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). 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 (AgNO
3) solution was standardised against 20 mL of 0.1% sodium chloride (NaCl) to confirm the molarity of AgNO
3 (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:
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:
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:
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 cm
3/m
2/24 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% O
2: 20% CO
2 (MAP80/20) or 70% N
2: 30% CO
2 (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.