Screening, Identification, and Application of Superior Starter Cultures for Fermented Sausage Production from Traditional Meat Products

Abstract

In this study, 43 strains of Staphylococcus spp. and 22 strains of lactic acid bacteria (LAB), isolated from six representative fermented meat products (domestic and international), were subjected to a comprehensive safety evaluation, including hemolytic activity, catalase test, hydrogen sulfide production, and antibiotic susceptibility screening. Nine strains were selected for secondary screening based on safety criteria, fermentation characteristics, and acid and salt tolerance tests. Two optimal strains were identified—Staphylococcus saprophyticus LH-5 and Latilactobacillus sakei OFN-11—demonstrating excellent compatibility and no mutual antagonism. Both strains were non hemolytic, catalase positive, susceptible to some of the antibiotic tested, and did not produce hydrogen sulfide, mucus, or gas. These favorable fermentation characteristics included lipase/protease production, amino acid decarboxylase negativity, and salt and acid tolerance. Application experiments in fermented sausages were analyzed for 55 volatile compounds, related to meaty, fruity, and fatty aroma profiles compared to commercial starter cultures. The formulation including the selected strains exhibited lower acidity than its commercial unterparts while maintaining superior sensory and physicochemical attributes. These findings suggest that the S. saprophyticus LH-5 and L. sakei OFN-11 consortium holds promising potential as a starter culture for fermented meat products, offering technological advantages to become a fermentation agent that meets the preferences of Chinese consumers.

1. Introduction

Fermented meat products are highly valued by consumers worldwide due to their unique flavor profiles and significant nutritional benefits [1]. Under natural or controlled conditions, these products undergo microbial fermentation, which enhances their distinctive flavor, color, and texture, while extending shelf stability [2,3]. The specific fermentation environment contributes significantly to the unique flavor profiles of fermented meat products. Multiple variations in terms of ingredients and processing are found around the world, thus generating a large variety of fermented meat sausages like the well-known salami, salchichón, saussiçon sec, and chorizo in Europe, and summer sausage and pepperoni in the USA [4]. Typical examples include Jinhua ham and Cantonese-style cured sausage, both widely favored by consumers [5]. The production process generally involves natural fermentation, post-fermentation, and drying, with fermentation durations often exceeding one year. These products harbor complex microbial communities, where bacteria, yeasts, and molds play essential roles in the fermentation process. Among these microorganisms, LAB and Staphylococcus spp. are predominant [6,7]. LAB rapidly produce acid, lowering the pH and inhibiting the growth of pathogenic and spoilage microorganisms. They also metabolize flavor compounds such as esters and alcohols, thereby contributing to the distinctive flavors of the product [8]. Staphylococcus spp. also play a crucial role in flavor and color development in fermented meat products while reducing biogenic amine content. During growth and metabolism, these bacteria break down fats into free fatty acids and ester compounds while degrading proteins into peptides and free amino acids, enhancing nutrient bioavailability for human absorption [9]. Furthermore, Staphylococcus spp. produce nitrate/nitrite reductases during their metabolism, facilitating the reduction of nitrate and nitrite to nitroso groups. These nitroso groups bind to myoglobin, forming stable, bright-red nitroso–myoglobin, contributing to color stabilization in fermented meat products [10,11].

Traditional fermented meat products primarily rely on natural fermentation conditions, making it difficult to scientifically monitor microbial growth dynamics during production. This phenomenon often results in inconsistent product quality and safety [12]. Consequently, the application of starter cultures (selected microbial inoculants) has shown promising potential for enhancing traditional fermentation processes [13,14]. Globally, the fermented meat industry has transitioned from spontaneous fermentation to targeted microbial inoculation followed by controlled fermentation [15]. Representative examples include Spanish fermented sausages, Italian fermented sausages and Belgian fermented sausages [16]. The predominant starter cultures typically consist of LAB and coagulase-negative Staphylococci (CoNS), with process parameters such as temperature, humidity, and fermentation duration varying depending on product specifications [17].

Starter culture-fermented meat products have yet to gain significant market traction in China due to flavor profiles that diverge from traditional Chinese dietary preferences. This study aims to address this challenge by screening superior microbial strains capable of producing fermented meat products that better align with Chinese consumers’ taste preferences while imparting distinct fermentation characteristics. The key research objectives included evaluating strain safety, fermentation characteristics, and functional properties. Furthermore, comparative analyses were conducted to assess differences in physicochemical properties, sensory attributes, and flavor profiles between sausages inoculated with the selected strains and those using commercial starter cultures. The findings are expected to provide a theoretical foundation for developing fermented meat products that align with Chinese consumer preferences.

2. Materials and Methods

2.1. Test Materials

2.1.1. Selection of Fermented Meat Samples and Isolation of Isolates

Six top-selling fermented meat products were purchased from a supermarket in Luohe, China: Jinhua Ham (Jinhua Ham Co., Ltd., Jinhua, China), Xuanwei Ham (Xuanwei Puji Ham Food Co., Ltd., Xuanwei, China), Pepperoni Salami Slices (Ma’anshan Bairui Food Co., Ltd., Ma’anshan, China), Spanish Espetec Salami (S.A. Co., Ltd., Vic, Spain), French Saucisson Sec Salami (Bastides Co., Ltd., Rodez, France), Spanish-style Salami (Shandong Xinjing Western Food Co., Ltd., Linyi, China). Among them, Jinhua ham and Xuanwei ham were fermented meat products without a starter culture, while the other four samples had fermenter added directly during the production process. Under aseptic conditions, the fermented meat sample was aseptically minced, and 5 g of the homogenized sample was transferred into 45 mL of sterile saline solution (0.85% w/v NaCl). The mixture was homogenized using a stomacher homogenizer, followed by serial decimal dilutions. Aliquots of appropriate dilutions were uniformly spread onto MRS Agar (de Man, Rogosa and Sharpe Agar) and TSA (Tryptic Soy Agar) (Merck, Darmstadt, Germany) plates using the spread-plate technique. All plates were incubated at 37 °C for 48 h. Distinct single colonies were isolated through iterative streak-plate purification until axenic cultures were obtained. Purified strains were preserved as LB broth (Luria-Bertani) (Merck, Darmstadt, Germany) stock cultures and cryopreserved in 20% (v/v) glycerol stocks stored at −80 °C.

2.1.2. 16S rRNA Genetic Analysis

Single colonies purified from MRS Agar and TSA plates were individually selected. Genomic DNA was extracted from each strain using an Omega DNA/RNA/protein isolation kit (Omega, Beijing, China) according to the manufacturer’s protocol, the DNA extraction procedure strictly followed the manufacturer’s instructions. Polymerase chain reaction (PCR) amplification was subsequently performed with universal bacterial 16S rDNA primers (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R: 5′-GGTTACCTTGTTACGACTT-3′) targeting the hypervariable regions of the 16S ribosomal RNA gene [18]. The amplified products were then analyzed by Sangon Biotech Co., Ltd., Shanghai, China. The sequence similarities of each contig were assessed by comparing their homologies in the GenBank database using Basic local alignment search tool (BLAST) (http://www.ncbi.nlm.nih.gov accessed on 25 October 2024), and analyzed for homology and constructed a phylogenetic tree using MEGA-X software (version 10.0.5), genetic similarity threshold of 95% for classification of bacterial species.

2.1.3. Preliminary Safety Screening of Isolates

The purified strains after Gram staining were identified for cell morphology using light microscopy. Safety screening of the selected strains was conducted through the following sequential assessments:

Mucoid phenotype assay: The single colony after purification was picked and streaked into TSA medium, which was incubated at 37 °C for 24 h. Single colonies were observed and recorded by picking with an inoculating needle; the presence of mucus was considered positive, and the absence of mucus production was considered negative.

Catalase test [19]: Bacterial cultures that were in the stationary phase were placed on slides containing 5% H₂O₂, and the absence of visible bubbles within 2 min was considered negative.

