Harbin Dry Sausages Research Articles

Impact and correlation of fermentation temperature on the bacterial community, flavor characteristics, and proteolysis of Harbin dry sausages

Proteins undergo degradation to produce peptides and free amino acids, which in turn promote the production of volatile compounds with important contributions to the taste and aroma. This study investigated the effect of fermentation temperatures (8, 15, and 25 °C) on the bacterial community, flavor profile, and protein degradation of Harbin dry sausages. The physical and chemical properties were improved at 25 °C compared with 8 and 15 °C. Staphylococcus xylosus increased with the increase in fermentation temperature, whereas Latilactobacillus sakei decreased. The degree of protein degradation increased, and the content and proportion of taste peptides and free amino acids increased. Similarly, a higher fermentation temperature led to an increase in volatile compounds, such as aldehydes, alcohols, and esters. In conclusion, there is an inseparable relationship between proteolysis and microbial proteases. The fermentation temperature of 25 °C best contributed to the sensory quality and flavor characteristics of Harbin dry sausages.

Effect of glycine and Pediococcus pentosaceus R1 combined application on the physicochemical properties, oxidative stability, and taste profile of Harbin dry sausages

This study investigated the effects of the application of glycine (Gly) and Pediococcus pentosaceus R1(Pp), alone or in combination, on the physicochemical properties, oxidative stability, and taste quality of Harbin dry sausages. The results demonstrated that after nine days of fermentation, the Gly + Pp group exhibited significantly (P < 0.05) lower moisture content (19.04%), water activity (0.686), and pH (4.78) values, alongside notably (P < 0.05) higher lactic acid bacteria count (8.11 log CFU/g sausage) and redness value (17.2), compared to the other three groups (P < 0.05). In addition, the dry sausages in the Gly + Pp group exhibited the lowest peroxide value (0.34 meq/kg sausage), thiobarbituric acid reactive substances (0.46 MAD/kg sausage), and protein carbonyl content (1.26 nmol/kg protein) during fermentation, followed by the Gly group, Pp group, and control group. Electronic tongue (e-tongue) and sensory evaluations revealed that the combined treatment with P. pentosaceus R1 and Gly resulted in superior taste characteristics. Besides, partial least squares regression (PLSR) analysis illustrated that the taste qualities characterized using the e-tongue were accordant with the sensory evaluation consequences, and total free amino acids (FAAs) and organic acids contributed to the dry sausages' taste properties. In conclusion, the combined application of Gly and P. pentosaceus R1 enhanced the physicochemical properties, oxidative stability, and taste profile of Harbin dry sausages.

Effect of Yeast Inoculation on the Bacterial Community Structure in Reduced-Salt Harbin Dry Sausages: A Perspective of Fungi-Bacteria Interactions.

Yeast strains are promising starters to compensate for the flavor deficiencies of reduced-salt dry sausages, but their influence on the bacterial community's structure has not yet been clarified. In this study, the effect of separately inoculating Pichia kudriavzevii MDJ1 (Pk) and Debaryomyces hansenii HRB3 (Dh) on the bacterial community structure in reduced-salt dry sausage was investigated. The results demonstrated that the inoculation of two yeast strains significantly reduced the pH, and enhanced the total acid content, lactic acid bacteria (LAB) counts, and total bacterial counts of reduced-salt sausages after a 12-day fermentation (p < 0.05). Furthermore, high-throughput sequencing results elucidated that the inoculation of yeast strains significantly affected the bacterial composition of the dry sausages. Especially, the relative abundance of bacteria at the firmicute level in the Pk and Dh treatments exhibited a significant increase of 83.22% and 82.19%, respectively, compared to the noninoculated reduced-salt dry sausage treatment (Cr). The relative abundance of Latilactobacillus, especially L. sakei (0.46%, 2.80%, 65.88%, and 33.41% for the traditional dry sausage (Ct), Cr, Pk, and Dh treatments, respectively), increased significantly in the reduced-salt sausages inoculated with two yeast strains. Our work demonstrates the dynamic changes in the bacterial composition of reduced-salt sausages inoculated with different yeast strains, which could provide the foundation for the in-depth study of fungi-bacteria interactions in fermented foods.

