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Article

Don’t Judge a Sausage by Its Cover: Effects of Inoculating Three Indigenous Lactic Acid Bacteria on Quality, Moisture Distribution, and Protein Structure in Fermentation

by
Yangyi Zheng
1,2,
Gaiming Zhao
1,2,
Shichang Zhao
1,3,
Xuan Li
1,2,
Wenming Cui
1,2,
Long Xu
1,2,
Chaozhi Zhu
1,2,* and
Lin Tong
4
1
Henan Key Lab of Meat Processing and Quality Safety Control, Henan Agricultural University, Zhengzhou 450002, China
2
College of Food Science and Technology, Henan Agricultural University, Zhengzhou 450002, China
3
International Education College, Henan Agricultural University, Zhengzhou 450002, China
4
National Beef Cattle and Yak Industry Technology System Kerchin Comprehensive Test Station, Tongliao 028100, China
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(3), 134; https://doi.org/10.3390/fermentation11030134
Submission received: 23 February 2025 / Revised: 6 March 2025 / Accepted: 6 March 2025 / Published: 11 March 2025
(This article belongs to the Section Fermentation for Food and Beverages)
Browse Figures
Versions Notes

Abstract

:
To produce products with standardized and optimal technical performance, probiotics, particularly Lactic Acid Bacteria (LAB), have long been utilized as fermentation starters in sausages, ensuring both the standardization and enhancement of product quality and safety. Microorganisms isolated from traditional meat products, due to their excellent adaptability to the fermentation environment and their ability to preserve desirable flavor, exhibit high potential as candidates for meat fermentation starters. Three indigenous LAB strains—Latilactobacillus sakei, Pediococcus pentosaceus, and Weissella cibaria, isolated from Yunnan ham—were applied in the fermentation of beef sausages to investigate the underlying factors responsible for quality changes. The results indicated that sausages fermented with L. sakei and P. pentosaceus exhibited the lowest pH (4.98) and aw (0.79), while displaying significantly higher hardness, cohesiveness, and chewiness. Additionally, LF-NMR measurements showed that L. sakei and P. pentosaceus promoted the transfer of immobilized water to free water, facilitating the drying and maturation process. Raman spectroscopy analysis revealed a reduction in α-helix content and an increase in disordered β-sheet and β-turn structures in the secondary protein structure. These findings suggest that L. sakei and P. pentosaceus improved quality attributes by modifying the secondary protein structure to enhance water migration and accelerate the ripening process. L. sakei and P. pentosaceus demonstrated desirable technological characteristics, indicating their efficacy for use in fermented sausage production. This study provides valuable insights into improving the production of fermented sausages using specific LAB strains.

1. Introduction

Fermented sausages, as a typical type of fermented meat product, are globally popular due to their unique flavor and extended shelf life. Traditional Chinese fermented sausages, which are produced without the inoculation of starter cultures or additives, rely on uncontrolled natural fermentation conditions. This, coupled with potential microbial contamination, can result in inconsistencies in product quality and safety [1].
To produce products with consistent and desirable technological properties, probiotics, primarily Lactic Acid Bacteria (LAB), have been employed as starter cultures in fermented sausages for an extended period [2,3,4]. Current research indicates that in meat fermentation, LAB facilitate the rapid acidification of the batter, resulting in lower pH values that define the final fermented product. This acidification improves microbial stability by inhibiting pathogenic activity through antibacterial metabolites, such as organic acids, bacteriocins, and hydrogen peroxide. Additionally, LAB induce physicochemical changes that enhance quality characteristics like texture [5] and flavor. During the ripening stage, microbiological, biochemical, and enzymatic alterations take place, including a decrease in starter viability, the release of intracellular enzymes, and the hydrolysis of proteins, carbohydrates, and lipids, leading to the formation of numerous volatile and non-volatile flavor compounds. Research over the past decade has suggested that protein changes are attributed not only to the activity of endogenous meat enzymes, but also to certain bacterial groups, notably LAB [1,6,7]. However, the relevant mechanisms need to be further clarified.
Commercial starter cultures may not always compete effectively with the native microbial flora, potentially leading to the loss of the product’s desired sensory characteristics [8]. Microorganisms isolated from traditional meat products show potential as meat starter culture candidates due to their excellent adaptability to the ecological environment of fermented meats [8]. Furthermore, LAB isolated from spontaneous fermentation processes could provide unique and appealing aromas owing to their specific fermentative metabolism [9,10]. Yunnan ham is a significant traditional fermented meat product in China, known for its distinctive flavor and abundant microbial resources. The natural fermentation process is supported the growth of indigenous microbiota, enhancing the microbiological safety and flavor profile of the final product [11]. Consequently, employing bacteria isolated from traditional meat products has the potential to improve the sensory properties and shelf-life of fermented sausages.
This study explores the effects of three indigenous LAB—L. sakei, P. pentosaceus, and W. cibaria—isolated from Yunnan ham on key quality parameters of fermented sausages, including pH, water activity (aw), texture, microbial profile, and total volatile basic nitrogen (TVB-N). Furthermore, low-field nuclear magnetic resonance (LF-NMR) and Raman spectroscopy are employed to investigate the underlying causes of the observed quality changes in the fermented beef sausages.