Hydrogen sulfide production test [19]: The above purified strains were respectively inoculated into lead acetate medium by stabbing. After being cultured in a biochemical incubator at 37 °C for 24 h, they were inspected, and the appearance of a black precipitate was considered positive, while its absence was considered negative.

Strain hemolytic assay [20]: The activated bacterial strain was streak-inoculated onto Columbia blood agar plates and cultured at 37 °C for 24 h. Colonies were observed for the presence of transparent hemolytic zones around them. The result was recorded as positive if clear hemolytic zones were observed, and negative if no hemolysis was detected.

Antibiotic sensitivity test [21]: The disk diffusion method on agar was used to conduct the antibiotic sensitivity experiment of common antibiotics for the strains. The strains were activated overnight in LB broth, then centrifuged at 6000 r/min for 10 min. After that, the supernatant was discarded, McFarland turbidity standards were used for inoculum preparation, and the pellet was diluted with normal saline to about 1 × 10⁷ CFU/mL. A total of 100 μL of the diluted suspensions of Staphylococcus spp. and LAB were spread on TSA and MRS solid media, respectively. Each antibiotic sensitivity disk was pasted on the surface of the medium, the types of antibiotics and antibiotic concentrations tested are shown in

Table 1. Antibiotic type and concentration.

Antibiotic	Concentration (μg /mL)
Erythromycin	150
Penicillin	100
Tetracycline	300
Ampicillin	100
Ciprofloxacin	50
Gentamicin	1200
Lincomycin	20
Ceftriaxone	300
Chloramphenicol	300
Cotrimoxazole	250

, each piece of paper contains a corresponding 0.1 mL. After culturing at 37 °C for 24 h, the diameter of the inhibition zone was measured. According to the size of the diameter, the results were classified into resistant (R), intermediate (I), and sensitive (S), and the antibiotic sensitivity was specifically determined according to the National Committee for Clinical Laboratory Standards (NCCLS) antibiotic sensitivity standard.

2.1.4. Stress and Enzyme Production Experiments of Isolates

Sodium chloride tolerance test [19]: Stationary phase cultures of the aforementioned bacterial strains were inoculated into LB and MRS broths supplemented with different NaCl concentrations (0%, 2%, 4%, and 6%) at a 1% inoculation rate. The cultures were incubated at 37 °C for 48 h, after which the optical density (OD) was measured at 600 nm wavelength.

Nitrite tolerance test [22]: Stationary-phase bacterial cultures (1% v/v) were inoculated into LB and MRS broths supplemented with different sodium nitrite (NaNO₂) concentrations (0, 50, 100, and 150 mg/kg). The cultures were incubated at 37 °C for 48 h, followed by measurement of optical density (OD) at 600 nm wavelength.

Bile salt resistance test: Stationary-phase bacterial cultures (1% v/v) were inoculated into LB and MRS broths containing varying concentrations of porcine bile salts (0%, 0.1%, 0.2%, and 0.4%). After 48 h incubation at 37 °C, the optical density (OD) was recorded at 600 nm wavelength.

Proteolytic activity assay [23]: Selected bacterial strains were streaked onto TSA agar medium containing 15% skim milk and MRS solid medium. The plates were incubated at 30 °C for 48 h. Colonies surrounded by transparent halos were considered positive.

Lipase-producing activity assay [23]: Neutral red (0.1 g) was dissolved in 20 mL distilled water and filtered through 0.45 μm microporous membranes. Polyvinyl alcohol was heated to prepare a 3% aqueous solution, which was mixed with olive oil (3:1 w/w) and emulsified using a high-speed emulsifier. The emulsion was combined with TSA/MRS agar (9:1 w/w) supplemented with neutral red indicator (1% w/v). After sterilization at 121 °C for 15 min, the media were poured into Petri dishes. The screened strains were streaked onto the prepared medium and incubated at 37 °C for 24 h. The appearance of red spots around colonies was interpreted as positive.

Amino acid decarboxylase assay [23]: Bromocresol violet (0.001 g/L) was incorporated into LB and MRS broth with respective additions of arginine (0.25% w/v), lysine (0.25% w/v), and tyrosine (0.5% w/v). Following sterilization at 121 °C for 15 min, the medium was dispensed into sterile tubes. Stationary-phase bacterial suspensions were inoculated into the medium, which was overlaid with sterile liquid paraffin. After 5-day incubation at 37 °C, color changes were evaluated: purple coloration was recorded as positive and yellow as negative, with amino acid-free medium serving as control.

Casein hydrolysis assay [24]: Stationary-phase bacterial suspensions (10 μL) were spot-inoculated onto casein agar plates. Following 48 h incubation at 37 °C, the formation of clear zones (hydrolysis zones) around colonies was observed, with their presence regarded as positive.

Gas production from glucose experiments: Bacterial strains were inoculated into glucose broth tubes containing inverted Durham tubes. After 48 h incubation at 37 °C, gas bubble accumulation in Durham tubes was considered indicative of positive results.

2.1.5. Growth Characterization and Acid Tolerance Tests of Isolates

The screened Staphylococcus strains were inoculated into LB broth and incubated at 37 °C with shaking at 180 r/min, while the LAB were inoculated into MRS broth and incubated at 37 °C under static conditions. Using the respective uninoculated liquid media as controls, the pH and optical density (OD) at 600 nm of the bacterial cultures were measured.

The screened Staphylococcus strains and LAB were inoculated into LB broth (for Staphylococcus) and MRS broth (for LAB) adjusted to pH 3.0, 3.5, 4.0, 4.5, and 5.0 to ensure that the OD_600nm of the liquid medium reaches approximately 0.2 after inoculation with each bacterial suspension. After incubation at 37 °C for 24 h, the OD_600nm of the cultures was measured.

2.1.6. Inter-Strain Antagonism Assay

Stationary-phase bacterial cultures were streak-inoculated linearly onto TSA, after colony formation, a secondary bacterial suspension was cross-streaked perpendicularly to the primary colonies using an inoculation loop. Plates were incubated at 37 °C for an additional 24 h to observe antibacterial activity (e.g., inhibition zones) between LAB and Staphylococcus strains. No antagonistic activity (colonies grow overlapped) if the two bacteria are cultured with a clear crosshatch, weak antagonistic activity (partial overlapping of colonies) if there is a clear line of colonies at the crosshatch and another line of colonies is not visible, and antagonistic activity (colonies cannot grow overlapped) if there is only one clear line of colonies at the crosshatch or no clear line at all.

2.2. Preparation of Fermented Sausages

Fermentation sausage production protocol based on Shao et al. [25], with key technical details:

Pretreatment of raw meat: Pork foreleg meat and pork back fat were selected as raw materials. They were thoroughly washed, cut into pieces, and set aside. Mincing of meat: The weight ratio of pork foreleg meat to pork back fat was adjusted to 7:3; both components were minced into particles of 4–6 mm.

The ingredients were calculated based on the raw meat weight, including 2.0% table salt, 1.8% glucose, 1.5% spices, 0.5% D-sodium erythorbate, and 0.5% monosodium glutamate. The raw meat was thoroughly mixed with the ingredients, followed by the addition of the screened starter culture strains at an inoculation level of 10⁷ CFU/g. The S. saprophyticus LH-5 and L. sakei OFN-11 isolates obtained in Section 2.1 were selected as starter cultures for subsequent fermented sausage experiments. Three distinct mixed starter culture formulations were developed, with strain specifications and inoculation ratios detailed in

Table 2. Inoculation of ingredients and starter culture.