Proteolysis and quality characteristics of Harbin dry sausages caused by the addition of Staphylococcus xylosus protease

This study aimed to investigate the effect of different concentrations of Staphylococcus (S.) xylosus protease on the proteolysis, quality characteristics, flavor development, and sensory attributes of dry sausages. The results indicated that S. xylosus protease significantly decreased (P < 0.05) the moisture content, water activity, shear force, pH value, lipid and protein oxidation of the dry sausages. Moreover, the addition of S. xylosus protease to Harbin dry sausages accelerated meat proteins proteolysis and development of key differential volatile compounds such as ketones, acids, and esters. The best sensory score was obtained at 1.2 g/kg. Additionally, molecular docking analysis suggested that hydrogen bonds and hydrophobic interactions force were the mainly driving forces in the S. xylosus protease-myosin complex. This study revealed that the addition of S. xylosus protease to Harbin dry sausages is a novel strategy for improving their quality and flavor.

Purification and Characterization of the Protease from Staphylococcus xylosus A2 Isolated from Harbin Dry Sausages.

The protease generated from Staphylococcus (S.) xylosus A2, which was isolated from Harbin dry sausages, was purified and characterized. The molecular weight of the purified protease was approximately 21.5 kDa, and its relative activity reached the highest at pH 6.0 and 50 °C. At pH 4.0–8.0 and temperatures of 20–50 °C, the protease was stable. Its activity was significantly improved by Ca2+ and Zn2+ ions (p < 0.05). The Michaelis constant and maximum velocity of the protease were 2.94 mg/mL and 19.45 U/mL·min, respectively. The thermodynamic parameters analysis suggested that the protease showed better catalytic properties at 40 °C. Moreover, the protease could hydrolyze meat proteins, and obtained hydrolysate is non-cytotoxic to the HEK-293 cells. These findings provide a theoretical basis for understanding the enzymatic characterization of S. xylosus A2 protease and its future application in fermented meat products.

Proteomic response strategies of Pediococcus pentosaceus R1 isolated from Harbin dry sausages to oxidative stress

The tolerance strategies of Pediococcus pentosaceus R1 to sublethal dose of H2O2 induced-oxidative stress were investigated with proteomic approaches. The results revealed that a total of 117 proteins were identified as differentially expressed proteins. Eight antioxidant enzymes were upregulated expression to defence the oxidative stress, especially manganese catalase. Besides, 14 proteins participating in carbohydrate and energy metabolism, were significantly upregulated expression (P < 0.05), in which six proteins involved in the two major pathways, pentose phosphate pathway and pyruvate metabolism. Furthermore, the results showed that the expression of four proteins related to the repair of oxidatively damaged proteins was upregulated (P < 0.05); and the upregulated expression was also found in five ATP-binding cassette transporter-related proteins, which involved in defence oxidative stress in P. pentosaceus R1 by regulating the transmembrane transport of many substrates (P < 0.05). Conversely, the expression of 13 proteins related to nucleotide biosynthesis, and four proteins participating in Fe–S cluster assembly, was significantly downregulated (P < 0.05). This work will facilitate the understanding of the antioxidative mechanisms in P. pentosaceus R1 and will provide valuable information for further engineering oxidation-tolerant strains as meat starter cultures.

Composite Gel Fabricated with Konjac Glucomannan and Carrageenan Could Be Used as a Cube Fat Substitute to Partially Replace Pork Fat in Harbin Dry Sausages.

The effect of the partial replacement of pork back-fat with a cube fat substitute (CFS) fabricated from konjac glucomannan and carrageenan on the physicochemical and sensory characteristics of Harbin dry sausages during 7 days of fermentation was investigated. There were the following five treatments: control (100% back-fat), FS1 (80% back-fat, 20% CFS), FS2 (60% back-fat, 40% CFS), FS3 (40% back-fat, 60% CFS) and FS4 (20% back-fat, 80% CFS). The results showed no significant differences (p > 0.05) in the physicochemical and sensory characteristics among the control, FS1 and FS2 treatments. However, higher replacement levels (60% and 80%) rendered higher degrees of change in the characteristics of the sausages, lowering the moisture content and aw and increasing the pH, hardness, chewiness and atypical appearance at the end of fermentation. Moreover, electronic nose analysis and hierarchical cluster analysis demonstrated that the FS3 and FS4 treatments destroyed the characteristic quality of the sausage. Overall, our results indicated that, to ensure the traditional characteristics of Harbin dry sausages, the upper limit of the fat replacement level with CFS should be set at 40%.

