.44 ± 0.38 cd	10.31 ± 0.81 c	2.57 ± 0.02 c	15.77 ± 0.56 d	60.21 ± 0.36 d	9.11 ± 0.15 d	2.64 ± 0.02 a
10f/0.5s	14.60 ± 0.25 bc	61.35 ± 0.38 d	9.07 ± 0.29 c	2.16 ± 0.09 d	14.81 ± 0.42 cd	60.81 ± 0.19 d	9.41 ± 0.19 d	2.10 ± 0.10 c
Overall effects (p values) of fixed (fat, salt, and fat × salt interaction) and random (batch) variables on seaweed sausages’ proximate composition.
Fat	<0.001 ***	<0.001 ***	<0.001 ***	0.137 ns	<0.001 ***	<0.001 ***	<0.001 ***	0.131 ns
Salt	0.581 ns	0.006 **	0.539 ns	<0.001 ***	0.008 **	0.023 *	0.463 ns	<0.001 ***
Fat * salt	0.100 ns	0.904 ns	0.863 ns	0.180 ns	0.320 ns	0.120 ns	0.098 ns	0.309 ns
Batch	0.471 ns	0.628 ns	0.675 ns	0.769 ns	0.384 ns	0.561 ns	0.982 ns	0.803 ns

^abcd For each seaweed, within each parameter, mean values (±standard error) in the same column bearing different superscripts are significantly different (p < 0.05). p values: ^ns—not significant; * p < 0.05–0.01 (significant); ** p < 0.01–0.001 (highly significant); *** p < 0.001 (extremely significant). %—g/100 g. C = control; f = fat; s = salt.

. The overall protein content of SS sausages was significantly influenced by the fat level, while IW sausages were significantly impacted by the fat and salt levels. Sausage treatments, SS and IW, containing 10% fat had higher (p < 0.05) protein content relative to the control. The addition of pork oyster muscle in low-fat and low-salt sausages may explain the variances in the protein content. Similar trends were noted in previous studies, where reducing fat and salt levels led to increased protein content [31,32].

The overall fat and salt levels had significant effects on the moisture content of SS and IW sausages. SS treatments, 20f/0.5s, 15f/0.5s, 10f/1.5s, 10f/1.0s, and 10f/0.5s, had higher (p < 0.05) moisture content than the control, while all IW treatments, except 20f/1.5s, had significantly higher moisture content than the control. For a given fat level, within each SS and IW treatment, there were no significant differences in moisture content; for example, 20f/1.5s, 20f/1.0s, and 20f/0.5s were not significantly different (p > 0.05). The moisture content of SS and IW sausages also increased (p < 0.05) with reduced fat addition. Previous studies reported the moisture contents of reduced fat frankfurter and sausages to be inversely proportional to the fat content, i.e., higher moisture in lower fat products [33,34].

The overall fat content of SS and IW-incorporated sausages was significantly influenced by the fat levels. The fat content of seaweed-incorporated sausages was lower (p < 0.05) than the control (Table 2). According to previous research on the seaweeds used in this study, their fat content is low (1.2% to 2%), and at an incorporation level of 2.5%, they contributed minimal fat to the various sausages [17,19,35]. López-López et al. [35] also reported that the fat content of beef patties was not influenced to any significant degree owing to the low fat content of the brown seaweed, wakame (0.88%).

The overall ash content of SS and IW sausages was significantly influenced by the salt level. SS and IW sausage treatments containing 1.5% salt contained significantly higher (p < 0.05) ash than the control. This was attributed to both seaweed species’ salt content (12–15%) and mineral contents [19]. Sea spaghetti sausage treatments with 1.0% and 0.5% salt and IW sausage treatments with 0.5% salt had significantly lower ash content than the control (

Table 3. pH, salt, water-holding capacity (WHC), and cook loss of reduced-fat and -salt pork sausages containing brown seaweeds.

Treatment	Sea Spaghetti (SS)				Irish Wakame (IW)
	pH	Salt (%)	WHC (%)	Cook loss (%)	pH	Salt (%)	WHC (%)	Cook Loss (%)
C25f/2s	5.81 ± 0.07 abc	1.82 ± 0.06 a	63.60 ± 1.10 a	22.65 ± 0.97 a	5.82 ± 0.02 a	1.79 ± 0.00 ab	62.79 ± 1.39 a	21.59 ± 0.61 a
20f/1.5s	5.71 ± 0.01 a	1.79 ± 0.01 a	64.96 ± 0.75 ab	27.04 ± 0.69 abc	5.89 ± 0.01 a	1.81 ± 0.02 a	64.52 ± 0.69 a	27.54 ± 1.34 c
20f/1.0s	5.88 ± 0.01 bc	1.22 ± 0.01 b	64.63 ± 1.11 ab	29.05 ± 0.29 c	5.85 ± 0.00 a	1.28 ± 0.02 c	66.33 ± 1.27 ab	29.72 ± 0.65 c
20f/0.5s	5.91 ± 0.01 bc	0.76 ± 0.01 c	66.97 ± 1.54 abc	29.15 ± 1.15 c	5.86 ± 0.00 a	0.79 ± 0.01 d	65.34 ± 1.82 a	29.28 ± 0.81 c
15f/1.5s	5.76 ± 0.04 ab	1.76 ± 0.01 a	66.52 ± 0.43 abc	27.62 ± 0.68 bc	5.86 ± 0.00 a	1.77 ± 0.01 ab	67.11 ± 0.74 ab	27.34 ± 0.53 bc
15f/1.0s	5.88 ± 0.01 bc	1.24 ± 0.01 b	67.31 ± 0.97 abcd	27.71 ± 0.64 bc	5.86 ± 0.00 a	1.25 ± 0.01 c	68.13 ± 3.17 ab	28.35 ± 0.71 c
15f/0.5s	5.93 ± 0.01 c	0.72 ± 0.01 c	66.94 ± 1.06 abc	29.66 ± 0.91 c	5.82 ± 0.00 a	0.76 ± 0.01 d	65.06 ± 1.06 a	31.22 ± 0.20 c
10f/1.5s	5.83 ± 0.01 abc	1.72 ± 0.02 a	69.56 ± 1.02 bcd	23.49 ± 0.86 ab	5.87 ± 0.00 a	1.74 ± 0.01 b	73.01 ± 0.81 b	23.15 ± 1.56 ab
10f/1.0s	5.89 ± 0.01 bc	1.21 ± 0.01 b	72.21 ± 1.00 d	27.81 ± 0.67 bc	5.84 ± 0.00 a	1.27 ± 0.01 c	73.70 ± 1.01 b	29.43 ± 0.88 c
10f/0.5s	5.91 ± 0.03 bc	0.72 ± 0.02 c	70.60 ± 0.92 cd	25.63 ± 1.56 abc	5.82 ± 0.00 a	0.74 ± 0.01 d	67.97 ± 1.09 ab	28.35 ± 0.23 c
Overall effects (p values) of fixed (fat, salt, and fat × salt interaction) and random (batch) variables on seaweed sausages’ pH, salt, WHC, and cook loss.
Fat	0.014 *	0.003 **	<0.001 ***	0.906 ns	<0.001 ***	0.002 **	<0.001 ***	0.021 *
Salt	<0.001 ***	<0.001 ***	0.430 ns	0.939 ns	<0.001 ***	<0.001 ***	0.046 *	<0.001 ***
Fat × salt	0.010 **	0.050 *	0.510 ns	0.749 ns	<0.001 ***	0.249 ns	0.389 ns	0.096 ns
Batch	0.975 ns	0.650 ns	0.797 ns	0.791 ns	0.205 ns	0.814 ns	0.953 ns	0.499 ns