	The Control Group		Self-Screening Strains Group			The Blank Group
	THM-17	VBL-60	Formulation 1	Formulation 2	Formulation 3
Inoculation of ingredients	2.0% of table salt, 1.8% of glucose, 1.5% of spices, 0.5% of D-sodium erythorbate, 0.5% of monosodium glutamate, 0.01% of sodium nitrite		2.0% of table salt, 1.8% of glucose, 1.5% of spices, 0.5% of D-sodium erythorbate, 0.5% of monosodium glutamate
Starter culture	S. xylosus, P. pentosaceus	S. xylosus, S. carnosus, P. pentosaceus, P. acidilactici	S. saprophyticus and L. sakei were inoculated at a 2:1 (v/v) ratio	S. saprophyticus and L. sakei were inoculated at a 1:1 (v/v) ratio	S. saprophyticus and L. sakei were inoculated at a 1:2 (v/v) ratio	None
	Total bacterial concentration of 1 × 107 CFU/g		The combined starter culture after Section 2.1 screening, total bacterial concentration of 1 × 107 CFU/g

.

In the blank group, no starter culture is added, and natural fermentation is carried out. In the control group, the commercial starter cultures, SACCO THM-17 and VBL-60 (SACCO Co., Ltd., Cadorago, Italy) for meat products are used. Commercial starters THM-17 (Staphylococcus xylosus and Pediococcus pentosaceus) and VBL-60 (S. xylosus, Staphylococcus carnosus, P. pentosaceus, and Pediococcus acidilactici) served as controls. The inoculation dosage followed the manufacturer’s instructions, with 20 g of starter added per 100 Kg of meat, ensuring a total bacterial concentration of 1 × 10⁷ CFU/g.

Enema: Thirty-millimeter pig casings were used, with knots tied every 12 cm, and removed the surface water stains and other impurities.

Fermentation and ripening: The sausages were placed in an environment with a constant temperature of 28 °C and a humidity of 90%, and ferment for 24 h. Subsequently, they were transferred to secondary fermentation conditions at 18 °C with 75% humidity for 10 days of ripening.

Packaging: The fermented sausages were removed from the ripening chamber. Surface debris and impurities were eliminated, followed by trimming to uniform dimensions. Finally, the products were vacuum-packed for storage.

2.3. Sensory Evaluation System of Fermented Sausages

According to the sensory evaluation protocol described by Zheng et al. [12], the fermented sausages were assessed through four key indicators: color, texture, odor, and taste. A panel of 30 trained evaluators (15 males and 15 females) participated in the unified evaluation process, and informed consent has been obtained from the participants involved in the sensory assessment. Their ages range from 20 to 70 years and are distributed as follows: 20–35 years (30%), 36–45 years (30%), 46–60 years (20%), and 61–70 years (20%). The sensory evaluation system is scored out of 100 points, divided into four categories: appearance (20 points), texture (20 points), aroma (30 points), and taste (30 points), additional overall rating option of 30 points. Based on preliminary surveys of professional panelists’ preferences, higher scores in each category indicate greater satisfaction with that attribute.

2.4. Determination of Physicochemical Indexes and Texture of Fermented Sausages

The pH values of different fermented sausages were measured using a handheld insertion pH meter (Testo205, Testo, Shanghai, China). Samples were subsequently sent to the Test Center of Food Laboratory of Zhongyuan for the following analyses: Moisture content: Determined gravimetrically according to GB 5009.3-2016 standards [26], China. Total volatile basic nitrogen (TVB-N): Quantified through micro-diffusion assay [27]. Nitrite content: Analyzed via N-(1-naphthyl) ethylenediamine spectrophotometric method (GB 5009.33-2016 [28]), China [29]. Color parameters (L*, a*, b*) were evaluated using a portable colorimeter (RM200QC, X-rite, Grand Rapids, MI, USA), with measurements taken at three randomized surface locations per sample.

For texture analysis the fermented sausages were placed on a chopping board and cut into cubes with a length, width, and height of 1 cm each, using a knife. A texture analyzer (CTX, Brookfield, MA, USA) with a P50 probe was used. The deformation amount was set to 50% and the measurement speed to 1 mm/s, and a two-cycle compression test was conducted [30]. All analyses were performed in triplicate to ensure data reliability.

2.5. Determination of Volatile Substances in Fermented Sausages

The volatile compounds in fermented sausages were analyzed using gas chromatography–ion mobility spectrometry (GC-IMS) following the protocol by Zhang et al. [31]. An amount of 2 g of each fermented sausage sample was accurately weighed into a 20 mL headspace bottle, and 2-octanol was added as internal standard; the sample was incubated at 80 °C for 15 min and then injected into the sample, and three sets of parallel were determined for each sample.

Incubation temperature: The sample was incubated at 80 °C for 15 min before injection—injection volume: 500 µL; incubation speed: 500 r/min; injection needle temperature: 110 °C. Column temperature: 60 °C; programmed ramp-up: initial flow rate of 2.0 mL/min held for 2 min, linearly increased to 10.0 mL/min within 8 min, linearly increased to 100.0 mL/min within 10 min, and then held for 10 min; chromatographic run time: 30 min; inlet temperature: 80 °C. Ionization source: tritium source (3 h); migration tube length: 53 mm; electric field strength: 500 V/cm; migration tube temperature: 45 °C; positive and negative ion modes: positive ion.

A mixed standard of six ketones was analyzed to establish calibration curves for retention time and retention index. The retention index of each target compound was calculated based on its retention time. Qualitative analysis was performed by searching and comparing against the GC retention index database (NIST 2020) and IMS migration time database built into VOCal 5.2.

2.6. Statistical Analysis

Data statistics and processing in this study were performed using Microsoft Excel 2019. Graphs were plotted and a one-way ANOVA analysis (significance level: p < 0.05) was conducted using Origin 2019 and SPSS 25. The principal component analysis (PCA) plugins in VOCal data processing software were utilized to generate fingerprint spectra and PCA plots, respectively, for comparing volatile organic compounds between samples. All experiments were performed in triplicate.

3. Results and Discussion

3.1. Isolation and Characterization of Microorganisms in Fermented Meat Products

A total of 67 isolates with distinct colony morphologies were isolated from six fermented meat products, including two types of ham and four types of salami. Based on 16S rDNA sequencing, the genus distribution was as follows: Staphylococcus (43 strains, 64.2%), Lactobacillus (15 strains, 22.4%), Pediococcus (7 strains, 10.4%), Corynebacterium (1 strain, 1.5%), and Enterococcus (1 strain, 1.5%). The detailed distribution is summarized in

Table 3. Isolates source and identification results of 16S rDNA.

Isolate Source	Isolates	Isolate Code	Isolates Source	Isolates	Isolate Code	Isolates Source	Isolates	Isolate Code
Xuanwei Ham	Staphylococcus saprophyticus	XW-1	Pepperoni Salami Slices	Staphylococcus nepalensis	BB-1	French Saucisson Sec Salami	Latilactobacillus curvatus	OFN-1
	Staphylococcus epidermidis	XW-2		Staphylococcus warneri	BB-2		Latilactobacillus sakei	OFN-2
	Staphylococcus pasteuri	XW-3		Staphylococcus saprophyticus	BB-3		Staphylococcus carnosus	OFN-3
	Staphylococcus pasteuri	XW-4		Staphylococcus pasteuri	BB-4		Latilactobacillus sakei	OFN-4
	Staphylococcus epidermidis	XW-5		Staphylococcus saprophyticus	BB-5		Latilactobacillus sakei	OFN-5
	Corynebacterium minutissimum	XW-6		Staphylococcus saprophyticus	BB-6		Latilactobacillus sakei	OFN-6
	Pediococcus acidilactici	XW-7		Staphylococcus warneri	BB-7		Latilactobacillus sakei	OFN-7
	Pediococcus acidilactici	XW-9		Pediococcus acidilactici	BB-8		Latilactobacillus sakei	OFN-8
	Pediococcus acidilactici	XW-10		Pediococcus acidilactici	BB-9		Latilactobacillus sakei	OFN-9
	Pediococcus acidilactici	XW-11		Staphylococcus epidermidis	BB-10		Latilactobacillus sakei	OFN-10
	Pediococcus acidilactici	XW-12		Staphylococcus warneri	BB-12		Latilactobacillus sakei	OFN-11
	—	—		—	—		Latilactobacillus sakei	OFN-12
Jinhua Ham	Staphylococcus warneri	JH-1	Spanish Espetec Salami	Staphylococcus saprophyticus	LH-1	Spanish-style Salami	Staphylococcus carnosus	YW-1
	Staphylococcus epidermidis	JH-2		Staphylococcus xylosus	LH-2		Staphylococcus carnosus	YW-2
	Staphylococcus saprophyticus	JH-3		Staphylococcus xylosus	LH-3		Staphylococcus condimenti	YW-5
	Staphylococcus epidermidis	JH-4		Staphylococcus saprophyticus	LH-4		Staphylococcus carnosus	YW-6
	Staphylococcus pasteuri	JH-5		Staphylococcus saprophyticus	LH-5		Staphylococcus xylosus	YW-7
	Staphylococcus warneri	JH-6		Staphylococcus xylosus	LH-6		Staphylococcus xylosus	YW-8
	Staphylococcus epidermidis	JH-7		Latilactobacillus sakei	LH-8		Latilactobacillus sakei	YW-9
	Staphylococcus warneri	JH-8		Staphylococcus saprophyticus	LH-9		Staphylococcus saprophyticus	YW-10
	Staphylococcus warneri	JH-9		Staphylococcus carnosus	LH-10		Staphylococcus xylosus	YW-11
	Staphylococcus epidermidis	JH-10		Enterococcus gilvus	LH-11		Latilactobacillus sakei	YW-12
	Staphylococcus warneri	JH-11		Latilactobacillus sakei	LH-12		—	—
	Staphylococcus saprophyticus	JH-12		—	—		—	—