2. Materials and Methods

2.1. Strains

L. sakei, P. pentosaceus, and W. cibaria were isolated from the traditional Chinese fermented product, Yunnan ham, and identified by 16S rDNA sequencing. They were stored at the College of Food Science and Technology, Henan Agricultural University. The bacteria were grown in de Man–Rogosa–Sharpe (MRS) broth (Qingdao Hope Bio-Technogy Co., Ltd., Qingdao, China) at 37 °C for 12 h, 12 h, and 14 h, respectively followed, by three generations of activation [12]. Subsequently, 1% (v/v) of the bacterial suspension from the seed culture was transferred into the amplification medium, and the bacterial count was adjusted to the desired level [1]. The cultures were then incubated at 37 °C for 12 h. After cultivation, cells were harvested by centrifugation at 10,000× g for 10 min at 4 °C, washed twice with sterile saline, and directly used for inoculating the meat batter [13].

2.2. Preparation of Sausages

Fresh beef and fat were obtained from Hengdu Food Co., Ltd. (Zhumadian, China). The cattle used in this study were slaughtered in accordance with the Ethical Guidelines for Animal Care, as stipulated by the Ministry of Science and Technology of the People’s Republic of China (2010). The sausages were formulated using beef (80 g), beef fat (20 g), sugar (4 g), salt (2.2 g), glucose (0.5 g), food-grade sodium nitrite (0.015 g), and food-grade sodium erythorbate (0.05 g) [14]. The prepared mixture was homogenized and subsequently categorized into four experimental groups: (1) a non-inoculated control group; (2) sausages inoculated with L. sakei; (3) sausages inoculated with P. pentosaceus; and (4) sausages inoculated with W. cibaria. Each bacterial strain was added to achieve a final concentration of approximately 107 colony-forming units (CFU)/g meat. The sausages underwent a fermentation process divided into several stages: first, they were fermented at 25 °C and 95 ± 2% relative humidity (RH) for 2 days; next, the temperature was reduced to 15 °C with an RH of 80 ± 2% for 6 days; then, they were kept at 15 ± 0.5 °C with an RH of 75% for another 6 days; and finally, they were matured at 12 °C and an RH of 70 ± 2% for the last 6 days. The total ripening period spanned 20 days. The sausages were produced in three separate batches at different time intervals, with each batch serving as a replicate.

2.3. Sensory Evaluation

Using a random sampling method, equal amounts of samples were taken from each group and sliced into 5 mm thick pieces. Ten professional sensory evaluators, comprising five men and five women, were invited to randomly assess the samples. The evaluators conducted their assessments individually and refrained from interacting with one another. To prevent sensory fatigue and maintain accuracy, water was provided for rinsing their mouths between evaluations. The sensory evaluation of the fermented beef sausages covered five criteria—texture, color and appearance, distinctive flavor, chewiness, and overall acceptability—with the specific assessment parameters outlined in Table 1.

2.4. Microbiological Analysis

Microbiological analysis was conducted to monitor the population dynamics during fermentation (days 0 and 2), ripening (days 8 and 14), and at the end of ripening (day 20). At each sampling point, sausage casings were aseptically removed, and 25 g samples were diluted in 225 mL of sterile normal saline, followed by homogenization for 120 s. LAB were counted on MRS agar following the method of [15] and incubated at 30 °C for 48 h. The total viable bacteria of Staphylococcus, Micrococcus, and Enterobacteriacea were determined using Plate Count Agar (PCA) (30 °C, 48 h), Mannitol Salt Agar (MSA) (37 °C, 48 h), and Violet Red Bile Dextrose Agar (VRBDA) (37 °C, 24 h), respectively (Hope Bio-Technogy Co., Ltd., Qingdao, China) [16].

2.5. Total Volatile Basic Nitrogen (TVB-N)

The 10 g sausage sample was mixed with 100 mL deionized water and homogenized. The subsequent filtrate was analyzed for TVB-N using an automatic Kjeldahl nitrogen analyzer (Hanon Instrument Co., Ltd., Jinan, China) following the manufacturer’s operating instructions. Results are expressed as mg/100 g, with three replicate measurements conducted per batch [17].

2.6. Texture Profile Analysis (TPA)

The fermented sausage samples were sliced into 10 mm thick segments and subjected to a two-cycle compression test using a TA.XT Plus texture analyzer (TMS-PRO, Food Technology Corp., Washington, DC, USA) equipped with a “T”-type probe. Each specimen was positioned centrally on the TPA platform and compressed at a constant speed of 60 mm/min until it reached 50% of its initial height, with a trigger force threshold of 5 N. The derived parameters, such as firmness, springiness, chewiness, cohesiveness, and adhesive properties, were calculated from the generated deformation curves [18]. Triplicate measurements were performed for each experimental batch to ensure reproducibility.