^abcd For each seaweed, within each parameter, mean values (±standard error) in the same column bearing different superscripts are significantly different (p < 0.05). p values: ^ns—not significant; * p < 0.05–0.01 (significant); ** p < 0.01–0.001 (highly significant); *** p < 0.001 (extremely significant). %—g/100g. C = control; f = fat; s = salt.

), presumably due to the lower salt levels in the sausages (Table 1). López-López et al. [10] and Kim et al. [36] also found that seaweeds affected the ash content of frankfurters and sausages, respectively.

3.1.2. pH, Salt, Water-Holding Capacity (WHC), and Cook Loss

The pH of SS and IW seaweeds was 5.55 and 6.29, respectively [19]. The pH values of SS and IW sausage treatments ranged from 5.71 to 5.93 and 5.82 to 5.89, respectively (Table 3). The overall pH of SS and IW sausages was significantly influenced by the fat and salt levels, and a fat × salt interaction was observed. In this study, the addition of seaweeds to reduced-fat and -salt sausages did not affect (p > 0.05) the pH of the sausage treatments compared to the control. However, within each fat treatment, the pH value of SS sausage treatments decreased with increasing salt levels. Puolanne et al. [37] also observed a reduction in pH values as the amount of salt applied increased in pork sausage batter. The impact of salt on the pH of meat is mostly attributed to the stronger affinity of the Cl− ion to proteins, such as myosin, compared to the Na+ ion. The Cl− ions are adsorbed by the positively charged groups of myosin, causing a change in the isoelectric point and thereby reducing the pH [38,39].

The overall salt content of SS sausage treatments was significantly influenced by the fat and salt levels, and a fat × salt interaction was observed, while fat and salt levels had a significant effect on the salt content of IW sausages. Fat and salt may interact, affecting water retention and the distribution and solubility of salt within the sausage, which may impact the salt content of the sausages [38]. This may also be due to the varying fat and salt levels in their formulations (Table 1). Seaweed sausages (SS and IW treatments) containing 1.5% salt had salt levels that were comparable (p > 0.05) to the control. The salt content of SS and IW sausages was higher than the projected levels for each formulation (0.5%, 1.0%, and 1.5% salt) (Table 1), presumably due to the salt levels present in both brown seaweed species (12–15%) [19].

The overall WHC of SS sausage treatments was significantly influenced by fat level, and IW samples were affected by fat and salt levels. The WHC of SS (10f/1.5s, 10f/1.0s, and 10f/0.5s) and IW (10f/1.5s and 10f/0.5s) treatments was significantly higher than the control. This increased WHC may be attributed to the increased pork meat content in sausages (Table 1). The overall cook loss of IW sausages was significantly impacted by the fat and salt levels. The SS treatments (20f/1.5s, 10f/1.5s and 10f/0.5s) had similar (p > 0.05) cook loss values to the control, while other treatments had higher (p < 0.05) values. Cook loss values were higher (p < 0.05) in all IW treatments compared to the control, except for treatment 10f/1.5s. Higher cook loss values can be attributed to the reduction in salt in seaweed sausages. Ruusunen et al. [8] noted that the reduction in salt in meat products increases cooking loss due to its water- and fat-binding properties.

3.1.3. Surface Colour (CIE L*a* b*) of Pork Sausages

The fat level did not influence the surface colour of SS sausages. However, the surface redness (a*) was significantly affected by the salt level (

Table 4. Surface colour (CIE L*a* b*) of reduced-fat and -salt pork sausages containing brown seaweeds.

