A double bacteria compound starter and application thereof

Abstract

The application discloses a double-bacterium compound starter and application thereof. The double-bacterium compound starter is composed of lactobacillus plantarum and staphylococcus equorum at a weight ratio of 1:1 to 5:1.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a dual-strain compound fermentation agent and its application. Background Technology

[0002] Longtian Zha Rou (Longtian braised pork) is a locally distinctive meat-vegetable co-fermented product, but currently it mainly relies on natural fermentation, which suffers from problems such as unstable microbial composition, quality fluctuations, and insufficient industrial control. Existing research mainly focuses on its quality and microbial community changes, and there is a lack of development of a native fermentation agent specifically for Longtian Zha Rou. This study screens functional strains suitable for this system and constructs a compound fermentation agent, which can improve fermentation controllability and enhance acidification, lipid oxidation, color stability, and textural quality. Summary of the Invention

[0003] The present invention aims to overcome the shortcomings of the prior art and provide a dual-strain compound fermentation agent and its application.

[0004] To achieve the above objectives, the technical solution provided by this invention is as follows: The dual-strain compound fermentation agent is composed of Lactobacillus plantarum and Staphylococcus equi in a weight ratio of 1:1 to 5:1, preferably 1:1.

[0005] Preferably, the *Lactobacillus plantarum* has the CGMCC No. 39555 and is named *Lactobacillus plantarum* (…). Lactobacillus plantarum LP 17 was deposited on July 3, 2026, at the China General Microbiological Culture Collection Center (Beijing); the *Staphylococcus equi* accession number is CGMCC No. 39554, and it is named *Staphylococcus equi*. Staphylococcus equorum SE 55 was deposited at the China General Microbiological Culture Collection Center (Beijing) on ​​July 3, 2026.

[0006] Preferably, the *Lactobacillus plantarum* and *Staphylococcus equi* are selected from Longtian braised pork.

[0007] The above-mentioned dual-strain compound fermentation agent can be used for fermenting Longtian braised pork.

[0008] The present invention will be further described below: This invention constructs a starter culture composed of *Lactobacillus plantarum* and *Staphylococcus equi*. The starter culture was selected primarily based on the protease and lipase activities of the strains and is suitable for the targeted fermentation of high-fat fermented meat products such as Longtian braised pork. Tests showed that the selected *Lactobacillus plantarum* exhibited strong protease activity and some lipase activity, while *Staphylococcus equi* showed good lipase and protease activity. The combined strains maintained their ability to degrade proteins and fats. When this compound starter culture was inoculated into the Longtian braised pork system, it promoted the formation of dominant bacterial communities of lactic acid bacteria and staphylococci during fermentation. Through acidification, moderate protein hydrolysis, and moderate fat hydrolysis, it promoted product maturation and flavor development.

[0009] Compared to the uninoculated naturally fermented group, this compound starter culture exhibited a more stable fermentation control effect in the non-fried Longtian braised pork system. In the mid-to-late stages of fermentation, the inoculated group showed reduced levels of volatile basic nitrogen and lipid oxidation, indicating that the promoted protein and fat degradation was primarily a controllable enzymatic maturation process rather than putrefactive decomposition. Simultaneously, the inoculation treatment improved product texture and color, increased lean meat maturity, more pronounced fat softening, and improved meat redness and overall fermentation color. Based on comprehensive microbiological, physicochemical, textural, and color indicators, this compound starter culture can improve the controllability and quality stability of the Longtian braised pork fermentation process, and can shorten the suitable fermentation cycle for non-fried Longtian braised pork from the traditional approximately 50 days to approximately 40 days, demonstrating application value in shortening the production cycle, improving product consistency, and enhancing fermentation quality. Attached Figure Description

[0010] Figure 1 The results of bacterial community α-diversity analysis during the fermentation process of Longtian braised pork in the fried and non-fried groups are presented.

[0011] Figure 2 The results of the bacterial community structure difference analysis during the fermentation process of Longtian braised pork in the fried group and the non-fried group.

[0012] Figure 3 The results of heatmap analysis of the total number of OTUs, dominant bacterial genera, and genera level during the fermentation process of Longtian braised pork.