2.7. pH and aw Analysis

For pH measurement, 10 g of the samples was homogenized in 100 mL of a saturated KCl solution [19]. The pH values were then determined with a pH meter (Mettler Toledo Instruments Co., Ltd., Shanghai, China). Aw was assessed using a water activity meter (Aqualab 4TE Duo, METER Group Inc., Pullman, Washington, DC, USA).

2.8. Low-Field Nuclear Magnetic Resonance (LF-NMR) Measurement

Approximately 2 g of fermented sausage was thinly sectioned and housed within cylindrical glass tubes (18 mm internal diameter). Transverse relaxation time (T2) measurements were conducted at 32 °C using a Niumag pulsed nuclear magnetic resonance analyzer (Model PO001, Niumag Corporation, Shanghai, China) operating at a proton resonance frequency of 22.7 MHz. The Carr–Purcell–Meiboom–Gill (CPMG) [20,21] pulse sequence was applied with a ζ value (interval between 90 °C and 180 °C pulses) of 100 μs. Data acquisition involved a 3 s repetition delay between scans, capturing 4000 echo signals through four sequential scan cycles. Signal processing utilized MultiExp Inv Analysis software (Version 4.08, Niumag Corporation, Shanghai, China) with multi-exponential fitting, revealing three distinct T2 relaxation components (T2b, T21, and T22) and their corresponding moisture distribution fractions (P2b, P21, and P22).

2.9. Raman Spectroscopic Analysis

Raman spectral data were obtained in the range of 400–4000 cm−1 with a LabRAM HR Evolution Laser Confocal Raman Spectrometer (Horiba Jobin Yvon, Longjumeau, France). A sausage slice, 0.5 mm thick, was positioned on a concave slide and then inserted into the Raman spectrometer. A 532 laser transmitter was used and the measurement parameters were set as follows: power attenuation 10%, three scans, 30 s acquisition time.
Each sample type was analyzed with at least three replicates. Spectra underwent smoothing, baseline correction, and normalization using Labspec version 6.0 software (Horiba Jobin Yvon, Longjumeau, France). The secondary structures of muscle proteins, specifically the percentages of α-helix, β-sheet, β-turn, and random coil were established according to Nawrocka et al. (2017) [22] and Zhu et al. (2020) [23].

2.10. Statistical Analysis

The results are represented as mean values accompanied by standard deviations derived from the replicates. Statistical analysis was carried out utilizing SPSS version 16.0.0 (SPSS Inc., Chicago, IL, USA). Duncan’s multiple comparison tests were used to compare mean values, and differences among the mean values were considered significant when p < 0.05. The pictures were drawn using Origin 2021 (Origin Lab Corporation, Northampton, MA, USA).

3. Results and Discussion

3.1. Sensory Evaluation

In the process of fermented sausage production, inoculation with a single strain of LAB generally enhanced the chewiness and overall acceptability of the sausage. As shown in Figure 1, the overall acceptability of beef sausages fermented with L. sakei and P. pentosaceus was higher than the control (uninoculated sausages).
Additionally, sausages fermented with L. sakei and P. pentosaceus exhibited higher chewiness scores compared to the control and those fermented with W. cibaria, aligning with the texture analysis results (Table 2). However, there were minor differences in color scores among the different groups of fermented sausages. The flavors of fermented beef sausages with L. sakei and P. pentosaceus were close to the unique flavor of naturally fermented sausages, consistent with the results obtained by Hu, Y. et al. (2022) [2] using a single lactic acid bacterium for fermentation.
The increased chewiness is likely attributed to the enhanced protein cross-linking induced by LAB fermentation [24,25]. Similar trends have been reported in other meat fermentation systems, where LAB modify texture through proteolysis and moisture redistribution [26,27]. The enhancement in flavor can be ascribed to the accumulation of organic acids, peptides, and free amino acids generated during fermentation, which intensify umami and sourness [28,29]. Additionally, the presence of volatile compounds such as esters and aldehydes further reinforces the characteristic aroma of fermented sausages [30,31,32]. The similarity in color scores among the different fermentation groups may result from the consistent pH levels, which contribute to myoglobin stabilization and prevent excessive pigment oxidation [33,34]. Previous studies have demonstrated that pH plays a critical role in regulating pigment oxidation and denaturation, thereby influencing meat color stability [35].