Effects of temperature and pH on the structure of a protease from Lactobacillus brevis R4 isolated from Harbin dry sausage and molecular docking of the protease to the meat proteins

Spectroscopy and molecular dynamics were utilized to evaluate the influence of pH and temperature on the structural characteristics of Lacobacillus brevis R4 protease isolated from Harbin dry sausage. The protease has a stable spatial structure at pH 7 and 40 °C, and the extended secondary structure of the protease was identified. The structure of the protease can be changed or denatured at pH 8 and 70 °C, the changes at which predominantly manifested as changes in the secondary structure, such as α-helices, β-sheets, β-turns and random coils. In addition, carbonyl vibration, –NH vibration, C–H stretching vibration and disulfide bonds were present in the L. brevis R4 protease at various pH and temperatures. Molecular docking showed that the protease can interact with the myosin light chain, myosin heavy chain and troponin, which demonstrates the protease may accelerate the degradation of meat proteins, promote meat tenderness and then improve the flavor and quality of Harbin dry sausages.

Physiological, Morphological and Antioxidant Responses of Pediococcus pentosaceus R1 and Lactobacillus fermentum R6 Isolated from Harbin Dry Sausages to Oxidative Stress.

As functional starter cultures and potential probiotics, the ability of lactic acid bacteria to resist oxidative stress is essential to maintain viability and functional properties. This study investigates the effects of H2O2 at different concentrations (0, 1, 2, and 3 mM) on the physiological, morphological, and antioxidant properties of Pediococcus pentosaceus R1 and Lactobacillus fermentum R6 isolated from Harbin dry sausages. The increase in H2O2 concentration induced a significant increase in reactive oxygen species and a decrease in intracellular ATP levels (p < 0.05). Based on scanning electron microscopy, transmission electron microscopy, and electric conductivity analysis, H2O2 stress caused cell deformation, the destruction of cell membrane integrity, partial loss of the cytoplasm, and an increase in the cell conductivity of both strains. H2O2 stress with 1 mM or 2 mM concentrations could effectively improve the scavenging rates of free radicals, the activities of superoxide dismutase and glutathione peroxide, and the total antioxidant capacity of both strains (p < 0.05). In conclusion, an appropriate oxidative stress contributed to the activation of the antioxidant defense system of both strains, conferred strains a better effect in inhibiting the oxidation of fermented foods, and improved the health of the host.

Biochemical properties of extracellular protease from Staphylococcus epidermidis isolated from Harbin dry sausages and its hydrolysis of meat protein

This study aimed to explore biochemical properties of extracellular protease from Staphylococcus (S.) epidermidis BP9 isolated from Harbin dry sausages and its future potential application in Harbin dry sausages. The protease was purified by 80% ammonium sulfate, ion exchange, and gel filtration chromatography to obtain a 24 kDa extracellular protease. The protease reached maximal activity at pH 6.0 and 50 °C and was stable at pH 4.0–9.0 and 20–40 °C. Its protease activity was inhibited in the presence of Fe2+. The enzymatic characterization of the protease revealed a Vmax 62.5 U/mL·min, Km 11.76 mg/mL, and the half-life = 62.44 min, ΔH*d = 72.11 kJ/mol, ΔG*d = 91.40 kJ/mol, and ΔS*d = −59.60 J/mol·K at 50 °C. In addition, the protease hydrolysed myofibrillar protein into small particles and produced soluble peptides. This study provides a basis for understanding the biochemical characteristics of the S. epidermidis BP9 protease and its future application for Harbin dry sausages.