[0013] Figure 4 This represents the results of changes in the horizontal bacterial community composition during the fermentation process of Longtian braised pork.

[0014] Figure 5 The results are from the blood plate test of candidate Staphylococcus equi strains.

[0015] Figure 6 The results of the rabbit plasma coagulase test for candidate Staphylococcus equi strains.

[0016] Figure 7To screen strains, the colony morphology, Gram staining morphology, growth curve, and antagonism test results were analyzed.

[0017] Figure 8 Phylogenetic tree of selected strains constructed based on 16S rRNA gene sequences.

[0018] Figure 9 The results show the changes in microbial counts during the inoculation and fermentation process of Longtian braised pork.

[0019] Figure 10 The results show the changes in basic physicochemical indicators of Longtian braised pork during the inoculation and fermentation process.

[0020] Figure 11 The results show the changes in the main textural indicators of lean and fatty meat in Longtian braised pork during the inoculation and fermentation process. Detailed Implementation

[0021] All experimental samples were taken from Ningxiang City, Changsha.

[0022] Group Z represents non-fried Longtian braised pork; Group N represents fried Longtian braised pork.

[0023] 1. Materials and Methods 1.1 Materials and Reagents Samples of Longtian Zha Rou (a type of braised pork) at different fermentation stages were collected from a local intangible cultural heritage preservation factory in Ningxiang County, Hunan Province, China (28.2774°N, 112.5518°E). Samples were collected every 10 days during fermentation from day 0 to day 50. Ningxiang Huazhu pork and shredded radish were purchased from a local supermarket. Camellia oil was purchased from Hunan Guitaitai Camellia Oil Technology Co., Ltd. (Hunan, China). De Man, Rogosa and Sharpe (MRS) agar, mannitol salt agar (MSA), MRS broth, nutrient broth, and total bacterial count agar (PCA) were purchased from Guangdong Huankai Microbial Technology Co., Ltd. Bromocresol purple was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Blood agar plates were purchased from Hunan Bickman Biotechnology Co., Ltd. Freeze-dried rabbit plasma was purchased from Qingdao Haibo Biotechnology Co., Ltd. Protease and lipase detection kits were purchased from Nuomin Keda Biotechnology Co., Ltd. Unless otherwise specified, all reagents were of analytical grade.

[0024] 1.2 Preparation of Longtian Braised Pork The preparation of deep-fried Longtian braised pork followed the method reported by Lou et al. (2025b), and this group was defined as Group N. The preparation of non-deep-fried Longtian braised pork involved slight modifications to the traditional process. Ningxiang flower pig pork was cut into pieces 3–5 cm wide and marinated with 2% salt for 2 days. Fresh white radishes were washed, cut into radish shreds approximately 0.2 cm thick, and sun-dried. Subsequently, the deep-fried pork was wrapped in the radish shreds at a meat-to-radish mass ratio of 4:6. The wrapped samples were placed in earthenware jars and naturally fermented under anaerobic conditions for 50 days; this group was defined as Group Z.

[0025] 1.3 High-throughput sequencing Genomic DNA was extracted from the meat and shredded radish portions of Longtian braised pork using the CTAB method. The quality and concentration of the extracted DNA were determined by 1% agarose gel electrophoresis. The V3–V4 regions of the bacterial 16S rRNA gene were amplified using primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) (SEQ ID NO.1) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (SEQ ID NO.2).

[0026] The total volume of the PCR reaction system was 20 μL. Each reaction system included 10 μL of 2× Pro Taq, 0.8 μL of forward primer (5 μM), 0.8 μL of reverse primer (5 μM), template DNA, and nuclease-free water, and was brought to a final volume of 20 μL with ddH2O. The template DNA concentration was 10 ng / μL. The amplification reaction was performed on an ABI GeneAmp® 9700 PCR instrument. The PCR conditions were as follows: 95 °C pre-denaturation for 3 min; followed by 30 cycles, including 95 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 45 s; and a final extension at 72 °C for 10 min. The reaction products were then held at 10 °C.