3.2. Microbiological Analysis

The viable counts of total bacteria [36], LAB, Staphylococcus and Micrococcus, and Enterobacteriaceae in the sausages during fermentation and ripening are shown in Figure 2.
The changes in the total bacterial count and Lactic Acid Bacteria count (LAB count) in all individual fermented sausages with inoculated fermentation showed the same trends during the fermentation process [37]. The contents of LAB increased dramatically during the initial 2 days of fermentation, reaching a peak on day 2, causing them to become the dominant microorganisms in the fermented sausages, consistent with a previous study [38]. The contents in the natural fermented sausages were significantly lower (p < 0.05) than the inoculated sausages. This result was due to the actions of bacteriocins and the adaptation of LAB to acidic environments [39,40]. LAB are beneficial microbes in fermented sausages, as they suppress harmful pathogens and spoilage organisms through acid production and antimicrobial compound synthesis [14]. LAB counts slightly decreased after day 2, with this slight decrease probably being because of the depletion of fermentable substrates. The counts in the natural fermentation group (control) were significantly lower (p < 0.05) than the inoculated sausages. The Staphylococcus and Micrococcus counts increased during the initial 2 days, but the increase in the natural fermentation group was significantly higher (p < 0.05) than in the inoculated sausages. Lower counts of Staphylococcus and Micrococcus were found in the three inoculated sausages; acidification caused by higher LAB counts was probably responsible for this phenomenon because Staphylococcus and Micrococcus are sensitive to environments with a reduced pH [17,41]. The dominance of Lactobacillus, Staphylococcus, and Micrococcus in fermented meat products improves the safety of meat products and imparts unique flavors to the final product [42]. Enterobacteriaceae counts were present at first, but their counts in all groups rapidly decreased during fermentation (p < 0.05). As for the inoculated fermentation sausages, the Enterobacteriacea counts decreased to 1.92 ± 0.03 log CFU/g, 2.14 ± 0.02 log CFU/g, and 2.53 ± 0.11 log CFU/g on the 20th day, respectively, while that in the naturally fermented sausage decreased to 3.05 ± 0.11 log CFU/g. When compared to natural fermentation, the decrease rate of the Enterobacteriacea count in the sausages fermented with inoculation was higher, indicating that LAB could inhibit Enterobacteriacea to improve the safety of fermented meat [37]. It is worth noting that Enterobacteriaceae counts were the lowest in the sausages inoculated with L. sakei and P. pentosaceus, indicating higher microbial safety.

3.3. TVB-N Analysis

The degradation of proteins and other nitrogenous compounds, driven by biochemical and microbial activities, leads to the accumulation of organic amines (cadaverine, putrescine, isobutylamine, and methylated amines), collectively referred to as total volatile basic nitrogen (TVB-N) [43]. TVB-N serves as an objective biomarker to assess the deterioration of freshness and safety in muscle foods [43,44]. The subsequent formation of TVB-N-related compounds, including trimethylamine-N-oxide (TMA-N-O), trimethylamine (TMA), dimethylamine (DMA), and formaldehyde, as well as the deamination of adenine nucleotides, may potentially exert adverse effects on human health. In general, TVB-N levels in meat and seafood tend to rise as storage time progresses [43,45,46]. Studies have shown that the TVB-N value should be less than 30 mg/100 g in high-quality fermented sausages.
In this study, the TVB-N values of all treatment groups during the whole fermentation and ripening process were less than 30 mg/100 g, i.e., within the safe range. The TVB-N values of all sausages increased rapidly during the sausage fermentation and ripening process. Lower TVB-N values were observed in sausages inoculated with P. pentosaceus and L. sakei. At day 20 of fermentation, the TVB-N values of the sausages fermented with P. pentosaceus and L. sakei were 22.30 mg/100 g and 25.54 mg/100 g, respectively, which were significantly (p < 0.05) lower than the sausages inoculated with W. cibaria (28.50 mg/100 g) and the natural fermented samples (29.04 mg/100 g). The results suggest that inoculating with P. pentosaceus and L. sakei may inhibit the growth of microorganisms with amino acid decarboxylase activity [14,47], such as Enterobacteriaceae, as shown in Figure 3A. Thus, this might reduce the production of amines in sausages [17,46], which is beneficial to ensure the quality of fermented beef sausages.

3.4. Texture Properties

Table 2 presents the texture profile analysis (TPA) results of the fermented sausages during the fermentation process. The results indicate that the hardness, cohesion, and chewiness of all treatment groups progressively increased throughout the ripening period (p < 0.05). By the end of the fermentation, the hardness values of the L. sakei and P. pentosaceus groups were significantly higher (p < 0.05) than the others, and the natural fermented group had significantly higher (p < 0.05) values than the samples inoculated with W. cibaria. At the end of the fermentation, there were no significant differences (p > 0.05) in cohesiveness and spring values between the natural fermented samples and the samples inoculated with L. sakei and P. pentosaceus, which were significantly higher (p < 0.05) than the samples inoculated with W.cibaria. The results for cohesion and chewiness were similar to hardness; there were no significant differences (p > 0.05) in the samples inoculated with L. sakei and P. pentosaceus, which had significantly higher (p < 0.05) values than the others. Furthermore, the values of cohesion and chewiness in the natural fermented samples were significantly higher (p < 0.05) than in the samples inoculated with W. cibaria. The changes may be related to the constant dehydration and drying during the sausage fermentation and ripening process. Hardness has been reported to exhibit an inverse correlation with moisture content in fermented sausages. The progressive increase in hardness during ripening is primarily attributed to moisture loss. Additionally, studies have indicated that drying plays a crucial role in determining the binding and rheological properties of sausages [18,48]. The incorporation of sugar into sausage formulations has been shown to enhance the hardness and chewiness of the final product, primarily due to pH reduction below the isoelectric point of myofibrillar proteins [18].