.

As shown in Table 3, Pediococcus acidilactici was dominant in Xuanwei ham. In Jinhua ham, S. warneri was the dominant bacteria species. These results indicated significant differences in the microbial compositions between the ham varieties. In French Saucisson Sec Salami, L. sakei was the dominant bacteria, whereas the other three types of salami were predominantly associated with Staphylococcus spp. However, significant variations in the dominant bacteria strains were observed among the salami samples, possibly due to differences in the starter cultures used during production. The selection of microbial strains plays a critical role in shaping the flavor characteristics of salami [32]. Additionally, the dominant bacteria in traditionally fermented hams (e.g., Xuanwei and Jinhua) differed significantly from those in commercially inoculated salami, suggesting distinct microbial-driven mechanisms of flavor formation. Further research is required to elucidate the relationship between microbial community composition and flavor profiles in these products.

3.2. Results of Preliminary Screening for the Safety of Excellent Fermentation Isolates

The results of the mucus production test showed that three of the 67 strains were mucus-producing, and they were subsequently eliminated. The H₂O₂ catalase test showed that all 43 strains of Staphylococcus spp. isolated from the samples were catalase positive, indicating the presence of the H₂O₂ enzyme; as a result, they were used in subsequent experiments. Additionally, 20 of the 22 LAB, comprising 15 Lactobacillus and 7 Pediococcus, were catalase positive. Among the 67 strains, 29 were hemolysis negative, including 14 Staphylococcus and 15 LAB strains. The hydrogen sulfide test identified nine hydrogen sulfide negative strains, including five Staphylococcus spp. and four LAB strains. After the initial screening for strain safety, nine strains with good fermentation characteristics and no mucus production, hemolysis, or hydrogen sulfide production were selected for subsequent screening. These strains were LH-5, LH-9, YW-1, YW-2, YW-5, OFN-7, OFN-9, OFN-11, and XW-7.

3.3. Rescreening Results of Selected Fermentation Isolates

3.3.1. Salt, Sodium Nitrite, and Bile Salt Tolerance of Isolates

Salt and nitrite tolerance are particularly important characteristics for a microorganism to proliferate and participate in fermentation. Bile tolerance is a key trait for a probiotic microorganism, enabling it to survive, colonize, and exert beneficial effects in the gut.

Fermented meat products generally contain high salt concentrations, making salt tolerance a crucial criterion for selecting starter cultures. As shown in

Table 4. Experimental results of Nacl resistance of isolates.

Isolates	Control Group	2%	4%	6%
LH-5	1.24 ± 0.03 a	1.23 ± 0.04 a	1.21 ± 0.06 b	1.29 ± 0.06 a
LH-9	1.27 ± 0.02 a	1.25 ± 0.04 a	1.28 ± 0.05 a	1.24 ± 0.07 a
YW-1	1.29 ± 0.04 a	1.28 ± 0.07 a	1.28 ± 0.08 a	1.12 ± 0.04 b
YW-2	1.18 ± 0.03 a	1.19 ± 0.03 a	1.14 ± 0.04 a	1.17 ± 0.06 a
YW-5	1.31 ± 0.09 a	1.31 ± 0.06 a	1.26 ± 0.03 b	1.29 ± 0.05 a
OFN-7	1.13 ± 0.02 a	1.14 ± 0.02 a	1.17 ± 0.08 a	1.14 ± 0.03 a
OFN-9	1.15 ± 0.01 a	1.14 ± 0.09 a	1.12 ± 0.02 a	1.15 ± 0.08 a
OFN-11	1.14 ± 0.03 a	1.16 ± 0.03 a	1.15 ± 0.08 a	1.16 ± 0.02 a
XW-7	1.20 ± 0.04 a	1.18 ± 0.09 a	1.20 ± 0.04 a	1.16 ± 0.08 a

Note: Different lowercase letters (a,b) in the same row indicate significant differences between samples. (p < 0.05).

, all nine tested strains exhibited robust growth in environments containing 2%, 4%, and 6% NaCl. At 6% NaCl, the growth performance of all isolates, except YW-1, showed no significant difference compared to the control group.

Nitrite plays a critical role in meat processing by enhancing preservation, stabilizing color, and exhibiting antioxidant activity [33]. As shown in

Table 5. Experimental results of NaNO₂ resistance of isolates.

Isolates	Control Group	50 mg/L	100 mg/L	150 mg/L
LH-5	1.24 ± 0.03 a	1.26 ± 0.02 a	1.20 ± 0.06 a	1.22 ± 0.07 a
LH-9	1.27 ± 0.02 a	1.26 ± 0.07 a	1.28 ± 0.05 a	1.25 ± 0.05 a
YW-1	1.29 ± 0.04 a	1.30 ± 0.06 a	1.24 ± 0.08 b	1.05 ± 0.04 c
YW-2	1.18 ± 0.03 a	1.20 ± 0.03 a	1.18 ± 0.04 a	1.18 ± 0.06 a
YW-5	1.31 ± 0.01 a	1.30 ± 0.07 a	1.28 ± 0.03 a	1.30 ± 0.03 a
OFN-7	1.13 ± 0.02 a	1.14 ± 0.02 a	1.12 ± 0.01 a	1.15 ± 0.02 a
OFN-9	1.15 ± 0.01 a	1.17 ± 0.04 a	1.15 ± 0.02 a	1.17 ± 0.04 a
OFN-11	1.14 ± 0.03 a	1.13 ± 0.03 a	1.14 ± 0.03 a	1.15 ± 0.04 a
XW-7	1.20 ± 0.04 a	1.18 ± 0.04 a	1.21 ± 0.04 a	1.10 ± 0.02 b

Note: Different lowercase letters (a–c) in the same row indicate significant differences between samples. (p < 0.05).

, the nitrite tolerance test revealed that strains YW-1 and XW-7 exhibited a significant decrease in OD values after 24 h of incubation in a liquid medium containing 150 mg/L sodium nitrite. In contrast, the other seven strains exhibited strong tolerance to this nitrite concentration.

Bile salts in animals facilitate the emulsification and absorption of dietary fats while disrupting the cell membranes of certain Gram-positive bacteria, leading to their elimination. Therefore, bile salt tolerance is essential for probiotic colonization in the human gastrointestinal tract [34]. As shown in

Table 6. Experimental results of hog bile salt resistance of isolates.