[0027] Amplification products were detected by 2% agarose gel electrophoresis, with a loading volume of 3 μL per sample. The target band was recovered using the AxyPrep DNA Gel Extraction Kit. Purified amplicones were quantified using the QuantiFluor™-ST blue fluorescence quantitative PCR system. After quantification, the purified PCR products were mixed in appropriate proportions according to the sequencing requirements of each sample. Sequencing libraries were constructed using the NEBNext® Ultra™ DNA Library Prep Kit. Library quality was assessed using an Agilent Bioanalyzer 2100 and Qubit; qualified libraries were used for subsequent high-throughput sequencing.

[0028] 1.4 Strain screening Lactic acid bacteria and staphylococci were isolated from group Z samples. The technical characteristics of the isolated strains were evaluated, including growth performance, safety, acidification ability, tolerance, antibacterial activity, and enzyme activity. The screened strains were identified by 16S rRNA gene sequencing. After identification, the strains were deposited at the Culture Collection Center on July 3, 2026, with the accession number CGMCC No. 39555, and named *Lactobacillus plantarum* (CGMCC). Lactobacillus plantarum LP 17; The *Staphylococcus equi* with accession number CGMCC No. 39554 is named *Staphylococcus equi* (LP 17). Staphylococcus equorum SE 55.

[0029] 1.5 Determination of extracellular protease activity Extracellular protease activity was determined using a protease activity assay kit (NMW0715, Nominco Biotechnology Co., Ltd., China). After centrifugation of the bacterial culture medium, the cell-free supernatant was collected for analysis. This method is based on the principle of casein hydrolysis and the release of tyrosine. Under alkaline conditions, the reaction products form a blue complex, and the absorbance was subsequently measured at 680 nm using a microplate reader. Protease activity was calculated according to the kit instructions.

[0030] 1.6 Determination of extracellular lipase activity Extracellular lipase activity was determined using a lipase activity assay kit (NMW1043, Novinco Biotechnology Co., Ltd., China). Cell-free supernatant was used as the enzyme sample. This method is based on the enzymatic hydrolysis of the chromogenic substrate by lipase to generate a methyl halogenated product. The amount of this red product generated was detected using a microplate reader at a wavelength of 580 nm. Lipase activity was calculated according to the kit instructions.

[0031] 1.7 Sample Preparation The two selected bacterial strains were activated for at least two generations in MRS broth and NB broth, respectively. After 18 h of culture, the bacterial cells were collected by centrifugation at 4 °C and 10,000 × g for 5 min. The bacterial pellet was washed twice with sterile physiological saline (0.9%, w / v). The concentration of the bacterial suspension was then adjusted to achieve a final inoculum level of 7 log CFU / g.

[0032] Samples from groups Z and N were used as raw materials. After cooling, the bacterial suspension was evenly sprayed onto the surface of pork and dried radish using a sterile sprayer. Four treatment groups were set up: uninoculated fried samples (NCK), uninoculated non-fried samples (ZCK), fried samples inoculated with the screening strain at an inoculation level of 7 log CFU / g (N-LP-SE), and non-fried samples inoculated with the screening strain at an inoculation level of 7 log CFU / g (Z-LP-SE). The treated samples were sealed in ceramic jars and fermented for 50 days at 10–20 °C and 60%–70% relative humidity. After sampling, the samples were stored at −80 °C and used for subsequent physicochemical index determination within 2 days.

[0033] 1.8 Physicochemical and Microbiological Analysis 1.8.1 Color Measurement The color characteristics of Longtian braised pork samples were determined using a CS-580A colorimeter (Shenzhen Fangyuan Instrument Co., Ltd., Shenzhen, China). Before measurement, the colorimeter was calibrated using a black calibration plate (L* = 0, a* = 0, b* = 0) and a white calibration plate (L* = 91.51, a* = −1.17, b* = 1.91). After calibration, the L*, a*, and b* values ​​of the samples were recorded. Each sample was measured three times.

[0034] 1.8.2 Moisture content and water activity Moisture content was determined according to the drying method in GB 5009.3—2016 standard. Samples were dried to constant weight at 105 ℃, and the moisture content was calculated based on the mass loss before and after drying. Water activity was determined using a water activity meter (Hangzhou Kefei Supply Chain Management Co., Ltd., Hangzhou, China).