3.5. pH and aw

Figure 3B illustrates the variations in pH and aw during the sausage fermentation and ripening process. The pH values for all fermented sausages sharply decreased (p < 0.05) within the initial 2 days of fermentation, subsequently decreasing at a slower rate and stabilizing after day 8, with a slight increase observed after day 14 in the natural and W. cibaria groups. The pH of the inoculated samples was significantly lower (p < 0.05) than that of the natural fermentation samples during the fermentation period [49,50]. The rise in pH at the final stage was associated with ammonia formation, reduced electrolyte dissociation, and an increased concentration of buffering proteins [51]. Lorenzo et al. (2014) [52] suggested that bacterial proteinases facilitate protein degradation, leading to the generation of peptides, amino acids, and amines, which enhance the buffering capacity. Additionally, the pH values in the L. sakei and P. pentosaceus groups were significantly lower than those of the other two groups during fermentation and ripening (p < 0.05), indicating that L. sakei and P. pentosaceus exhibited higher lactic acid production activity [36]. The lower pH was important to inhibit undesirable microbial growth in the fermented sausages, which is consistent with the microbiological analysis results (Figure 2). L. sakei and P. pentosaceus were more effective in controlling the number of Enterobacteriaceae counts.
The aw values for all sausages showed no significant changes (p > 0.05) during the first 2 days of fermentation, and the aw values of the inoculated samples showed no significant differences (p > 0.05) from the uninoculated samples. This stability can be attributed to the high humidity in the fermentation chamber, which minimized evaporative water loss by maintaining a saturated vapor pressure environment [53]. After 2 days of fermentation, the aw of all the sausages decreased sharply (p < 0.05); final aw values of 0.79~0.81 were observed, at which most microorganisms would cease to grow. The aw values in the samples inoculated with L. sakei and P. pentosaceus were significantly lower than in the other two groups after 8 days (p < 0.05). Previous studies have shown that the rapid growth of inoculated bacteria can accelerate the dehydration of sausages [17]. Furthermore, studies have indicated that a lower pH in inoculated sausages may contribute to a subsequent reduction in aw [14]. This decrease is facilitated by the pH-induced denaturation of myofibrillar proteins, which enhances water expulsion from the protein matrix through capillary action [54,55]. This is consistent with the results of this study; the pH values in the sausages fermented with L. sakei and P. pentosaceus were the lowest, and the aw values were also the lowest. Lower pH values were shown to suppress the growth of some undesirable microbes (Figure 2 and Figure 3B); thus, the TVB-N values in the products could be controlled (Figure 3A). The texture changes were mainly related to the constant dehydration during the ripening process [18,48]. How low pH promotes the dehydration and drying of fermented sausages, resulting in lower aw values and better texture characteristics, is further illustrated by the moisture distribution and protein conformation in this study.

3.6. LF-NMR Analysis

LF-NMR can reflect changes in the mobility and distribution of the moisture inside the myofibrillar gel of sausages [23]. The shortest relaxation time component (T2b) was categorized as water tightly bound to macromolecular structures, predominantly proteins. Water entrapped within the dense myofibrillar protein network was assigned to the intermediate relaxation time (T21), whereas extracellular free water occupying interstitial spaces corresponded to the longest relaxation component (T22) [56].
As shown in Figure 4A–D, during the fermentation process, T2b, T21, and T22 in every group moved to a low relaxation time. The relaxation time suggests that water bound to protein was less stable and more likely to be released [57]. There was no significant difference in P2b between the different treatments during the sausage fermentation and ripening process (Figure 4E–H), which reflects the stability of the bound water closely held by the muscle proteins [58]. P21 in the natural fermented sample and the samples inoculated with L. sakei and P. pentosaceus decreased, while P22 increased, suggesting that part of the immobilized water was converted into free water. For the groups fermented with L. sakei and P. pentosaceus, the immobilized water transferred to free water more obviously (Figure 4F,G), which was conducive to the drying and maturation process of the fermented sausages, and thus could accelerate the drying and ripening process of fermented sausages. However, the change was not observed when fermented with W. cibaria (Figure 4H), indicating that these bacteria are not good for the dehydration and drying process of fermented sausages.

