1. Introduction
Sausages became one of the most favored meat products in the world thanks to their nutritional value and attractive sensory profiles. However, emulsified sausages generally contain high fat contents (20–30%), which are associated with obesity, high blood pressure, cardiovascular, non-alcoholic fatty liver diseases and other chronic diseases [
1,
2]. The recommended dosage of fat intake in the diet is 15–30%, and the healthy intake of saturated fatty acids should be less than 10% [
3]. Based on the recent increase in demand for healthier diets, the production of reduced-fat sausages with a healthier nutritional composition has become a hot topic in the meat products industry. However, reducing the fat content in meat products without considering composition can lead to a decrease in the flavor and quality of meat products with poor texture parameters [
4]. Therefore, to meet current consumer needs, developing suitable fat replacers to replace animal fat in meat products without sacrificing sensory and textural quality is a major challenge for meat industries.
At present, fat replacers are mainly divided into the three categories: fat-based, carbohydrate-based, and protein-based products [
5]. Various types of plant-based oil emulsions have been applied as fat replacers in processed reduced-fat meat products. Meanwhile, plant-based oils, such as soy, olive, peanut, tea seed, coconut, palm and maize oils, exhibit a positive impact on nutritional composition by reducing cholesterol content and improving fatty acid composition [
6]. However, the applications of the oil-in-water emulsions are limited in food systems due to physical instability, soft texture and oxidation problems [
7]. To overcome these problems, several strategies have been developed, including the utilization of proteins (soy proteins, sodium caseinate, whey proteins, corn germ protein), carbohydrates (starches, konjac glucomannan, cellulose, inulin, oat fiber, polysaccharide), hydrocolloids (xanthan, locust bean gum) and even natural antioxidants (curcumin, carotenes, annatto bixin, capsorubin) [
8,
9,
10]. Zhang et al. [
11] used water-soluble β-glucan to make low-fat sausages with the presence of peanut protein isolate or soy protein isolate, which significantly improved the water-holding capability and increased the hardness, chewiness and cohesiveness of prepared sausages. Li et al. [
12] reported that the utilization of stabilized emulsion containing zein/carboxymethyl dextrin and carrageenan could enhanced the texture and improved the oxidative stability of sausages. Therefore, composite-based emulsions display better functionality than those with a single matrix, such as protein–polysaccharide composite emulsion, in which polysaccharides and proteins could be combined via the complexation process to improve the texture and network structures [
13].
Mannoproteins are glycoproteins mainly isolated from yeast, which consist of a backbone formed by mannose units linked by α-(1-6) bonds and side chains composed of oligosaccharides [
14]. Mannoproteins mainly contain 80–90% of carbohydrates and 5–20% of protein, and the contained essential amino acids are higher than those of plant proteins [
15,
16]. Mannoproteins have found great applications in the wine making industry for their techno-functional properties, such as their reduction of free ochratoxins and phenolic compounds, their growth promotion of malolactic bacteria and their limitation of tartrate salt crystallization [
17]. Due to their amphiphilic nature, i.e., their consisting of hydrophobic proteins and hydrophilic mannose polymers, mannoproteins have been regarded as effective bio-emulsifiers, widely used in several food applications, such as French salad dressing [
18]. Ashraf et al. [
19] reported that mannoproteins exhibited an effective emulsifying property compared to whey protein concentrate under a broad range of conditions. Furthermore, mannoproteins are regarded as a novel prebiotic food ingredient, and recent studies reported that mannoproteins show anticancer, antioxidant, antimicrobial and prebiotic properties [
20,
21], which exhibit positive effects on obesity and gut microbiota dysbiosis induced by high-fat diets [
22]. These results indicate that mannoproteins present great potential in food applications due to their excellent emulsifying and prebiotic properties.
In our previous study, mannoprotein MP112 was produced based on a gentle and environmentally friendly approach from baker’s yeast using a myxobacterial β-1,6-glucanase GluM, which exhibited high purity of 90% and molecular weight of 112.7 kDa [
23]. The starting materials and isolation methods determine the composition and structure of mannoproteins, resulting in different functional properties [
24,
25]. Mannoprotein MP112 exhibited different chemical and morphological characterizations, with higher content of hydrophilic residues and 4.14% methionine, and had favorable emulsifying and emulsion-stabilizing properties. The application of MP112-stabilized emulsion (6% mannoprotein MP112 content in emulsion formulation) in myofibrillar protein composite gel could form a fine-stranded gel network and thereby improve the strength and water holding ability of composite gels.