Treatment	Sea Spaghetti (SS)			Irish Wakame (IW)
	L*	a*	b*	L*	a*	b*
C25f/2s	70.65 ± 0.32 a	7.52 ± 0.31 a	14.63 ± 0.42 a	73.41 ± 0.32 a	6.29 ± 0.19 a	13.26 ± 0.13 a
20f/1.5s	66.28 ± 0.11 b	4.15 ± 0.27 b	14.02 ± 0.14 a	63.70 ± 0.63 bc	−0.20 ± 0.10 bc	11.25 ± 0.23 b
20f/1.0s	64.61 ± 1.25 b	3.81 ± 0.35 b	13.61 ± 0.41 a	61.77 ± 0.37 c	−0.57 ± 0.07 c	11.30 ± 0.11 b
20f/0.5s	65.23 ± 0.02 b	5.24 ± 0.50 b	12.84 ± 0.32 a	65.23 ± 0.02 b	−0.44 ± 0.16 bc	12.84 ± 0.32 ac
15f/1.5s	64.79 ± 0.27 b	4.53 ± 0.51 b	13.36 ± 0.36 a	61.41 ± 0.41 cd	−0.18 ± 0.07 bc	11.86 ± 0.19 bc
15f/1.0s	65.44 ± 0.25 b	4.42 ± 0.32 b	13.70 ± 0.25 a	61.24 ± 0.24 cd	−0.13 ± 0.11 bc	11.48 ± 0.14 b
15f/0.5s	65.47 ± 0.56 b	5.42 ± 0.29 b	12.94 ± 0.27 a	65.47 ± 0.56 b	−0.30 ± 0.17 bc	12.94 ± 0.27 a
10f/1.5s	63.66 ± 0.97 b	4.82 ± 0.51 b	13.69 ± 0.48 a	57.91 ± 0.58 e	−0.08 ± 0.07 bc	11.56 ± 0.18 b
10f/1.0s	62.90 ± 0.53 b	4.30 ± 0.23 b	13.09 ± 0.23 a	58.99 ± 0.65 de	0.35 ± 0.07 b	11.24 ± 0.12 b
10f/0.5s	64.76 ± 1.85 b	5.19 ± 0.66 b	13.95 ± 0.62 a	57.58 ± 0.98 e	0.02 ± 0.38 bc	11.35 ± 0.25 b
Overall effects (p values) of fixed (fat, salt, and fat × salt interaction) and random (batch) variables on seaweed sausages’surface colour (CIE L*a* b*).
Fat	0.065 ns	0.473 ns	0.712 ns	<0.001 ***	0.005 **	0.003 **
Salt	0.486 ns	0.015 *	0.355 ns	<0.001 ***	0.633 ns	<0.001 ***
Fat × salt	0.544 ns	0.911 ns	0.136 ns	<0.001 ***	0.226 ns	0.004 **
Batch	0.850 ns	0.840 ns	0.570 ns	0.937 ns	0.374 ns	0.566 ns

^abcde For each seaweed, within each parameter, mean values (±standard error) in the same column bearing different superscripts are significantly different (p < 0.05). p values: ^ns—not significant; * p < 0.05–0.01 (significant); ** p < 0.01–0.001 (highly significant); *** p < 0.001 (extremely significant). C = control; f = fat; s = salt.

). Conversely, the lightness (L*) and yellowness (b*) values of IW sausages were significantly influenced by the fat and salt levels, and a significant fat × salt interaction was observed.

The a* values of IW sausages were also influenced by fat level. In comparison to the control, the addition of SS and IW seaweeds to sausages reduced (p < 0.05) the lightness (L*) and redness (a*) of fresh pork sausages (Table 4). However, no significant effect on surface yellowness (b*) was observed. Other than the fat and salt levels having effects on the colour changes of SS and IW sausages, colour changes are also presumably due to pigments such as fucoxanthin (brown or olive green), α-carotene (yellow and green), chlorophyll a (blue-green), and phaephytin a (olive-brown) present in SS and IW [40,41]. These pigments may have a substantial impact on the sensory perception of these sausages, affecting customers’ purchasing behaviour and preferences. Similarly, the incorporation of sea spaghetti and konjac into frankfurters and sea spaghetti in gel/emulsion systems decreased L* and a* values [11,42].

IW sausage’s negative a* values indicated an associated greenness in pork sausage treatments. This may be due to the higher chlorophyll a (blue-green), which is predominant in green plants, present in IW (172 mg/100 g) than in SS (150.14 mg/100 g) [40,43]. The b* values for treatments IW 20f/0.5s and 15f/0.5s treatments were similar (p > 0.05) to the control, while other treatments had lower (p < 0.05) b* values (

Table 5. Texture profile analysis (TPA) of reduced-fat and -salt pork sausages containing brown seaweeds.