3.7. Protein Structure Based on Raman Spectroscopy Analysis

Figure 5 displays the observed protein secondary structure changes that occurred during the sausage fermentation process. Secondary protein structures, such as amides, were among which those of amide I (1645~1685 cm−1) and amide III (1200~1350 cm−1) were the most useful. Amide I includes α-helix, β-sheet, β-turn, and random coil structures [59].
Increasing the fermentation time significantly (p < 0.05) decreased α-helix contents, but increased β-sheet and β-turn structures (Figure 5); this is similar to the previous results reported by Kang et al., (2016) [60], which may be attributed to the transformation from α-helix structures to β-sheet and β-turn structures. During fermentation for 0~2 days, the α-helix contents in the inoculated groups decreased significantly, which may be related to the dramatic decrease in pH [56]. According to Dumetz et al. (2008) [61], pH can affect the protonation state of charged amino acids and a-carboxyl and a-amino terminal groups at the surface of proteins. By changing the protonation state of the charged residues, the pH impacted the detailed nature of protein interactions. The structure of α-helix represents the regularity of protein molecules, while the structures of β-sheet and β-turn reflect the looseness of protein molecules [62]. The α-helix decreased, accompanied by an increase in β-sheet and β-turn structures, indicating that the protein structure was transformed from orderly to disorderly [63]. This will lead to the exposure of hydrophobic groups, and the water-holding capacity of proteins will be reduced and the dehydration will be accelerated [63,64]. The α-helix decreased more obviously throughout the fermentation process in the samples inoculated with L. sakei and P. pentosaceus. On the one hand, low pH directly led to changes in protein secondary structures; on the other hand, low pH promoted dehydration and drying, resulting in a higher salt concentration that promoted changes in protein secondary structures. At the end of the fermentation, the α-helix of the samples inoculated with W. cibaria was the highes. The results suggest that protein molecular unfolding and peptide chain breaking or structural stretching can be promoted by L. sakei and P. pentosaceus. As a result, the water holding capacity of the proteins decreased because of the exposure of the hydrophobic groups and the immobilized water was transferred to free water [65], which is in agreement with the results observed by LF-NMR (Figure 4). Thus, the dehydration and drying of the sausages were accelerated and the products had better texture characteristics (Table 2). In addition, studies have shown that the β-sheet is the base for and a key factor in gel formation, so an increase in β-sheet percentage during the sausage fermentation and ripening process could improve the gel structure of proteins and increase the hardness of the gel [66]. Herrero et al. (2008) [67] also reported a positive correlation between the hardness of meat and the content of β-turn and β-sheet, which is consistent with the results of TPA changes in this study (Table 1).

4. Conclusions

The results of the current study demonstrated that the autochthonous strains of L. sakei and P. pentosaceus could be used in dry-fermented sausage manufacturing, since they led to better technological characteristics and sensory qualities of fermented sausages, while the strain of W. cibaria was not suitable as a starter culture. More importantly, it was found that the effects of L. sakei and P. pentosaceus on the fermented sausages did not only inhibit harmful microorganisms, as previously reported, but resulted in beneficial changes in the texture characteristics of the fermented sausages. The results suggested that the decrease in pH might have caused changes in the secondary structures of proteins, reducing their water-holding capacity. Bound water could have been converted into free water, which led to faster moisture loss during maturation, resulting in a reduction in aw and an improvement in texture. Furthermore, the sensory evaluation showed that sausages fermented with these strains had enhanced flavor and overall acceptability. This study provides valuable information promoting the development of fermented beef sausages.