[0035] 1.8.3 Determination of colony count and pH Under aseptic conditions, 10 g of Longtian braised pork sample was weighed and added to 90 mL of sterile 0.9% physiological saline. The mixture was homogenized for 3 min using a sterile homogenizer (Scientz-04, Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China). The resulting homogenate was serially diluted with sterile physiological saline. Appropriately diluted sample solutions were then spread onto corresponding selective culture media.

[0036] Total bacterial count was determined using total colony count agar (PCA); lactic acid bacteria were counted using Man, Rogosa and Sharpe (MRS) agar; and staphylococci were counted using mannitol agar (MSA). All plates were incubated at 36 ± 1 °C for 48 h. Microbial counts are expressed as log CFU / g. The pH of the filtrate was determined using a calibrated pH meter (FiveEasyPlus, Mettler Toledo, Shanghai, China), following a method adapted from Wang et al. (2023).

[0037] 1.9 Determination of textural properties Following the method of Akhtar et al. (2025) with slight modifications, the marinated Longtian braised pork was cut into 1 cm × 1 cm × 1 cm cubes. Texture analysis was performed using a texture analyzer (Stable Micro Systems Corporation, UK) in TPA mode. The parameters were set as follows: probe model P50; trigger force 5 N; pre-test speed, test speed, and post-test speed all 1 mm / s; test time 3 s; compressibility deformation 50%.

[0038] 1.10 Oxidation Index Analysis 1.10.1 Determination of TVB-N TVB-N was determined according to GB 5009.228—2016 standard. The determination was performed using a fully automated Kjeldahl nitrogen analyzer (Hainan Energy Future Technology Group Co., Ltd., K9860, China). 10 g of lean meat sample was weighed, 1 g of light magnesium oxide and 75 mL of distilled water were added, and the sample was soaked for 30 min. The titration endpoint was set at pH 4.65; titration was performed using 0.1 mol / L hydrochloric acid standard titrant, with 30 mL of boric acid solution as the absorbent, and the distillation time was 3 min. The calculation formula is as follows: (Formula 1)

[0039] In the formula, X is the volatile basic nitrogen content in the sample; V1 is the volume of hydrochloric acid standard titrant consumed in the sample titration; V2 is the volume of hydrochloric acid standard titrant consumed in the reagent blank titration; c is the concentration of hydrochloric acid standard titrant; 14 is the mass equivalent of nitrogen corresponding to the titration; and m is the sample mass.

[0040] 1.10.2 Determination of MDA MDA content was determined according to GB 5009.181—2016. 5 g of sample was weighed and added to 50 mL of 37.5 g / L trichloroacetic acid solution, and the mixture was shaken at 50 ℃ for 30 min. After filtration, 5 mL of the filtrate was mixed with 5 mL of TBA reagent and reacted at 90 ℃ for 30 min. After cooling, the absorbance was measured at 532 nm using a microplate reader (Thermo Fisher, USA). The MDA content was calculated based on the 1,1,3,3-tetraethoxypropane standard curve. The calculation formula is as follows: (Formula 2)

[0041] In the formula, X is the malondialdehyde content; c is the malondialdehyde concentration obtained from the standard curve; V is the final volume of the sample solution; and m is the sample mass.

[0042] 1.11 Statistical Analysis All analyses were performed in triplicate. Results are expressed as mean ± standard deviation (SD). One-way ANOVA was performed using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). A p-value < 0.05 was considered statistically significant.

[0043] Figure 1 This figure shows the results of bacterial community α-diversity analysis during the fermentation of Longtian braised pork in the fried and non-fried groups. In the figure, (a) represents changes in the ACE index; (b) represents changes in the Chao1 index; (c) represents changes in the Sobs index; (d) represents changes in the Good's coverage index; (e) represents changes in the Simpson index; and (f) represents changes in the Shannon index. This figure illustrates the impact of different processing methods on bacterial richness, diversity, and sequencing coverage during the fermentation of Longtian braised pork.