Treatment	Sea Spaghetti (SS)				Irish Wakame (IW)
	Hardness (N)	Springiness (mm)	Cohesiveness	Chewiness (N*mm)	Hardness (N)	Springiness (mm)	Cohesiveness	Chewiness (N*mm)
C25f/2s	43.07 ± 2.48 a	0.881 ± 0.01 a	0.707 ± 0.01 ab	26.62 ± 1.87 a	45.50 ± 1.30 a	0.921 ± 0.01 a	0.700 ± 0.00 ab	29.34 ± 0.75 a
20f/1.5s	47.65 ± 2.46 a	0.893 ± 0.03 a	0.685 ± 0.01 ab	29.06 ± 1.28 ab	59.73 ± 1.70 bc	0.930 ± 0.01 a	0.720 ± 0.01 b	39.85 ± 0.76 c
20f/1.0s	56.12 ± 2.46 a	0.835 ± 0.01 a	0.613 ± 0.01 c	28.82 ± 0.86 ab	61.24 ± 1.67 bc	0.877 ± 0.01 a	0.705 ± 0.01 ab	37.81 ± 1.09 bc
20f/0.5s	47.42 ± 4.13 a	0.848 ± 0.02 a	0.661 ± 0.01 bc	26.34 ± 1.21 a	45.51 ± 1.70 a	0.874 ± 0.03 a	0.668 ± 0.01 a	26.64 ± 1.35 a
15f/1.5s	55.12 ± 3.65 a	0.898 ± 0.01 a	0.692 ± 0.01 ab	34.19 ± 2.27 ab	59.35 ± 2.96 bc	0.914 ± 0.01 a	0.691 ± 0.01 ab	37.39 ± 1.25 bc
15f/1.0s	56.71 ± 5.49 a	0.866 ± 0.01 a	0.670 ± 0.01 ab	32.86 ± 3.25 ab	42.44 ± 2.79 a	0.896 ± 0.01 a	0.692 ± 0.01 ab	26.13 ± 1.19 a
15f/0.5s	49.94 ± 4.83 a	0.872 ± 0.01 a	0.674 ± 0.01 ab	29.17 ± 2.10 ab	43.92 ± 3.12 a	0.884 ± 0.01 a	0.694 ± 0.00 ab	26.87 ± 2.00 a
10f/1.5s	59.48 ± 1.56 a	0.921 ± 0.01 a	0.718 ± 0.01 b	39.33 ± 0.77 b	64.96 ± 2.36 c	0.925 ± 0.01 a	0.704 ± 0.01 ab	42.30 ± 1.32 c
10f/1.0s	52.21 ± 7.36 a	0.855 ± 0.03 a	0.684 ± 0.02 ab	30.61 ± 4.47 ab	51.05 ± 1.70 ab	0.893 ± 0.01 a	0.703 ± 0.00 ab	32.00 ± 0.89 ab
10f/0.5s	52.80 ± 2.62 a	0.864 ± 0.04 a	0.662 ± 0.01 bc	30.44 ± 2.96 ab	46.41 ± 3.91 a	0.922 ± 0.01 a	0.708 ± 0.01 b	30.20 ± 2.56 a
Overall effects (p values) of fixed (fat, salt, and fat × salt interaction) and random (batch) variables on seaweed sausages’TPA.
Fat	0.414 ns	0.373 ns	0.004 **	0.037 *	0.009 **	0.166 ns	0.157 ns	<0.001 ***
Salt	0.331 ns	0.013 *	<0.001 ***	0.037 *	<0.001 ***	0.009 **	0.086 ns	<0.001 ***
Fat × salt	0.475 ns	0.918 ns	0.046 *	0.451 ns	0.010 **	0.274 ns	0.012 *	0.006 **
Batch	0.721 ns	0.950 ns	0.824 ns	0.869 ns	0.625 ns	0.726 ns	0.831 ns	0.637 ns

^abc For each seaweed, within each parameter, mean values (±standard error) in the same column bearing different superscripts are significantly different (p < 0.05). p values: ^ns—not significant; * p < 0.05–0.01 (significant); ** p < 0.01–0.001 (highly significant); *** p < 0.001 (extremely significant). C = control; f = fat; s = salt. N—Newton. mm—millimetre. N*mm—Newton*millimetre.

). Kim et al. [36] also reported a reduction in the lightness of sausages with increasing levels of sea tangle. These authors concluded that brown seaweed pigments such as xanthophyll, carotenes, and chlorophyll have adverse effects on the colour of raw sausages. The changes in the L*, a* and b* values of SS and IW sausages may affect the visual acceptability of these sausages by consumers.

3.1.4. Texture Profile Analysis (TPA) of Cooked Pork Sausages

The overall cohesiveness of SS sausages was significantly influenced by the fat and salt levels, and a fat × salt interaction was observed, and chewiness by the fat and salt levels. The springiness of both SS and IW sausages was affected by the salt level (Table 5). The hardness and springiness values of SS sausage treatments were not different (p > 0.05) from the control. The SS20f/1.0s treatment had significantly lower cohesiveness, while SS10f/1.5s had significantly higher chewiness values than the control. The overall hardness and chewiness of IW sausages were significantly affected by the fat and salt levels, and a fat × salt interaction was observed. The hardness and chewiness of IW sausage treatments: 20f/1.5s, 20f/1.0s, 15f/1.5s, and 10f/1.5s, were higher (p < 0.05) than the control (Table 5). The springiness and cohesiveness of IW seaweed sausages were similar (p > 0.05) to the control.

Cofrades et al. [42] and Fernández-Martín et al. [9] observed increasing hardness and chewiness and decreasing springiness by adding sea tangle in low-salt gel/emulsion meat systems and sea spaghetti in pork meat batter. However, some reports have observed softer patties being generated by adding wakame seaweed [35]. The variances in research findings can most likely be attributed to the seaweed type, the seasonality of the seaweed harvesting, and the gross composition of the seaweed utilised, etc. Also, the reduction in fat in meat products such as meat batters and sausages can yield harder products [31,44]. Salt also acts as a solubilising agent in emulsified products, stabilising the fat within the protein matrix (protein–fat interaction) and contributing to juiciness and texture [45]. According to Laranjo et al. [46], reduced amounts of sodium can decrease the ability of protein to retain water, which leads to changes such as tougher and chewier products. Changes in the texture profile (hardness and chewiness) of SS and IW sausages, when compared to the control, may have an impact on consumer sensory acceptance.

3.1.5. Sensory Analysis (Sensory Acceptance Testing (SAT)) on Cooked Pork Sausages

The effects of the fat, salt, fat × salt, treatment, batch, panellist, and session on SAT results are shown in

Table 6. Sensory acceptance testing (SAT) (mean hedonic scores) of reduced-fat and -salt pork sausages containing brown seaweeds.