Author Contributions

Conceptualization, G.Z. and C.Z.; Methodology, Y.Z., W.C., L.X. and C.Z.; Software, Y.Z.; Validation, Y.Z., S.Z., X.L., W.C., L.X., C.Z. and L.T.; Formal analysis, G.Z., S.Z., X.L., W.C., L.X., C.Z. and L.T.; Investigation, Y.Z.; Resources, G.Z.; Data curation, Y.Z.; Writing—original draft, Y.Z.; Writing—review & editing, Y.Z. and C.Z.; Visualization, Y.Z., S.Z. and X.L.; Supervision, G.Z., S.Z., X.L., W.C., L.X. and L.T.; Project administration, G.Z. and C.Z.; Funding acquisition, G.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Beef Cattle Industrial Technology System (project No. CARS-37) and the Joint Fund of Science and Technology Research and Development Plan of Henan (222103810019).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Research data are available if needed.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effect of different LAB on sensory evaluation of fermented beef sausage. Control: uninoculated sausages; L. sakei: sausages inoculated with L. sakei; P. pentosaceus: sausages inoculated with P. pentosaceus; W. cibaria: sausages inoculated with W. cibaria.
Figure 1. Effect of different LAB on sensory evaluation of fermented beef sausage. Control: uninoculated sausages; L. sakei: sausages inoculated with L. sakei; P. pentosaceus: sausages inoculated with P. pentosaceus; W. cibaria: sausages inoculated with W. cibaria.
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Figure 2. Evolution of microflora during fermentation and ripening of fermented sausages. (A) Lactic Acid Bacteria counts, (B) Staphylococcus and Micrococcus counts, (C) Total viable bacteria counts, (D) Enterobacteriaceae counts. Control: uninoculated sausages; L. sakei: sausages inoculated with L. sakei; P. pentosaceus: sausages inoculated with P. pentosaceus; W. cibaria: sausages inoculated with W. cibaria.
Figure 2. Evolution of microflora during fermentation and ripening of fermented sausages. (A) Lactic Acid Bacteria counts, (B) Staphylococcus and Micrococcus counts, (C) Total viable bacteria counts, (D) Enterobacteriaceae counts. Control: uninoculated sausages; L. sakei: sausages inoculated with L. sakei; P. pentosaceus: sausages inoculated with P. pentosaceus; W. cibaria: sausages inoculated with W. cibaria.
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Figure 3. Evolution of TVB-N value (A) and pH and Aw values (B) during fermentation and ripening of fermented sausages. Control: uninoculated sausages; L. sakei: sausages inoculated with L. sakei; P. pentosaceus: sausages inoculated with P. pentosaceus; W. cibaria: sausages inoculated with W. cibaria.
Figure 3. Evolution of TVB-N value (A) and pH and Aw values (B) during fermentation and ripening of fermented sausages. Control: uninoculated sausages; L. sakei: sausages inoculated with L. sakei; P. pentosaceus: sausages inoculated with P. pentosaceus; W. cibaria: sausages inoculated with W. cibaria.
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Figure 4. Evolution of water populations of three components during fermentation and ripening of fermented sausages. Means with different uppercase letters (A–D) indicate significant differences between different treatments at the same time points (p < 0.05) and means with different lowercase letters (a–e) indicate significant differences at different times for the same treatment (p < 0.05). (A): fermented for 2 days; (B): fermented for 8 days; (C): fermented for 14 days; (D): fermented for 20 days; (E): control, uninoculated sausages; (F): L. sakei, sausages inoculated with L. sakei; (G): P. pentosaceus, sausages inoculated with P. pentosaceus; (H): W. cibaria, sausages inoculated with W. cibaria.
Figure 4. Evolution of water populations of three components during fermentation and ripening of fermented sausages. Means with different uppercase letters (A–D) indicate significant differences between different treatments at the same time points (p < 0.05) and means with different lowercase letters (a–e) indicate significant differences at different times for the same treatment (p < 0.05). (A): fermented for 2 days; (B): fermented for 8 days; (C): fermented for 14 days; (D): fermented for 20 days; (E): control, uninoculated sausages; (F): L. sakei, sausages inoculated with L. sakei; (G): P. pentosaceus, sausages inoculated with P. pentosaceus; (H): W. cibaria, sausages inoculated with W. cibaria.
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Figure 5. Evolution of secondary structure components during fermentation and ripening of fermented sausages. Means with different uppercase letters (A, B) indicate significant differences between different treatments at the same time points (p < 0.05) and means with different lowercase letters (a–d) indicate significant differences at different times for the same treatment (p < 0.05). (A): control, uninoculated sausages; (B): L. sakei, sausages inoculated with L. sakei; (C): P. pentosaceus, sausages inoculated with P. pentosaceus; (D): W. cibaria, sausages inoculated with W. cibaria.
Figure 5. Evolution of secondary structure components during fermentation and ripening of fermented sausages. Means with different uppercase letters (A, B) indicate significant differences between different treatments at the same time points (p < 0.05) and means with different lowercase letters (a–d) indicate significant differences at different times for the same treatment (p < 0.05). (A): control, uninoculated sausages; (B): L. sakei, sausages inoculated with L. sakei; (C): P. pentosaceus, sausages inoculated with P. pentosaceus; (D): W. cibaria, sausages inoculated with W. cibaria.
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Table 1. Scoring criteria for the sensory evaluation of fermented sausages.
Table 1. Scoring criteria for the sensory evaluation of fermented sausages.
Evaluation Criteria
(Weight)		StandardScore
Organization condition
(20%)		The meat is firm in texture, with a smooth and even surface, and distinct separation between lean meat and fat particles.15~20
The meat is relatively firm in texture, with a relatively smooth and even surface.10~15
The meat has a somewhat loose texture, allowing for slicing, but lacks smoothness and evenness on the surface.5~10
The meat is not densely compacted, making it impossible to form slices.1~5
Color and lustre
(20%)		The lean meat presents a deep red hue, while the fat appears pristine white with a lustrous sheen.15~20
The lean meat is slightly red, appearing slightly dull, while the fat is whiter.10~15
The lean meat is a dark red, while the fat has a slight yellowish hue.5~10
The lean meat darkens or takes on a greenish hue, while the fat becomes yellowish, with indistinct boundaries.1~5
Special flavor
(20%)		The unique flavor is rich and free from any off-notes.15~20
The fermented aroma is pronounced, with no off-notes present.10~15
The fermented aroma is subtle, with slight off-notes present.5~10
There are no distinctive flavors, but there are off-notes present.1~5
Chewiness
(20%)		Firm and resilient with good chewiness.15~20
There is a certain level of firmness and resilience, with good chewability.10~15
Too hard or somewhat loose, with average chewability.5~10
Difficult to chew.1~5
Overall acceptability
(20%)		Strong appetite.15~20
Intense appetite.10~15
Has appetite.5~10
Essentially no appetite.1~5
Table 2. Changes in texture during fermentation and ripening of fermented sausages.
Table 2. Changes in texture during fermentation and ripening of fermented sausages.
TimeTreatmentHardnessCohesivenessSpringCohesionChewiness
2 dControl31.40 ± 2.60 b0.20 ± 0.00 a1.33 ± 0.09 b7.47 ± 0.76 b10.07 ± 1.50 b
L. sakei53.65 ± 4.98 a0.30 ± 0.00 a2.37 ± 0.17 a15.20 ± 1.31 a36.13 ± 4.74 a
P. pentosaceus56.33 ± 3.03 a0.30 ± 0.00 a2.38 ± 0.11 a15.67 ± 0.85 a37.33 ± 2.00 a
W. cibaria27.03 ± 3.78 c0.20 ± 0.00 b1.47 ± 0.41 b5.24 ± 1.05 c7.70 ± 1.55 c
8 dControl78.00 ± 2.45 b0.30 ± 0.00 a1.89 ± 0.05 b26.37 ± 1.01 c52.33 ± 2.61 b
L. sakei109.27 ± 2.96 a0.30 ± 0.00 a2.10 ± 0.10 a32.17 ± 0.40 a67.60 ± 2.42 a
P. pentosaceus110.77 ± 3.99 a0.27 ± 0.06 a2.37 ± 0.07 a27.73 ± 0.87 b62.57 ± 2.97 a
W. cibaria31.30 ± 0.56 c0.20 ± 0.00 b1.27 ± 0.03 c6.87 ± 0.25 d8.70 ± 0.56 c
14 dControl95.70 ± 2.42 b0.30 ± 0.00 a2.72 ± 0.08 a26.80 ± 1.64 b72.73 ± 4.44 b
L. sakei152.98 ± 14.43 a0.30 ± 0.00 a2.84 ± 0.13 a40.30 ± 3.91 a114.30 ± 9.16 a
P. pentosaceus151.65 ± 5.06 a0.30 ± 0.00 a2.80 ± 0.11 a38.78 ± 1.50 a108.50 ± 4.86 a
W. cibaria46.75 ± 2.16 c0.20 ± 0.00 b1.25 ± 0.08 b8.18 ± 0.36 c10.20 ± 0.81 c
20 dControl150.00 ± 24.85 b0.20 ± 0.00 a2.42 ± 0.35 a31.43 ± 8.32 b73.93 ± 13.11 b
L. sakei208.48 ± 17.80 a0.23 ± 0.05 a2.53 ± 0.17 a50.30 ± 3.02 a126.95 ± 3.82 a
P. pentosaceus222.90 ± 12.10 a0.20 ± 0.00 a2.69 ± 0.22 a49.50 ± 4.55 a132.85 ± 13.69 a
W. cibaria61.43 ± 3.24 c0.13 ± 0.05 b1.38 ± 0.04 b8.88 ± 0.10 c12.23 ± 0.33 c
The symbols represent mean ± standard deviation obtained from three independent experiments. “a–d” indicate significant at the same ripening time (n = 3, p < 0.05). Control: uninoculated sausages; L. sakei: sausages inoculated with L. sakei; P. pentosaceus: sausages inoculated with P. pentosaceus; W. cibaria: sausages inoculated with W. cibaria.
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Zheng, Y.; Zhao, G.; Zhao, S.; Li, X.; Cui, W.; Xu, L.; Zhu, C.; Tong, L. Don’t Judge a Sausage by Its Cover: Effects of Inoculating Three Indigenous Lactic Acid Bacteria on Quality, Moisture Distribution, and Protein Structure in Fermentation. Fermentation 2025, 11, 134. https://doi.org/10.3390/fermentation11030134