Biochemical properties of extracellular protease from Staphylococcus carnosus RT6 isolated from Harbin dry sausages, and its hydrolysis of meat proteins

The characteristics of the extracellular protease, produced by Staphylococcus carnosus RT6 isolated from Harbin dry sausages, and its hydrolysis of meat proteins were investigated. The protease was purified by ammonium sulfate, ion exchange, and gel filtration chromatography to obtain a 20.0kDa extracellular protease. The protease reached maximal activity at pH 9.0 and 50 °C and was stable at pH 7.0 to 11.0 and 20 to 40 °C. Its protease activity was easily inhibited in the presence of Zn2+ , Fe2+ , and Fe3+ . The enzymatic characterization of the protease revealed a Vmax 49.50 U/ml·min, Km 8.19mg/ml, and the half-life=28.06min, ΔH* d =114.11kJ/mol, ΔG* d =89.24kJ/mol, and ΔS* d =77.00 J/mol·K at 50 °C. In addition, the protease hydrolyzed meat protein into small particles and produced soluble peptides. This study provides a basis for understanding the biochemical characteristics of the S. carnosus RT6 protease and its future application for fermented meat products.

Effects of temperature and pH on the structure of a metalloprotease from Lactobacillus fermentum R6 isolated from Harbin dry sausages and molecular docking between protease and meat protein.

Microbial protease can interact with meat protein in fermented meat products at a certain pH and temperature. To investigate the effects of various pH values and temperatures on the structural characteristics of Lactobacillus fermentum R6 protease, which was isolated from Harbin dry sausages, spectroscopy techniques and molecular dynamics were utilized to evaluate structural changes. The protease exhibited a stable spatial structure at pH 7 and 40 °C, and the extension of the protease structure was also promoted. Although the structure of the protease could be changed or destroyed by pH 8 and 70 °C, it was mainly determined by the changes of secondary and tertiary structures such as α-helix, β-sheet, β-turn and random coil. In addition, carbonyl vibration, -NH vibration, C-H stretching vibration and disulphide bonds were present in L. fermentum R6 protease under various pH and temperature conditions. Molecular docking showed that the protease can interact with myosin light chain, myosin heavy chain, actin and myoglobin. The protease can maintain stable structure and interact with meat protein, which reflected certain application prospects in the fermentation of Harbin dry sausages. © 2021 Society of Chemical Industry.

Fungal community succession and volatile compound dynamics in Harbin dry sausage during fermentation

This study investigated the fungal community succession and volatile compound dynamics of Harbin dry sausage during a twelve-day fermentation using high-throughput internal transcribed spacer amplicon sequencing and headspace solid-phase microextraction gas chromatography-mass spectrometry. Aspergillus pseudoglaucus was found to be the primary species in the sausages during fermentation, whereas Lasiodiplodia theobromae, Alternaria alternata, Aspergillus caesiellus, and Trichosporon asahii were also prevalent. Additionally, a total of 72 volatile compounds were identified in the dry sausages, of which 24 key compounds (odor activity value > 1) dominated flavor development, including 3 aldehydes, 1 ketone, 4 alcohols, 9 esters, 4 alkenes, and 3 other compounds. Furthermore, correlation analysis suggested that most of the core fungi were positively correlated with the key volatile compounds, particularly A. pseudoglaucus, Aspergillus gracilis, Trichosporon caseorum, Debaryomyces hansenii, and T. asahii. Our findings provide novel insights into the fungal ecology and flavor development of Harbin dry sausages.

The succession and correlation of the bacterial community and flavour characteristics of Harbin dry sausages during fermentation

This study was to investigate the succession of the bacterial community in Harbin dry sausages during fermentation and its correlation to the sausage flavour. The results showed that Staphylococcus count increased but the moisture content and water activity decreased (P < 0.05). The lactic acid bacteria count first increased and then decreased, while the pH showed an opposite trend during a twelve-day fermentation (P < 0.05). According to a single molecule real time sequencing analysis, Lactobacillus sakei and Staphylococcus xylosus were the predominant bacterial species over the entire fermentation period. A total of 79 volatile compounds were detected using the headspace solid phase microextraction gas chromatography-mass spectrometry. Furthermore, the correlation between the core bacteria (top 30) and the major volatile compounds was explored, which highlighted that three bacterial species (Lb. sakei, S. xylosus and Weissella hellenica) were significantly correlated with the major volatile compounds, such as 2,3-butanediol, ethanol, 3-hydroxy-2-butanone, ethyl hexanoate and ethyl lactate. The results may provide a theoretical basis for improving the flavour and overall quality of dry sausage.