Currently, whether mannoprotein-based emulsion could be applied as a fat replacer in reduced-fat sausage production is unknow. Therefore, the goal of this study is to use the MP112-stabilized emulsion to reduce fat content in sausages and evaluate its effect on the nutritional composition, oxidative stability and textural and sensory quality of emulsified sausages.
2. Materials and Methods
2.1. Preparation of MP112 Emulsion
According to the method reported by Qiao et al. [
26], mannoprotein MP112 was prepared from baker’s yeast by the β-1,6-glucanase GluM enzymolysis (
Figure S1). According to the published emulsion formulation in our previous studies [
23], the emulsion with the addition of mannoprotein MP112 (MP112 emulsion) was prepared as described below. A total of 5 g sodium caseinate was dissolved in 50 mL deionized water, followed by the addition of same mass ratio olive oil blend (Betis, Torres Y Ribelles S.A.), which contains 20% olive oil and 80% sunflower oil. Then, these mixtures were vortexed to homogeneity in ice water bath using homogeneous mixer (Ultra Turrax T-25 Basic, IKA Co., Staufen, Germany). A total of 6 g mannoprotein and appropriate deionized water was added into the prepared solutions with vortexing at 15,000 rpm three times (1 min each time at an interval of 30 s) to a constant volume of 100 mL. The mass fraction of the oil phase, sodium caseinate and mannoprotein in emulsion was 50%, 5% and 6% (
w/v), respectively. The preparation of MP112 emulsion was replicated thrice.
2.2. Reduced-Fat Emulsified Sausage Making
The ratio of lean fat in traditional emulsified sausage was 70:30 (wt/wt). A total of 24% pork backfat was added in control samples (ME0), while in other formulations, pork backfat was substituted with MP112 emulsion at levels of 25% (ME25), 50% (ME50), 75% (ME75) and 100% (ME100). Reduced-fat emulsified sausages with different replacement ratio of MP112 emulsion were prepared according to the designed formulations shown in
Table 1. Pork leg muscle (all visible fat and connective tissue were trimmed) and back fat were purchased from a local commercial meat processing company (Sushi Group, Nanjing, China); the obtained pigs were slaughtered according to the requirements of National Standards of China “Pig Slaughter and Quarantine Regulations”. The required amount of pork leg muscle, salt, sugar, phosphate, and pepper were added and chopped at high speed with 1/3 iced water for 1.5 min using a vacuum chopper (BZBJ-40, Hangzhou Aibo Technology Engineering Ltd., Hangzhou, China). The pork back fat was added and chopped for 1.5 min with 1/3 the iced water, followed by addition of the MP112 emulsion with the rest of the iced water chopping for 1.5 min. The temperature of all meat batter samples was kept below 16 °C.
The mixtures were stuffed into hog gut casings (24 mm diameter) using a sausage stuffer (EB-12, Mainca UK Ltd., Berks, PA, USA) to form sausages (60 g, 15 cm long), followed by cooking at low temperature of 80 °C in a water bath for 40 min with an internal temperature of around 74 °C. Sausages were then taken and cooled to room temperature with plastic bag packaging. The production process of sausages was replicated thrice. For subsequent analysis, sausages of each treatment were kept at 4 °C without access to light and randomly taken within a week of sausage production.
2.3. Physicochemical Analysis
Color measurements: After equilibration to room temperature for 30 min, color parameters were assessed on the fresh surface of 2 cm thick sausage slices at room temperature using a CR-400 colorimeter (Minolta Camera Co., Osaka, Japan) and Illuminate C, calibrated with a white plate. Color parameters from six replicate samples (L*, lightness; a*, redness; b*, yellowness) were recorded using the average values of four measurements taken from different locations on each sausage slice.
Cooking loss and proximate analysis: The cooking loss of emulsified sausages from different treatments was determined by recording the weight difference between the uncooked and cooked sausages, which was expressed as a percentage of initial weight. Each sausage sample weight was measured after equilibration to room temperature.