	Sea Spaghetti (SS)					Irish Wakame (IW)
	Appearance	Aroma	Texture	Flavour	Overall Acceptability	Appearance	Aroma	Texture	Flavour	Overall Acceptability
C25f/2s	6.91 ± 0.40 a	7.25 ± 0.34 a	7.24 ± 0.38 a	7.57 ± 0.31 a	7.18 ± 0.35 a	6.58 ± 1.12 a	7.03 ± 0.75 a	7.03 ± 0.90 a	6.98 ± 0.82 a	6.67 ± 1.00 a
20f/1.5s	6.94 ± 0.26 a	7.61 ± 0.95 a	6.77 ± 0.25 a	7.75 ± 1.45 a	6.62 ± 0.30 a	5.95 ± 0.93 a	6.36 ± 0.72 a	6.13 ± 0.79 ab	6.26 ± 0.73 ab	6.10 ± 0.81 ab
20f/1.0s	6.95 ± 0.30 a	8.46 ± 1.60 a	6.40 ± 0.38 a	6.97 ± 0.32 a	6.78 ± 0.33 a	5.92 ± 0.89 a	6.48 ± 0.71 a	5.57 ± 0.84 ab	6.25 ± 0.72 ab	5.73 ± 0.74 ab
20f/0.5s	6.87 ± 0.31 a	6.68 ± 0.33 a	6.36 ± 0.33 a	6.13 ± 0.31 a	6.07 ± 0.31 a	5.75 ± 0.69 a	6.13 ± 0.65 a	4.97 ± 0.83 b	5.53 ± 0.79 b	5.42 ± 0.69 ab
15f/1.5s	6.89 ± 0.27 a	8.39 ± 1.44 a	6.49 ± 0.34 a	6.80 ± 0.26 a	6.73 ± 0.27 a	6.09 ± 0.82 a	6.11 ± 0.88 a	5.73 ± 0.82 ab	6.03 ± 0.82 ab	5.80 ± 0.81 ab
15f/1.0s #	6.82 ± 0.33 a	6.66 ± 0.39 a	6.15 ± 0.41 a	6.73 ± 0.35 a	6.49 ± 0.34 a	6.24 ± 0.60 a	6.19 ± 0.79 a	6.00 ± 0.71 ab	5.89 ± 0.56 ab	6.00 ± 0.64 ab
15f/0.5s	6.93 ± 0.27 a	6.61 ± 0.37 a	6.38 ± 0.40 a	6.14 ± 0.33 a	6.16 ± 0.30 a	6.30 ± 0.61 a	5.91 ± 0.65 a	5.19 ± 0.79 b	5.45 ± 0.82 b	5.34 ± 0.97 ab
10f/1.5s	6.96 ± 0.26 a	6.78 ± 0.35 a	5.85 ± 0.37 a	6.39 ± 0.32 a	6.35 ± 0.35 a	5.90 ± 0.78 a	5.88 ± 0.72 a	5.38 ± 0.95 b	5.85 ± 0.69 ab	5.59 ± 0.63 ab
10f/1.0s	6.97 ± 0.32 a	6.76 ± 0.37 a	5.99 ± 0.35 a	6.59 ± 0.36 a	6.46 ± 0.35 a	6.17 ± 0.53 a	6.13 ± 0.81 a	4.89 ± 0.89 b	5.45 ± 0.72 b	5.21 ± 0.69 b
10f/0.5s#	7.09 ± 0.28 a	6.76 ± 0.28 a	5.97 ± 0.36 a	6.10 ± 0.37 a	5.94 ± 0.34 a	5.81 ± 0.88 a	5.70 ± 0.89 a	5.18 ± 0.98 b	5.16 ± 0.89 b	5.31 ± 1.00 ab
Overall effects (p values) of fixed (fat, salt, and fat × salt interaction and treatment) and random (batch, panellist, and session) variables on seaweed sausages’ sensory descriptors.
Fat	0.639 ns	0.783 ns	0.198 ns	0.508 ns	0.459 ns	0.113 ns	0.034 *	0.252 ns	0.773 ns	0.743 ns
Salt	0.990 ns	0.388 ns	0.923 ns	0.333 ns	0.356 ns	0.890 ns	0.650 ns	0.431 ns	0.325 ns	0.521 ns
Fat × salt	0.844 ns	0.572 ns	0.251 ns	0.435 ns	0.716 ns	0.752 ns	0.997 ns	0.120 ns	0.405 ns	0.216 ns
Treatment	0.921 ns	0.581 ns	0.053 ns	0.274 ns	0.051 ns	0.392 ns	0.062 ns	<0.001 ***	<0.001 ***	0.002 **
Batch	0.557 ns	0.567 ns	0.926 ns	0.282 ns	0.638 ns	0.088 ns	0.971 ns	0.231 ns	0.612 ns	0.189 ns
Panellist	<0.001 ***	0.365 ns	<0.001 ***	0.024 *	<0.001 ***	0.002 **	0.011 *	<0.001 ***	0.002 **	<0.001 ***
Session	0.843 ns	0.788 ns	0.778 ns	0.498 ns	0.667 ns	0.828 ns	0.895 ns	0.433 ns	0.870 ns	0.497 ns

^ab For each seaweed, within each parameter, mean values (±standard error) in the same column bearing different superscripts are significantly different (p < 0.05). p values: ^ns—not significant; * p < 0.05–0.01 (significant); ** p < 0.01–0.001 (highly significant); *** p < 0.001 (extremely significant). ^# Seaweed sausages selected for shelf-life analysis. C = control; f = fat; s = salt.

. The overall SAT results of SS sausages were not influenced by the fat and salt levels and no fat × salt interaction was observed. The treatment, batch, and session had no effects on the SS sausages’ overall SAT results. However, the panellists affected SS sausages’ SAT descriptors, except aroma.

TPA results (Section 3.1.4) reveals that hardness and chewiness of sausages containing SS and IW were higher compared to the control sausages. However, when considering sensory texture evaluations, the seaweed-incorporated sausages scored lower than the control sausages. This discrepancy suggests that while the addition of seaweeds may enhance certain textural properties of sausages, it could negatively impact overall consumer sensory acceptance due to the perceived increase in hardness and chewiness.