Ultrasonic-assisted extraction of polyphenol from the seeds of Allium senescens L. and its antioxidative role in Harbin dry sausage

The ultrasonic-assisted extraction of total polyphenol from Allium senescens L. (ASL) seeds was conducted, and the antioxidant efficacy of ASL seed extract (ASLSE) was assessed in Harbin dry sausages. ASLSE extracted with 1:30 g/mL water at 150 W for 15 min had the highest antioxidant capacities (P < 0.05). Subsequently, different addition levels (0, 2, 4, 6 and 8 g/kg) of freeze-dried ASLSE were applied in dry sausages during a twelve-day fermentation, and BHT treatment was the positive control. The lower pH values and carbonyl contents were detected in the treatments with 6 and 8 g/kg ASLSE than those in the other treatments at 12 d (P < 0.05). However, there was no difference in the water activity, lipid oxidation and color among the treatments with 6 and 8 g/kg ASLSE and 0.2 g/kg BHT (P > 0.05) at 12 d. These results indicated that 6 g/kg ASLSE could be effective in inhibiting lipid and protein oxidation and reducing color deterioration of dry sausages.

Beneficial effects of Jerusalem artichoke powder and olive oil as animal fat replacers and natural healthy compound sources in Harbin dry sausages

This study aimed to improve the nutritional quality of Harbin dry sausages using natural plant-based Jerusalem artichoke powder (JAP) and olive oil as animal fat replacers. Low-fat Harbin dry sausages were manufactured with 2 different formulations containing JAP and olive oil as pork fat replacers. The texture, rheological properties, microstructure, water holding capacity, muscle protein structure, physicochemical indices, microbiological characteristics, and sensory evaluation of the sausages were analyzed. The result showed that Harbin dry sausages with JAP and olive oil were healthier than control sausages based on the lower fat content and improved fatty acid composition. Scanning electron microscopy showed gel network formation in sausages with a high JAP content. Low-field nuclear magnetic resonance illustrated that the water-holding capacity of the modified sausages was improved, suggesting that the replacers enhanced protein gel formation by changes in C–H stretching and bending vibrations, a reduction in α-helixes, and increases in β-sheets and random coils accompanying the exposure of reactive groups and microenvironment of the tertiary structure. Dynamic rheological and texture tests indicated that the replacers improved the elasticity of sausages. The reduction of fat and addition of replacers significantly enhanced lipid oxidative resistance. Overall, JAP and olive oil improved the fatty acid composition, gel structure, lipid oxidative resistance, and sensory quality of the sausages. These results may contribute to the development of healthy meat products to further reduce animal fat.

Metabolomics profiling reveals defense strategies of Pediococcus pentosaceus R1 isolated from Harbin dry sausages under oxidative stress

The physiological performance and whole-cell metabolomic response of Pediococcus pentosaceus R1 to oxidative stress were investigated. The response to a sublethal concentration (3 mM) of H2O2 resulted in a time-dependent decrease in viable cells. After 1 h of oxidative stress, the level of reactive oxygen species (ROS) and the activities of antioxidant enzymes significantly increased (P < 0.05). Metabolomics analysis identified 74 metabolites as significant differentially metabolites (P < 0.05). The increased levels of amino acids were mainly involved in the supply of energy, and two major amino acids (l-glutamine and l-histidine) can act as antioxidants to protect the bacteria against oxidative stress. The accumulation of saccharides (d-galactose, d-glucose and d-maltose) may be an energy conservation strategy for the bacteria. The levels of saturated fatty acids (myristic acid, caproic acid and dodecanoic acid), which could prevent the entry of H2O2 by decreasing membrane fluidity, significantly increased (P < 0.05). This research facilitates understanding of the antioxidative mechanisms of P. pentosaceus R1 and will benefit the construction of engineering probiotics that can better tolerate stress factors when applied in food industrial processes.