Crude protein, fat, moisture and ash content were determined by referring to the AOAC official methods [
27]. The moisture of sample content was determined by drying at 105 °C in an oven until a constant weight, and the crude protein content of the samples was determined using the Kjeldahl method (Kjeltec™ 8000, Foss Co., Hillerod, Denmark). The conversion factor of total nitrogen converted to the crude protein was designed as 6.25. Crude fat content was determined using a Soxhlet system (GY-ZFCDY-6P Soxhlet extractor, Shanghai Guiyong Co., Shanghai, China) after ether extraction. Ash content was determined by burning the samples at 550 °C in a muffle furnace for 8 h.
Texture profile analysis: After equilibration to room temperature for 30 min, sausages were cut into cylindrical segments with flat cut surfaces (diameter 24 mm, height 10 mm) and subjected to texture profile analysis using TA. XT PlusC (Stable Micro Systems Ltd., Goldaming, UK), equipped with a 50 mm diameter cylindrical probe (P/50). The parameters are used as follows: the test speed, 2 mm/s; compression rate, 50%; the return speed, 5 mm/s. Texture parameters (hardness, springiness, cohesiveness and chewiness) were recorded with six repetitions.
2.4. Fatty Acid Analysis
The fat of emulsified sausages was extracted with a chloroform-methanol (2:1
v/v) solution as previously described [
28]. After saponification treatment in 0.5 mol/L sodium hydroxide methanol solution at 60 °C for 30 min, fatty acids were then converted into methyl esters with 14% boron trifluoride methanol solution. Then, fatty acid methyl esters were dissolved in 1.5 mL of hexane. Fatty acid composition was analyzed by a gas chromatograph (Trace GC Ultra, Thermo Fisher Scientific Inc., Cleveland, OH, USA) and a 100-m capillary column (SP 2560, Supelco, Bellefonte, PA, USA) with a split ratio of 100:1. The temperature program was designed as follows: ramped oven temperature at 140 °C for 2 min, followed by increasing to 225 °C at 5 °C/min and maintained at 225 °C for 17 min; the inlet temperature and the detector temperature was 240 °C. The injection volume was 1 μL with nitrogen as carrier gas. The fatty acid methyl esters of samples were identified by comparing the retention times to standard fatty acid methyl ester mixtures (Supelco 37 Component FAME mix, Sigma, Bellefonte, PA, USA).
2.5. Lipid Oxidation Measurement
For analysis of the evolution of lipid oxidation, emulsified sausage samples were implemented in triplicate from each batch from 0, 3, and 7 days stored at 4 °C. Lipid oxidation was analyzed by the 2-thiobarbituric acid-reactive substances (TBARS) method with minor modifications [
29]. Samples (±5 g each) were minced by the vacuum chopper and vortex-mixed with 25 mL of 7.5% TCA solution containing 0.1% EDTA. A total of 2 mL of the supernatant was aspirated, followed by mixing with 2 mL 0.02 mol/L MDA and heating at 95 °C for 30 min. After cooling to room temperature, the absorbance of the clear pink layer was recorded at 532 nm using a spectrophotometer UV Mini 1240 (Shimadzu Corp., Kyoto, Japan), and the treatments without the sample were used as control. The result was expressed in milligram of malondialdehyde (MDA) per kg of meat.
2.6. Sensory Evaluation
Sensory evaluation was performed by 12 trained panelists from Nanjing Agricultural University, consisting of 5 males and 7 females (age range 23 to 45) in a quiet room under a mixture of fluorescent and natural light without communication. Sausage samples were warmed on the plate after being cut into pieces and encoded with random numbers. Each panelist was required to use distilled water for gargling between each sample evaluation. A 9-point hedonic scale was used to assessed the appearance, flavor, texture, juiciness and overall acceptability of sausage samples from different treatments with a numerical value ranging from 1 = dislike extremely to 9 = like extremely [
30]. After deducting the outliers, the sensory score was averaged.
2.7. Statistical Analysis
Data were analyzed using the SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) by one-way ANOVA. ANOVA mean comparisons were performed in terms of the Duncan’s multiple-range test at the significance level of p < 0.05. The correlations among variables were determined by two-tailed Pearson’s correlation coefficient.