Isolates	Control Group	0.1%	0.2%	0.4%
LH-5	1.24 ± 0.03 a	1.24 ± 0.05 a	1.10 ± 0.06 b	0.59 ± 0.07 c
LH-9	1.27 ± 0.02 a	1.24 ± 0.06 ab	1.14 ± 0.05 b	0.46 ± 0.10 c
YW-1	1.29 ± 0.04 ab	1.34 ± 0.01 a	1.07 ± 0.05 b	0.64 ± 0.08 c
YW-2	1.18 ± 0.03 a	1.10 ± 0.08 a	1.00 ± 0.09 c	0.28 ± 0.05 d
YW-5	1.31 ± 0.01 a	1.32 ± 0.04 a	1.07 ± 0.05 b	0.42 ± 0.07 c
OFN-7	1.13 ± 0.02 b	1.26 ± 0.03 a	1.10 ± 0.02 c	0.28 ± 0.05 d
OFN-9	1.15 ± 0.01 a	1.15 ± 0.04 a	1.13 ± 0.05 a	0.54 ± 0.05 b
OFN-11	1.14 ± 0.03 b	1.19 ± 0.04 a	1.18 ± 0.07 a	0.51 ± 0.08 c
XW-7	1.20 ± 0.04 a	1.16 ± 0.02 ab	1.19 ± 0.06 a	0.37 ± 0.02 b

Note: Different lowercase letters (a–d) in the same row indicate significant differences between samples. (p < 0.05).

, all the strains showed satisfactory growth, although significant differences were noted at bile salt concentrations of ≤0.2% compared to the control group. However, exposure to 0.4% bile salt resulted in significant OD value reductions across all strains. Notably, YW-2, OFN-7, and XW-7 showed the most significant decreases, with OD values dropping below 0.4.

3.3.2. Rescreening Results of Fermentation Characteristics

Bacterially derived lipases can hydrolyze fats in meat products, releasing fatty acids such as linoleic acid, which play a key role in flavor development. Similarly, proteases degrade meat proteins into free amino acids, significantly enhancing the formation of desirable flavor profiles in fermented meat products [35,36]. Rescreening results showed that seven of the nine tested strains exhibited lipase-producing activity, while six produced proteases, and five showed casein-hydrolyzing ability. In amino acid decarboxylase tests, six isolates tested negative, as they generated an acidic environment during cultivation, potentially inhibiting the growth of pathogenic bacteria. Additionally, gas production by microorganisms in fermented meat products can compromise product quality, texture, and appearance, and even result in the formation of toxic gases. As shown in

Table 7. Results of fermentation characterization rescreening of isolates.

Isolates	Amino Acid Decarboxylase	Protease Activity	Lipase Activity	Casein Hydrolase	Gas Production Assay
LH-5	−	+	+	+	−
LH-9	+	−	+	+	−
YW-1	+	+	+	+	−
YW-2	−	+	+	+	−
YW-5	+	+	+	+	−
OFN-7	−	+	−	−	−
OFN-9	−	+	+	−	−
OFN-11	−	−	+	−	−
XW-7	−	−	−	−	−

Note: “+”: positive, “−”: negative.

, none of the nine strains exhibited gas-producing activity.

Four isolates with excellent fermentation performance and safety profiles were selected based on a comprehensive evaluation of salt tolerance, nitrite tolerance, bile salt tolerance, and fermentation characteristics. These isolates included S. saprophyticus LH-5 (isolated from Spanish Espetec Salami), S. carnosus YW-2 (from Spanish-style Salami), and L. sakei OFN-9 and L. sakei OFN-11 (from French Saucisson Sec salami). These four isolates were chosen for subsequent identification and growth characterization experiments.

3.3.3. Results of Resistance Assay of Isolates Screened for Fermentation Characteristics

The antibiotic resistance of these strains was evaluated using 10 common antibiotics to ensure the safety of the selected strains. As shown in

Table 8. Antibiotic susceptibility of isolates.

Antibiotic	Concentration (/disc)	Isolates
		LH-5	YW-2	OFN-9	OFN-11
Erythromycin	15 μg	I	I	S	S
Penicillin	1 μg	S	S	S	S
Tetracycline	30 μg	R	I	S	S
Ampicillin	10 μg	S	S	I	I
Ciprofloxacin	5 μg	S	S	R	R
Gentamicin	120 μg	R	I	R	R
Lincomycin	2 μg	R	R	S	S
Ceftriaxone	30 μg	S	S	S	S
Chloramphenicol	30 μg	S	S	S	S
Cotrimoxazole	25 μg	R	R	R	R

Note: S: sensitive; I: intermediate; R: resistant.

, all strains exhibited strong sensitivity to penicillin, ceftriaxone, and chloramphenicol. After treatment with erythromycin and lincomycin, the two Staphylococcus strains exhibited higher resistance than the two Lactobacillus strains, S. saprophyticus LH-5 showed resistance to tetracycline, and both strains of LAB were resistant to ciprofloxacin. All strains exhibited weak sensitivity to gentamicin and trimethoprim-sulfamethoxazole, indicating strong resistance to these antibiotics. Studies suggest that although strain-specific variations exist, most LAB are intrinsically resistant to ciprofloxacin, gentamicin, and cotrimoxazole [37]. The two Staphylococcus strains exhibited resistance to cotrimoxazole and varying levels of tetracycline resistance. Notably, YW-2 demonstrated some susceptibility to tetracycline compared to LH-5. These findings align with previous studies showing that clinically significant Staphylococcus like Staphylococcus aureus frequently develop resistance to both tetracycline and cotrimoxazole in clinical settings [38]. In dairy microbiology research, lactobacilli generally exhibit resistance to aminoglycoside antibiotics targeting protein synthesis. However, substantial interspecies variation in resistance phenotypes has been documented [39,40], Additionally, the two Staphylococcus strains showed low sensitivity to erythromycin, gentamicin, and lincomycin, consistent with findings reported by Wang et al. [41].

3.4. Taxonomic Identification of Fermentation Isolates

3.4.1. Morphological Characterization of the Isolates

As shown in Figure 1, after 24 h of incubation at 37 °C, S. saprophyticus LH-5 and S. carnosus YW-2 formed milky-white, circular colonies with glossy, opaque surfaces. These colonies measured approximately 1–2 mm in diameter and exhibited a convex morphology. In contrast, L. sakei OFN-9 and OFN-11, incubated for 48 h under the same conditions, developed white, circular colonies with opaque and convex structures. These colonies were also 1–2 mm in diameter, had a viscous texture, and emitted a distinctive lactic acid bacterial aroma.

3.4.2. 16S rDNA Gene Sequence Analysis of Selected Isolates

As shown in Supplementary Figure S2, the 16S rDNA sequences of the tested strains (LH-5, YW-2, OFN-9, and OFN-11) were successfully amplified, with the products exhibiting clear, bright bands at approximately 1500 bp on 1% agarose gel electrophoresis. Subsequent BLAST analysis of these sequences against the GenBank database revealed the following homologies: LH-5 shared 93% similarity with S. saprophyticus, YW-2 showed 91% similarity with S. carnosus, and OFN-9 and OFN-11 displayed 95% similarity with L. sakei. Phylogenetic tree construction using MEGA-X software further confirmed these relationships. Based on these results, LH-5 was identified as S. saprophyticus, YW-2 as S. carnosus, and OFN-9/OFN-11 as L. sakei.

3.5. Growth Characteristics and Acid-Producing Capacity of the Isolates

This study evaluated the growth profiles and pH changes in the culture environment of the Staphylococcus isolates (LH-5 and YW-2) and LAB isolates (OFN-9 and OFN-11), selected based on their superior fermentation performance. The data were analyzed and plotted to generate corresponding curves, characterizing their metabolic activity and environmental adaptability.