As stated above, the control sausages scored higher for texture and overall acceptability than reduced-fat and -salt SS sausages, SAT results were not significantly different (Table 6). Therefore, all SS treatments were deemed acceptable by panellists. This result may be attributed to the concentration of sea spaghetti (2.5%) in the reformulated sausages, masking the variation in fat and salt levels amongst treatments without overwhelming the flavour of the sausages. Wilkin et al. [47] noted that SS concentrations above 15% usually express the dominant fishy flavours and concluded that the use of SS at lower concentrations would result to less overpowering in the product, hence leading to more desirable products. Therefore, without negatively influencing the sensory acceptance by panellists, the SS treatment with the lowest fat and salt levels (10f/0.5s), which provides 60% less fat and 75% less salt than the control, was selected for packaging and shelf-life analysis trials (lipid oxidation and microbiological analysis). This fat and salt reduction surpasses the target of reducing fat and salt by 10% by 2025, as set by FSAI [3].

The overall SAT results, except aroma, of IW sausages were not influenced by the fat and salt levels and no fat × salt interaction was observed. The aroma was influenced by the fat level. The treatment affected IW sausage’s overall texture, flavour, and overall acceptability. The batch and session did not affect SAT descriptors, while the panellists influenced the SAT results (Table 6). The addition of IW to sausages did not significantly affect the liking of appearance and aroma of reduced-fat and -salt sausages compared to the control. The texture and flavour sensory descriptors scores for 20f/0.5s, 15f/0.5s, 10f/1.0s, and 10f/0.5s treatments were lower (p < 0.05) than the control. Also, the texture descriptor score for treatment 10f/1.5s was lower (p < 0.05) than that of the control. Treatment 10f/1.0s had the lowest liking (p < 0.05) for flavour and overall acceptability (Table 6). The IW sausage treatment 15f/1.0s contained the lowest fat and salt levels, which were not significantly different from the control with respect to each sensory descriptor. Vilar et al. [48] reported a higher odour intensity for 1-Octen-3-ol in Irish wakame as 3.3 compared to sea spaghetti (2), following gas chromatography olfactometry (GC-O) analysis. This compound has been shown to contribute to the formation of a seafood aroma [49]. In combination with the reduced fat and salt contents, panellists may have highlighted differences in samples and preferred IW sausages containing higher fat and salt contents, in comparison to SS sausages. This may explain the reduced acceptability of IW-containing pork sausages. Therefore, IW treatment (15f/1.0s), which provides 40% less fat and 50% less salt than the control, was selected for the packaging and shelf-life study. This fat and salt reduction also exceeds the target of reducing fat and salt by 10% by 2025, as set by FSAI [3].

3.2. Shelf-Life Stability of Packaged Selected Pork Sausages

Factors such as high fat content, comminuted structure, and lack of thermal processing render pork sausages susceptible to quality deterioration and spoilage by lipid oxidation and microbial contamination. In the present study, the sausage seasoning (see Section 2.2) contains antioxidant (E301—sodium ascorbate) and preservative (E223—sodium metabisulphite) agents which serve to enhance sausage quality and shelf-life.

The shelf-life extension of processed meats such as pork sausages is complex due to the multi-functional properties of the ingredients used in products. For example, salt is used positively as a flavour enhancer and binder and has preservative effects with respect to microbial growth [50]. However, salt can also negatively impact lipid stability in processed meats by accelerating lipid oxidation, resulting in “off” odours and flavours [51]. Similarly, dried ground seaweeds contain a range of functional ingredients and health-promoting bioactive compounds [10], which would enhance product quality from a technological perspective, i.e., improve water binding, extend shelf life, and increase consumer appeal by presenting healthier products. Seaweed also contains minerals such as iron and copper [52], which are known to promote lipid oxidation in muscle foods.

In the current study, several packaging formats were examined to minimise the potential negative effects of ingredient reformulation on pork sausage quality and shelf-life. Based on the sensory analysis results, the control (C25f/2s) and two seaweed-containing sausages (SS10f/0.5s and IW15f/1.0s) were stored in aerobic packaging (AP), 80% O₂: 20% CO₂ modified atmosphere packaging (MAP80/20), 70% N₂: 30% CO₂ (MAP70/30), and vacuum-packaged (VP) conditions. These packaging conditions were selected to determine a suitable packaging condition for reduced-fat and -salt sausages containing brown seaweeds.

Shelf-life indicators examined included lipid oxidation and microbiological analysis of packaged sausages. AP is the most used packaging method utilised for sausages due to its cost effectiveness and ability to maintain sausage appearance [53]. The high oxygen in MAP80/20 is used to maintain a desirable colour, which is appealing to consumers, while CO₂ inhibits bacteria growth and nitrogen reduces lipid oxidation, which helps prevent rancidity and maintains the quality of meat over time [52]. VP is a packaging condition with the absence of any gas and is not widely used in sausage packaging as it can distort sausage shape, making it less appealing to consumers. However, by removing air from VP, it creates an oxygen-free environment, which is crucial for lipid oxidation reduction and retarding microbiological growth. In the absence of oxygen, lipid oxidation is inhibited, and the proliferation of aerobic bacteria and fungi is significantly slowed down, which helps delay spoilage. However, certain anaerobic bacteria can tolerate these conditions, but their proliferation is much slower compared to aerobic conditions [54,55].

3.2.1. Lipid Oxidation

The lipid oxidation (TBARS) values of the control, SS10f/0.5s, and IW15f/1.0s sausages stored under aerobic packaging (AP), modified atmosphere packaging (MAP), and vacuum-packaged (VP) conditions over 27 days at 4 °C are shown in

Table 7. Lipid oxidation (TBARS in mg MDA/kg sample) of control and selected seaweed sausages during storage at 4 °C.