Production, purification and biochemical characterization of the microbial protease produced by Lactobacillus fermentum R6 isolated from Harbin dry sausages

This study investigated the purification and biochemical characterization of the protease produced by Lactobacillus fermentum R6 isolated from Harbin dry sausages. The optimized fermentation conditions were as follows: a fermentation time of 48 h, an initial pH of 6 and a fermentation temperature of 37 °C. The 37.7 kDa extracellular protease was purified using ammonium sulphate deposition, an ion exchange layer system and gel filtration. The protease produced by L. fermentum R6 had the highest initial velocity and kcat/Km at pH 6, 40 °C. The microbial protease activity could be inhibited by ethylene diamine tetraacetic acid disodium salt (EDTA). The Vmax and Km of the protease were 58.2 ± 1.42 mg/min and 17.3 ± 0.85 mg/mL, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) reflected the ability of the protease to hydrolyse myofibrillar and sarcoplasmic proteins, in particular, myosin heavy chain, paramyosin, phosphorylase and creatine kinase-M type. In conclusion, L. fermentum R6 can be used as a starter culture or an enzyme-producing strain for the inoculation of Harbin dry sausages.

Quality characteristics and flavor profile of Harbin dry sausages inoculated with lactic acid bacteria and Staphylococcus xylosus

Pediococcus pentosaceus (Pp), Lactobacillus curvatus (Lc), Lactobacillus sakei (Ls) and Staphylococcus xylosus (Sx) were evaluated to determine their roles in the quality characteristics and flavor formation in Harbin dry sausage. The inoculation of dry sausages with bacterial strains, especially mixed strains (Pp + Sx + Lc and Pp + Sx + Ls), significantly decreased the moisture content and water activity. Compared with the sausages inoculated with a single strain and non-inoculated, those inoculated with mixed strains had shorter transverse relaxation time T21 that represents the immobilized water (P < 0.05) after a nine-day fermentation. The texture profile analysis showed that inoculation with bacterial strains increased the hardness and springiness of sausages. The sausages inoculated with mixed strains showed higher a*-values (P < 0.05). Furthermore, the percentages of some aldehydes, ketones, alcohols, acids and esters in the sausage inoculated with Pp + Sx + Lc were significant higher than that in control sausages (P < 0.05), which may be derived from carbohydrate catabolism (such as 3-hydroxy-2-butanone), amino acid metabolism (such as 3-phenylpropanol, 2,3-butanediol, phenylethyl alcohol, 2-methyl-propanal, 2-methyl-butanal and 3-methyl-butanal), β-lipid oxidation (such as 2-pentanone, 2-heptanone and 2-nonanone) and esterification (such as ethyl esters).

Purification and biochemical characteristics of the protease from Lactobacillus brevis R4 isolated from Harbin dry sausages

This study investigated the purification and biochemical characteristics of the protease secreted by Lactobacillus brevis R4 that was isolated from Harbin dry sausages. The optimized fermentation conditions were a fermentation time of 36 h, an initial pH of 5 and a fermentation temperature of 42 °C. A 27.9 kDa extracellular protease was purified using ammonium sulfate precipitation, ion exchange layer and gel filtration and was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protease produced by L. brevis R4 reached the highest relative protease activity at pH 7, 35 °C and 36 h. The microbial protease activity was inhibited by Zn2+, Cu2+ ions and ethylene diamine tetraacetic acid disodium salt (EDTA). The maximum velocity of the reaction (Vmax) and Michaelis constant (Km) of the protease were 65.9 mg/min and 17.1 mg/mL, respectively. SDS-PAGE demonstrated the ability of the protease to hydrolyse myofibrillar and sarcoplasmic proteins, especially on the myosin heavy chain, paramyosin, troponin, myosin light chain and glyceraldehyde dehydrogenase. The study provides a basis for understanding the enzymatic properties of the L. brevis R4 protease. In conclusion, L. brevis R4 has the potential to be used as a starter culture or protease-producing strain to produce Harbin dry sausages.