As shown in Figure 2, Staphylococcus isolates (LH-5 and YW-2) and LAB isolates (OFN-9 and OFN-11) exhibited normal growth in LB broth and MRS broth, respectively. LH-5 and YW-2 demonstrated similar growth rates and dynamics, reaching the stationary phase at approximately 15 h with an OD_600nm of 1.2. In contrast, OFN-9 and OFN-11 exhibited highly comparable growth patterns, reaching the stationary phase at 18 h with higher OD_600nm values of 1.4. All four isolates demonstrated robust and consistent growth under these conditions.

As shown in Figure 3, all strains demonstrated acid-producing capacity during cultivation, leading to a gradual decrease in the pH of the medium. The pH decrease trend closely aligned with the growth curve in Figure 3, gradually decreasing as the OD_600nm values increased. Specifically, the final pH values of OFN-9 and OFN-11 reached approximately 3.7 at 24 h, significantly lower than those of the Staphylococcus isolates LH-5 and YW-2, which remained at pH 5.0. All strains exhibited a rapid decrease in pH and favorable acid production within the 3–15 h interval.

Figure 4 shows the acid tolerance test results for the four isolates under different pH conditions. The two LAB isolates, OFN-9 and OFN-11, exhibited strong acid tolerance, maintaining OD_600nm values higher than 0.8 across all five tested acidity levels of pH ≥ 3.5. In contrast, the Staphylococcus isolates LH-5 and YW-2 showed a gradual decrease in OD_600nm values as acidity increased (pH decreased). Their growth was significantly inhibited at pH < 3.5, indicating poor acid tolerance under highly acidic conditions.

3.6. Inter-Isolate Antagonism Assay

As shown in

Table 9. Results of antagonism test between isolates.

Isolates	OFN-9	OFN-11
LH-5	−	−
YW-2	+	−

Note: “−” indicates no antagonistic activity (colonies grow overlapped); “+” denotes weak antagonistic activity (partial overlapping of colonies).

, by growth antagonism assay between isolates, weak antagonistic activity (+) was observed between S. carnosus YW-2 and L. sakei OFN-9, whereas no antagonistic activity (−) were detected among the other three co-culture combinations. This suggests that S. carnosus YW-2 and L. sakei OFN-9 cannot co-exist in a viable system as combined starter cultures for fermented meat products. However, the absence of antagonism between S. saprophyticus LH-5 and L. sakei OFN-11 showed compatibility between them, suggesting their potential as combined starter cultures for fermented meat product applications.

3.7. Analysis of Physicochemical Indexes of Fermented Sausages

3.7.1. Effect of Different Starters on pH, Moisture Content, Volatile Basic Nitrogen, Sodium Nitrite, and Color of Fermented Sausages

As indicated in

Table 10. Physicochemical indexes of fermented sausages.

Sample	pH	Moisture Content %	TVB-N mg/100 g	Sodium Nitrite mg/Kg	L*	a*	b*
Formulation 1	5.70 ± 0.05 a	19.6 ± 0.1 b	24.8 ± 0.34 c	4.00 ± 0.07 b	41.16 ± 1.23 b	13.00 ± 0.48 ab	10.53 ± 0.26 b
Formulation 2	5.65 ± 0.04 ab	18.9 ± 0.2 c	24.1 ± 0.55 c	3.54 ± 0.05 c	41.26 ± 1.22 ab	12.90 ± 0.16 ab	11.50 ± 0.44 ab
Formulation 3	5.61 ± 0.08 b	20.3 ± 0.1 a	19.2 ± 0.45 d	3.62 ± 0.12 c	41.83 ± 1.02 ab	12.53 ± 0.29 b	10.33 ± 0.48 b
THM-17	5.42 ± 0.11 c	19.6 ± 0.3 b	31.0 ± 0.21 a	5.11 ± 0.07 a	37.43 ± 0.98 c	12.20 ± 0.18 c	9.90 ± 0.36 c
VBL-60	5.34 ± 0.10 d	19.2 ± 0.2 bc	27.1 ± 0.65 b	5.23 ± 0.07 a	44.63 ± 1.35 a	13.50 ± 0.15 a	11.73 ± 0.23 a

Note: Different lowercase letters (a–d) in the same row indicate significant differences between samples. (p < 0.05).

, compared to fermented sausages produced with commercial starter cultures (THM-17 and VBL-60), those made with the combined S. saprophyticus LH-5 and L. sakei OFN-11 starter exhibited higher pH values and comparable moisture content. However, elevated pH levels may increase the risk of pathogenic microbial proliferation, as inhibiting the growth of foodborne pathogens generally requires lower pH conditions [42]. The combined culture group also demonstrated significantly lower Total Volatile Basic Nitrogen (TVB-N) and sodium nitrite residue. The color parameters of sausages fermented with the combined culture showed intermediate redness (a*) and brightness (L*) values relative to the THM-17 and VBL-60 groups, which fell within the typical range for fermented sausages. These findings suggest that the combined starter culture achieves a balanced acid production profile (as indicated by pH), improved nitrogen metabolism (evidenced by reduced TVB-N and nitrite levels), and desirable color characteristics comparable to commercial starters.

3.7.2. Effect of Different Fermentation Agents on the Textural Properties of Fermented Sausages

As shown in

Table 11. Textural analysis of fermented sausages.

Sample	Hardness/g	Springiness/mm	Cohesiveness	Chewiness/g·mm
Formulation 1	4687 ± 335 bc	1.88 ± 0.03 ab	0.76 ± 0.11 a	5228 ± 349 b
Formulation 2	4804 ± 426 b	1.85 ± 0.06 ab	0.66 ± 0.09 b	6308 ± 485 a
Formulation 3	4523 ± 230 c	1.94 ± 0.03 a	0.55 ± 0.12 c	6104 ± 479 a
THM-17	5104 ± 457 b	1.80 ± 0.11 b	0.66 ± 0.16 b	5421 ± 851 b
VBL-60	5801 ± 689 a	1.74 ± 0.16 c	0.56 ± 0.07 c	4808 ± 575 c

Note: Different lowercase letters (a–c) in the same row indicate significant differences between samples. (p < 0.05).

, all the formulations exhibited significantly lower hardness than the commercial starter VBL-60, while Formulation 3 exhibited the lowest hardness, significantly lower than both commercial starters THM-17 and VBL-60 (22% reduction compared to VBL-60). The formulations demonstrated higher springiness indices compared to the commercial starters, with Formulation 3 showing a statistically significant increase over both THM-17 and VBL-60. Conversely, Formulation 3 paradoxically exhibited the lowest cohesiveness (0.55 ± 0.12), inversely correlating with its reduced hardness. Notably, the chewiness scores of Formulations 2 and 3 were significantly higher than those of their commercial counterparts. This textural profile aligns with sensory analysis in Section 3.8, where Formulation 3 received the highest texture scores in consumer panels (n = 30), indicating optimal compliance with Chinese consumers’ preference for tender-fermented meat products. These findings suggested that combined starter cultures, such as S. saprophyticus and L. sakei, may enhance textural balance by modulating microbial metabolism, including lactic acid production and proteolytic activity, which affect protein degradation and moisture retention.

3.8. Results of Sensory Evaluation of Fermented Sausages

The comprehensive evaluation presented in Table 10, Table 11 and

Table 12. Effect of different formulas on sensory scores of fermented sausages.