Treatment	Packaging	Storage Time at 4 °C (days)
		1	6	13	20	27
C25f/2.0s (Control)	AP	0.96 ±0.01 abcdx	1.65 ± 0.04 axy	2.50 ± 0.46 axy	3.30 ± 0.41 ay	3.33 ± 0.55 ay
	MAP80/20	0.88 ± 0.02 bcdex	1.20 ± 0.09 bcxy	1.37 ± 0.06 bcxy	1.61 ± 0.20 bcdxy	2.07 ± 0.41 aby
	MAP70/30	0.67 ± 0.05 ex	0.65 ± 0.03 fx	0.72 ± 0.03 cx	0.71 ± 0.01 dx	0.76 ± 0.01 bx
	VP	0.89 ± 0.00 bcdex	1.12 ± 0.08 bcdx	1.46 ± 0.21 bcx	2.11 ± 0.47 bxy	3.02 ± 0.37 ay
SS10f/0.5s	AP	0.79 ± 0.03 dex	1.00 ± 0.01 cdexy	1.19 ± 0.11 bcxy	1.24 ± 0.09 bcdxy	1.36 ± 0.21 by
	MAP80/20	0.85 ± 0.07 cdex	0.94 ± 0.04 cdefx	1.06 ± 0.05 bcx	1.09 ± 0.16 bcdx	1.10 ± 0.16 bx
	MAP70/30	0.70 ± 0.02 ex	0.75 ± 0.03 efxy	0.88 ± 0.03 bcz	0.86 ± 0.03 cdyz	0.94 ± 0.01 bz
	VP	0.84 ± 0.05 dex	0.95 ± 0.02 cdex	1.01 ± 0.09 bcx	1.03 ± 0.08 bcdx	1.24 ± 0.28 bx
IW15f/1.0s	AP	1.15 ± 0.10 ax	1.69 ± 0.10 ax	1.71 ± 0.14 abx	1.93 ± 0.29 bcx	2.03 ± 0.29 abx
	MAP80/20	1.08 ± 0.04 abcx	1.14 ± 0.06 bcdxy	1.32 ± 0.03 bcyz	1.33 ± 0.06 bcdyz	1.49 ± 0.03 bz
	MAP70/30	0.87 ± 0.01 cdex	0.89 ± 0.02 defx	1.25 ± 0.08 bcyz	1.16 ± 0.01 bcdy	1.38 ± 0.03 bz
	VP	1.10 ± 0.04 abx	1.36 ± 0.07 bxy	1.69 ± 0.14 abxy	1.75 ± 0.13 bcdxy	1.91 ± 0.32 aby
Overall effects (p values) of batch on seaweed sausages’ lipid oxidation.
	Batch	0.050 *	0.145 ns	0.076 ns	0.032 *	0.066 ns

^abcdef Effect of packaging: within each treatment, mean values (±standard error) in the same column bearing different superscripts are significantly different (p < 0.05). ^xyz Effect of storage time (days): mean values in the same row bearing different superscripts are significantly different (p < 0.05). p values: ^ns—not significant; * p < 0.05–0.01 (significant); AP—aerobic packaging; MAP80/20—80% O₂ 20% CO₂; MAP70/30—70% N₂ 30% CO₂; VP—vacuum packaging. SS = sea spaghetti; IW = Irish wakame; C = control; f = fat; s = salt.

. The lipid oxidation results were influenced by the batch on day 1 and 20. In the control treatment, on days 6, 13, and 20, AP-stored samples had higher (p < 0.05) levels of lipid oxidation than the other packaging systems used in this study. The presence of high oxygen in the AP-stored control samples accelerated the lipid oxidation, as oxygen is a major factor in the lipid oxidation process. On day 27, the MAP70/30 control samples had lower (p < 0.05) TBARS values than the AP-stored and VP control sausage samples. In the SS10f/0.5s treatment group, no significant differences were observed between packaging conditions on days 1, 6, 13, and 20. In the IW15f/1.0s sausage samples stored in MAP70/30, lower (p < 0.05) TBARS values were observed than in the AP-stored and VP samples.

The lipid oxidation values of the control, SS10f/0.5s, and IW15f/1.0s ranged from 0.65 to 3.33 mg MDA/kg sausage, 0.70 to 1.36 mg MDA/kg sausage, and 0.87 to 2.03 mg MDA/kg sausage, respectively, stored under AP, MAP80/20, MAP70/30, and VP conditions. The minimum threshold required to detect negative changes, such as rancidity, in the organoleptic characteristics of meat products is approximately 2.0 mg MDA/kg of meat [56]. In the control (C25f/2.0s) group, AP, MAP80/20, and VP treatments exceeded this threshold on days 13, 27, and 20, respectively. The MAP70/30-stored control samples were still below the threshold on day 27 of storage. The threshold was not exceeded in the SS10f/0.5s sausage samples; however, IW15f/1.0s stored under AP condition exceeded this threshold on day 27. Samples exceeding this limit (2.0 mg MDA/kg) may also possess negative characteristics such as generation of odour, colour change, and changes in taste and texture, negatively altering functional qualities such as protein solubility and water-holding capacity and lowering the bioavailability of some nutrients [57].

Storage of meat in high-oxygen atmospheres (up to 80% oxygen) is useful for maintaining colour but can degrade meat quality over time [58,59]. A naturally occurring pigment known as myoglobin imparts a purplish colour in fresh meat. However, when myoglobin is exposed to air, it reacts with oxygen to generate bright-red oxymyoglobin, which is the colour consumers associate with fresh meat. Further exposure to oxygen transforms oxymyoglobin into brownish metmyoglobin, which has much lower consumer appeal, despite not indicating spoilage. Nitrogen used in MAP70/30 reduces lipid oxidation and pack collapse [60]. Hence, MAP70/30-stored sausages had lower lipid oxidation values.

3.2.2. Microbiological Analysis

Salmonella and E. coli remained absent throughout the storage period in all sausage sample treatments (

Table 8. TVC (log₁₀ CFU/g) of control and selected seaweed sausages during storage at 4 °C.