AMA Style

Zheng Y, Zhao G, Zhao S, Li X, Cui W, Xu L, Zhu C, Tong L. Don’t Judge a Sausage by Its Cover: Effects of Inoculating Three Indigenous Lactic Acid Bacteria on Quality, Moisture Distribution, and Protein Structure in Fermentation. Fermentation. 2025; 11(3):134. https://doi.org/10.3390/fermentation11030134

Chicago/Turabian Style

Zheng, Yangyi, Gaiming Zhao, Shichang Zhao, Xuan Li, Wenming Cui, Long Xu, Chaozhi Zhu, and Lin Tong. 2025. "Don’t Judge a Sausage by Its Cover: Effects of Inoculating Three Indigenous Lactic Acid Bacteria on Quality, Moisture Distribution, and Protein Structure in Fermentation" Fermentation 11, no. 3: 134. https://doi.org/10.3390/fermentation11030134

APA Style

Zheng, Y., Zhao, G., Zhao, S., Li, X., Cui, W., Xu, L., Zhu, C., & Tong, L. (2025). Don’t Judge a Sausage by Its Cover: Effects of Inoculating Three Indigenous Lactic Acid Bacteria on Quality, Moisture Distribution, and Protein Structure in Fermentation. Fermentation, 11(3), 134. https://doi.org/10.3390/fermentation11030134

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