Sample	Appearance	Texture	Aroma	Taste	Overall Rating
Formulation 1	15.93 ± 1.78 a	16.00 ± 1.52 b	21.13 ± 2.62 a	21.09 ± 2.00 a	19.09 ± 2.14 a
Formulation 2	15.87 ± 1.88 a	15.73 ± 2.95 c	20.93 ± 3.01 a	21.20 ± 1.78 a	18.60 ± 3.02 b
Formulation 3	15.62 ± 2.01 a	16.20 ± 3.20 a	21.20 ± 1.02 a	22.06 ± 2.01 a	19.35 ± 1.89 a
THM-17	15.23 ± 1.26 ab	14.80 ± 2.51 e	17.20 ± 3.58 c	17.40 ± 2.44 b	16.95 ± 1.99 c
VBL-60	14.93 ± 3.32 b	15.13 ± 3.11 d	19.26 ± 2.15 b	15.00 ± 3.29 c	15.86 ± 2.54 d

Note: Different lowercase letters (a–e) in the same row indicate significant differences between samples. (p < 0.05).

indicated that fermented sausages with lower acidity, a reddish color, good elasticity, reduced hardness, rich flavor, and the absence of meaty off-odor are more likely to align with the preference of Chinese consumers. These findings were consistent with those of Liu et al. [43]. Increased acidity in fermented sausages can cause significant irritation in the oral cavity, leading to discomfort, whereas a more reddish color enhances appetite, and decreased hardness broadens consumer acceptance [43,44]. Chinese consumers typically exhibit lower acceptance of European-style fermented sausages than their European counterparts, particularly in terms of distinctive flavors, acidity, and texture. This discrepancy stems from cultural and sensory preferences: Chinese consumers generally favor low-salt, low-fat foods with mild flavors, while the smoky, spicy, or complex fermented notes of European-style sausages often exceed traditional taste expectations in China [45]. These results suggested that fermented sausages produced with the combined starter culture (Formulation 1, 2, and 3) more closely align with consumer expectations than those made with commercial starter cultures. Notably, Formulation 3, with a 1:2 ratio of LH-5 to OFN-11, achieved the highest overall score, significantly outperforming the commercial starter formulations in terms of sensory attributes and textural properties. Consequently, Formulation 3 was selected as the optimal treatment group for subsequent volatile compound analysis to explore its flavor-enhancing mechanisms. These findings aligned with studies demonstrating that mixed starter cultures (e.g., combinations of Lactobacillus and Staphylococcus) improve protein degradation, lipid oxidation control, and volatile flavor diversity, ultimately enhancing sensory acceptance.

3.9. Effect of Different Formulas on Volatile Compounds in Fermented Sausages

Volatile flavor compounds in fermented sausages from four treatment groups, including the blank group (without starter culture), commercial starter cultures (THM-17 and VBL-60), and the combined starter culture (Formulation 3), were analyzed using GC-IMS. As shown in

Table 13. Volatile compound content in fermented sausages.

	Relative Content (μg/Kg)
Compound	Blank Group	THM-17	VBL-60	Formulation 3
Acids
Acetic acid	2396.41 ± 5.11	3882.29 ± 91.24	4165.68 ± 175.11	3264.19 ± 3.32
2-Methyl propanoic acid	24.51 ± 0.65	17.18 ± 2.62	16.44 ± 3.23	16.74 ± 1.2
1-Butanoic acid	57.69 ± 1.91	118.88 ± 6.5	107.91 ± 25.73	104.27 ± 0.8
3-Methyl butyric acid	149.31 ± 1.5	199.59 ± 30.26	215.37 ± 44.09	184.37 ± 12.83
Aldehyde
Nonanal	67.24 ± 7.6	93.63 ± 10.08	147.23 ± 14.56	99.76 ± 3.36
(E)-2-Pentenal	33.39 ± 4.86	24.35 ± 0.96	28.6 ± 1.25	79.86 ± 1.15
Hexanal	99.77 ± 4.43	44.98 ± 6.5	45.83 ± 0.83	322.06 ± 0.48
(E)-2-Heptenal	54.66 ± 1.66	21.12 ± 2.5	24.63 ± 1.02	62.69 ± 0.63
3-Methyl butanal	160.34 ± 2.71	127.02 ± 8.96	156.12 ± 10.46	150.12 ± 1.62
(E)-2-Hexen-1-al	31.25 ± 1.92	21.78 ± 3.57	24.82 ± 2.95	21.57 ± 0.56
Heptaldehyde	12.25 ± 1.28	13.55 ± 6.84	6.62 ± 1.23	11.42 ± 3.52
3-(methylsulfanyl)Propanal	19.95 ± 0.68	14.02 ± 1.3	14.96 ± 1.96	17.19 ± 12.9
3-Methyl-2-butenal	5.45 ± 3.89	6.11 ± 1.34	3.72 ± 0.09	3.92 ± 7.01
Butyraldehyde	94.95 ± 0.59	113.79 ± 4.93	85.88 ± 5.37	155.88 ± 2.06
(E)-2-Pentenal	6.8 ± 0.75	23.07 ± 6.93	12.23 ± 2.91	12.89 ± 1.71
n-Pentanal	40.41 ± 4.92	79.1 ± 6.6	156.03 ± 18.22	149.73 ± 7.67
(E)-2-Methylpent-2-enal	14.44 ± 0.98	46.97 ± 16.57	63.59 ± 16.48	68.21 ± 2.96
Propanal	123.37 ± 16.86	77.71 ± 13.55	207.3 ± 19.77	251.01 ± 10.43
Octanal	14.93 ± 0.93	16.57 ± 3.42	27.53 ± 7.55	25.29 ± 4.1
Ketone
2-Heptanone	34.03 ± 7.41	21.24 ± 1.11	52.66 ± 7.29	16.16 ± 6.24
2,3-Butanedione	58.92 ± 0.82	43.98 ± 1.23	52.93 ± 4.58	79.48 ± 0.03
4-Heptanone	42.24 ± 1.37	8.16 ± 2.14	11 ± 0.44	11.95 ± 1.46
Acetone	910.05 ± 296.83	862.85 ± 57.69	794.58 ± 32.46	910.21 ± 19.74
1-Hydroxy-2-propanone	52.18 ± 0.10	78.09 ± 4.36	24.38 ± 4.45	23.58 ± 0.16
2-Butanone	51.12 ± 0.81	87.77 ± 5.88	92.21 ± 7.07	91.74 ± 1.89
3-Nonanone	10.68 ± 0.92	15.43 ± 1.33	40.77 ± 6.47	37.15 ± 6.34
Acetoin	1638.1 ± 200.65	789.6 ± 74.87	160.77 ± 59.37	120.77 ± 16.58
Alcohol
Butanol	131.79 ± 4.55	202.49 ± 1.78	235.6 ± 11.3	194.32 ± 23.23
Isobutanol	30.24 ± 2.94	58.74 ± 2.58	70.76 ± 3.79	78.92 ± 5.96
1-Pentanol	149.31 ± 3.19	99.56 ± 7.8	121.37 ± 10.77	73.15 ± 1.95
Isoamyl alcohol	516.06 ± 18.38	508.79 ± 13.15	590.59 ± 14.97	232.74 ± 7.54
Ethanol	1299.57 ± 0.95	1013.2 ± 87.22	946.65 ± 31.41	1278.65 ± 0.53
3-Methyl-3-buten-1-ol	42.20 ± 2.54	10.20 ± 0.69	10.21 ± 1.24	12.08 ± 0.52
1-Penten-3-ol	61.94 ± 3.07	21.76 ± 8.02	24.97 ± 3.22	28.84 ± 1.08
(Z)-2-Penten-1-ol	29.53 ± 7.37	14.44 ± 3.46	13.56 ± 0.22	13.78 ± 10.03
2-Pentanol	14.52 ± 115.13	40.64 ± 6.06	37.45 ± 3.93	45.25 ± 104.35
Propanol	18.59 ± 0.5	73.69 ± 3.49	52.43 ± 2.6	59.84 ± 0.37
2-Propanol	12.51 ± 6.27	32.09 ± 3.67	31.08 ± 1.55	31.07 ± 10.36
2-Heptanol	22.06 ± 2.85	25.92 ± 2.34	81.5 ± 11.84	88.12 ± 11.36
Ester
Ethyl butyrate	62.95 ± 3.05	81.73 ± 1.4	80.95 ± 2.71	98.63 ± 0.91
Butanoic acid 3-methyl, ethyl	21.46 ± 0.97	14.29 ± 1.77	9.29 ± 0.08	11.20 ± 0.38
1-Butanol, 3-methyl-, acetate	14.47 ± 11.49	6.71 ± 1.86	2.20 ± 0.23	2.01 ± 16.8
Ethyl acetate	175.06 ± 1.89	209.02 ± 22.62	122.75 ± 1.9	244.75 ± 2.44
Butanoic acid, 3-methylbutyl	15.82 ± 2.44	10.07 ± 1.19	7.31 ± 1.19	5.48 ± 2.1
Hexyl formate	13.24 ± 1.07	24.69 ± 4.33	16.68 ± 2.38	14.53 ± 0.75
1,2-Propanediol diacetate	243.41 ± 0.49	466.68 ± 59.25	340.49 ± 47.63	447.25 ± 0.27
2-Methylbutanoic acid, methyl	1.85 ± 9.93	4.93 ± 1.12	2.96 ± 0.61	2.84 ± 1.04
Methyl benzoate	426.63 ± 3.45	579.27 ± 165.08	555.84 ± 301.16	599.45 ± 6.29
Hexyl propanoate	15.83 ± 3.43	15.13 ± 2.87	30.42 ± 2.97	31.56 ± 1.01
Other
α-Pinene	87.83 ± 4.31	80.07 ± 5.22	71.9 ± 11.16	157.71 ± 0.86
δ-3-carbene	42.22 ± 9.57	35.18 ± 3.18	36.45 ± 4.22	63.72 ± 6.1
2-Methylpyrazine	13.36 ± 14.63	13.73 ± 1.67	12.72 ± 0.92	18.12 ± 37.39
(Z)-2-Pentenal	7.53 ± 0.53	10.01 ± 0.36	8.55 ± 0.4	4.55 ± 1.77
2-Acetylfuran	16.48 ± 4.48	32.5 ± 5.16	30.8 ± 3.46	35.89 ± 12.89
(+)-Limonene	3.09 ± 1.16	3.7 ± 0.26	2.08 ± 0.2	2.89 ± 2.13
α-Terpinene	27.87 ± 1.06	43.34 ± 7.82	21 ± 2.2	21.07 ± 1.88
β-Thujene	29.33 ± 0.86	48.98 ± 6.4	34.15 ± 4.44	34.15 ± 1.36
Tetrahydrofuran	112.21 ± 2.1	326.28 ± 31.49	275.59 ± 32.4	175.19 ± 2.79
β-Cubebene	33.48 ± 0.36	77.31 ± 22.03	64.20 ± 10.68	64.28 ± 0.37
2,3-Diethyl-5-methylpyrazine	16.4 ± 0.83	51.61 ± 3.14	45.53 ± 0.89	52.07 ± 2.71