Treatment	Packaging	Storage Time at 4 °C (days)
		1	6	13	20	27
C25f/2.0s (Control)	AP	4.09 ± 0.02 ax	4.27 ± 0.03 axy	5.01 ± 0.05 axy	5.50 ± 0.40 ay	6.88 ± 0.47 az
	MAP80/20	4.06 ± 0.15 ax	4.15 ± 0.03 axy	4.18 ± 0.39 axy	4.22 ± 0.37 axy	5.44 ± 0.32 ay
	MAP70/30	4.08 ± 0.29 ax	4.20 ± 0.50 ax	4.16 ± 0.48 ax	4.35 ± 0.42 ax	5.11 ± 0.56 ax
	VP	4.18 ± 0.02 ax	4.18 ± 0.06 ax	4.09 ± 0.04 ax	4.89 ± 0.68 ax	5.16 ± 0.15 ax
SS10f/0.5s	AP	4.10 ± 0.12 ax	4.80 ± 0.79 ax	5.30 ± 0.75 ax	6.56 ± 0.86 ax	6.23 ± 0.06 ax
	MAP80/20	4.09 ± 0.13 ax	4.01 ± 0.20 ax	4.14 ± 0.55 ax	4.59 ± 0.94 ax	5.34 ± 0.73 ax
	MAP70/30	4.10 ± 0.45 ax	4.08 ± 0.04 ax	4.12 ± 0.17 ax	4.12 ± 0.36 ax	5.29 ± 0.66 ax
	VP	4.03 ± 0.13 ax	4.05 ± 0.13 ax	4.12 ± 0.32 ax	4.35 ± 0.11 ax	4.85 ± 0.25 ax
IW15f/1.0s	AP	4.13 ± 0.12 ax	5.00 ± 0.61 axy	5.25 ± 0.00 axy	6.54 ± 0.86 ay	6.38 ± 0.08 ay
	MAP80/20	4.06 ± 0.11 ax	3.97 ± 0.08 ax	4.54 ± 0.93 ax	5.09 ± 0.91 ax	5.08 ± 0.99 ax
	MAP70/30	4.10 ± 0.87 ax	3.97 ± 0.09 ax	4.08 ± 0.09 ax	4.54 ± 0.32 ax	5.05 ± 0.02 ax
	VP	4.07 ± 0.12 ax	4.07 ± 0.11 ax	4.05 ± 0.16 ax	4.14 ± 0.12 ax	4.60 ± 0.30 ax
Overall effects (p values) of batch on seaweed sausages’ TVC.
	Batch	<0.001 ***	0.013 *	0.003 **	0.078 ns	0.024 *

^a Effect of packaging: within each treatment, mean values (±standard error) in the same column bearing different superscripts are significantly different (p < 0.05). ^xyz Effect of storage time (days): mean values in the same row bearing different superscripts are significantly different (p < 0.05). p values: ^ns—not significant; * p < 0.05–0.01 (significant); ** p < 0.01–0.001 (highly significant); *** p < 0.001 (extremely significant). AP—aerobic packaging; MAP80/20—80% O₂ 20% CO₂; MAP70/30—70% N₂ 30% CO₂; VP—vacuum packaging. SS = sea spaghetti; IW = Irish wakame; s = salt; f = fat.

). No differences (p > 0.05) in the TVC were observed between packaging for each treatment and storage day. The microbiological results were influenced by the batch on day 1, 6, 13, and 27.

The Commission Regulation No. 2073/2005 set the acceptability limits applied to minced and mechanically separated meat examined at the end of the manufacturing process and are as follows: total viable counts (TVC): <5 × 10⁶ CFU/g (log₁₀ 6) of the product; E. coli: <500 CFU/g (log₁₀ 2) of product; Salmonella: absent in 10 g of product throughout the shelf-life [61]. According to these limits, the control and seaweed sausages (SS10f/0.5s and IW15f/1.0s) stored under AP exceeded the limit of acceptability (log₁₀ 6) on days 27 and 20, respectively (Table 8). These results suggest that salt reduction significantly contributes to the microbial stability of seaweed-containing sausages. The salt reduction was expected to increase spoilage without additional preservative factors, i.e., packaging. Salt has a preservative effect on processed meat by lowering water activity, which helps control microbial growth. The reduction can have a negative impact on the shelf-life [13]. The direct toxicity of Cl⁻, the elimination of oxygen from the media, the organisms’ sensitization to CO₂, and the disruption of the fast activity of proteolytic enzymes are among the elements contributing to the preservation qualities of NaCl [62].

On day 27 (end of storage), the TVC of each treatment stored under MAP80/20, MAP70/30, and VP did not exceed the acceptable limit. MAP and VP are known to greatly contribute to preserving and extending meat products’ shelf-life, due to their reduced oxygen content, in comparison to AP.

The French guidelines state the acceptable limit for dried seaweed products, as follows: TVC: ≤105 CFU/g (log₁₀ 5) of the product; faecal coliforms: ≤10; Salmonella: absent in 25 g of product throughout the shelf-life [63]. According to these guidelines for dried seaweed products, SS10f/0.5s stored under AP and MAP (MAP80/20 and MAP70/30) surpassed the recommended limits for TVC by day 13 and 27, respectively. Irish wakame sausage (IW15f/1.0s) stored under AP, MAP80/20, and MAP70/30 reached this acceptable limit on days 6, 20, and 27, respectively. These results suggest that the high oxygen in the aerobic conditions accelerated bacteria growth.

4. Conclusions

The study’s analysis highlights the effects of incorporating brown seaweeds, sea spaghetti (SS), and Irish wakame (IW) into reduced-fat and salt pork sausages. Significant impacts were observed on the sausages’ proximate composition, texture, and sensory properties. SS treatments were well-received by consumers, while IW treatments had lower sensory scores due to their having a stronger odour. The selected formulations were SS10f/0.5s and IW15f/1.0s. Shelf-life stability showed that MAP70/30 packaging was effective in preventing lipid oxidation, and vacuum packaging maintained acceptable microbiological levels. This suggests SS and IW as viable ingredients for enhancing nutritional value and creating novel sausage variations.