, 55 compounds with odor activity values (OAV) ≥ 1 were identified, and categorized into 4 acids, 15 aldehydes, 8 ketones, 12 alcohols, 10 esters, and 11 other volatile compounds.

Principal component analysis (PCA) was performed on the volatile flavor compounds to visually compare flavor differences in fermented sausages, as shown in Figure 5A. The two principal components, PC1 and PC2, accounted for 61% and 21% of the total variance, respectively. A distinct separation was observed between the blank group (CK) and the combined starter culture group formulation 3 (LH-5 + OFN-11), with both groups showing significant divergence from the commercial starter groups (THM-17 and VBL-60). Notably, Formulation 3 clustered exclusively on the left side of the x-axis, whereas the blank group occupied the negative y-axis region, reflecting its distinct flavor profile. The THM-17 and VBL-60 groups exhibited overlapping features on the PCA plot, indicating similar volatile flavor characteristics. However, both groups significantly differed from Formulation 3 along the x-axis. This spatial distribution highlights that the combined starter culture uniquely shaped the volatile compound profile, distinguishing it from the commercial starters. This finding was consistent with studies that examined microbial-driven lipid metabolism and flavor enhancement in fermented sausages [46].

The analysis of the volatile organic compound fingerprint profiles (Figure 5B) revealed distinct differences in signal peak intensities across samples under the same migration time, with brighter colors indicating higher compound concentrations. Each treatment group was analyzed in triplicate, with three replicates per sample. As shown in Table 13 and Figure 5, fermented sausages inoculated with Formulation 3 exhibited significantly lower pH than the commercial starter cultures (THM-17 and VBL-60). Specifically, acetic acid and 3-methyl butyric acid levels in the combined starter group were reduced by 15.92% and 7.63%, respectively, compared to the highest values observed in commercial starter culture groups. This finding aligns with the pH variations reported in Table 10, where the lower acidity in the combined starter group correlated with reduced pH [47]. Additionally, several compounds increased in concentration in Formulation 3 compared to the commercial starter culture groups. Hexanal, butyraldehyde, and propanal increased by 602.73%, 36.99%, and 21.09%, respectively. Isobutanol, ethanol, and 2-pentanol increased by 11.53%, 26.20%, and 11.34%, respectively. Ethyl butyrate, ethyl acetate, and methyl benzoate increased by 20.68%, 17.09%, and 3.48%, respectively, compared to the highest values in the commercial fermenter group. Other aromatic hydrocarbons, such as δ-3-carbene and 2-acetylfuran, also increased. Among them, hexanal, ethyl butyrate, (E)-2-Octenal, and acetone are commonly used as indicators of lipid oxidation and contribute grassy, citrus, and fatty flavors to the sausages [48]. Compounds such as ethyl acetate, δ-3-carbene, 1,2-propanediol diacetate, and butyraldehyde impart fruity, floral, and fresh aroma notes to the product [49]. Heterocyclic aromatic hydrocarbons, such as tetrahydrofuran and δ-3-carbene, are also associated with strong meaty flavor characteristics [50]. Therefore, the addition of Formulation 3 significantly enhances the content of volatile compounds such as aldehydes, alcohols, and esters, enriching the product with fruity, meaty, and fatty aroma notes and intensifying its overall flavor profile.

4. Conclusions

This study subjected 67 Gram-positive bacterial strains isolated from six types of traditional fermented meat products (including Chinese fermented ham and European salami) to sequential safety screening, antibiotic resistance testing, stress tolerance evaluation, and fermentation performance assessment. S. saprophyticus LH-5 and L. sakei OFN-11 were selected as combined starter cultures for fermented meat production. Comparative analysis with non-inoculated fermentation and commercial starter cultures revealed that Formulation 3 (S. saprophyticus LH-5:L. sakei OFN-11 = 1:2, total inoculation 1 × 10⁷ CFU/g) demonstrated superior acceptance among Chinese consumers. Previous sensory evaluations in our experiments indicated that Chinese consumers generally dislike the higher hardness, lower pH, and spicy flavor typical of European-style fermented sausages. They prefer softer texture, moderately higher pH, salty-sweet taste profiles, and express skepticism toward the distinct acidic flavor of fermented sausages—factors significantly influencing their overall preference judgments. GC-IMS analysis confirmed that Formulation 3 enhanced the richness and stability of volatile compounds in fermented sausages, particularly by increasing key flavor contributors, such as esters and aldehydes. These compounds are critical for generating grassy, citrus-like, and fatty aroma notes, which are considered highly desirable in fermented meat products. Therefore, combined fermentation using the selected isolates S. saprophyticus LH-5 and L. sakei OFN-11 can effectively enhance the distinctive flavor characteristics of fermented sausages, better aligning the product with the preferences of Chinese consumers. Formulation 3 holds significant potential as a dedicated starter culture for Chinese fermented meat products. The relatively slow market penetration of European-style fermented sausages in China may stem from consumer skepticism regarding acidity levels, appearance-related concerns, and textural divergence from traditional Chinese fermented meat products. Industry players, particularly Chinese enterprises, could consider product optimization through starter culture modification and processing parameter adjustments to better align with domestic sensory preferences. Since consumer preferences for food vary from region to region, we recommend that food companies conduct thorough market research targeting consumers in different regions and formulate corresponding product formulations based on the findings.