[0044] Figure 2 The results of bacterial community structure difference analysis during the fermentation process of Longtian braised pork in the fried and non-fried groups are shown. (a) represents the Pan-OTU analysis results; (b) represents the Core-OTU analysis results; (c) represents the Rank-abundance curve; (d) represents the principal coordinate analysis results based on Bray–Curtis distance; (e) represents the box plot of distances between samples; and (f) represents the LEfSe differential bacterial community analysis results. This figure is used to illustrate the differences in bacterial community composition and successional stability between the non-fried and fried groups.

[0045] Figure 3This figure presents the results of heatmap analysis on common OTUs, dominant bacterial genera, and genus-level associations during the fermentation process of Longtian braised pork. (a) shows the distribution of common and unique OTUs at different fermentation stages in the non-fried group; (b) shows the distribution of common and unique OTUs at different fermentation stages in the fried group; (c) shows the Circos analysis results of dominant bacterial genera in the non-fried group; (d) shows the Circos analysis results of dominant bacterial genera in the fried group; and (e) shows the genus-level bacterial community heatmap. This figure illustrates that the non-fried group retains more common OTUs and is enriched with more fermentation-related bacterial communities during fermentation.

[0046] Figure 4 This figure shows the changes in the horizontal bacterial community composition during the fermentation of Longtian braised pork. (a) represents the horizontal bacterial composition at different fermentation stages in the non-fried group; (b) represents the horizontal bacterial composition at different fermentation stages in the fried group. This figure illustrates the dynamic changes of dominant bacterial genera under the two processing methods and provides a basis for determining the source of the original starter culture strain.

[0047] Figure 5 This figure shows the blood plate test results for the candidate Staphylococcus equi strain. (a) represents SE 7; (b) represents SE 8; (c) represents SE 10; (d) represents SE 55; and (e) represents the Staphylococcus aureus positive control. This figure is used to illustrate the hemolytic safety evaluation results of the candidate Staphylococcus equi strain.

[0048] Figure 6 The results of the rabbit plasma coagulase assay for the candidate Staphylococcus equi strain are shown. In the figure, (a) represents the Staphylococcus aureus positive control; (b) represents SE 7; (c) represents SE 8; (d) represents SE 10; and (e) represents SE 55. This figure is used to illustrate the coagulase safety evaluation results of the candidate Staphylococcus equi strain.

[0049] Figure 7 To screen strains, the colony morphology, Gram staining morphology, growth curves, and antagonism test results were analyzed. (a) shows the colony morphology of LP 17; (b) shows the colony morphology of SE 55; (c) shows the growth curves of LP 17 and SE 55 based on viable cell count; (d) shows the Gram staining morphology of LP 17; (e) shows the Gram staining morphology of SE 55; and (f) shows the antagonism test results of the selected strains. This figure illustrates the good growth characteristics and basic application potential of the selected strains.

[0050] Figure 8This is a phylogenetic tree constructed based on the 16S rRNA gene sequence of the selected strains. The left side shows the phylogenetic tree for LP 17, and the right side shows the phylogenetic tree for SE 55. This figure illustrates that LP 17 was identified as *Lactobacillus plantarum*, and S.E55 was identified as *Staphylococcus equi*.

[0051] Figure 9 The figure shows the changes in microbial counts during the fermentation process of Longtian braised pork. (a) represents the change in total colony count; (b) represents the change in Staphylococcus count; and (c) represents the change in lactic acid bacteria count. This figure illustrates the effect of the inoculation starter culture on the changes in the number of major functional microorganisms during the fermentation of Longtian braised pork.

[0052] Figure 10 The figure shows the changes in basic physicochemical properties of Longtian braised pork during the inoculation and fermentation process. (a) represents the change in water activity; (b) represents the change in moisture content; (c) represents the change in pH; (d) represents the change in TVB-N content; and (e) represents the change in TBARS content. This figure illustrates the effects of the inoculation starter on the moisture state, acidification degree, protein degradation, and lipid oxidation of Longtian braised pork.

[0053] Figure 11 The figure shows the changes in the main textural parameters of lean and fat meat in Longtian braised pork during the inoculation and fermentation process. (a) represents the change in lean meat firmness; (b) represents the change in lean meat chewiness; (c) represents the change in lean meat resilience; (d) represents the change in fat firmness; (e) represents the change in fat chewiness; and (f) represents the change in fat resilience. This figure illustrates the effect of the inoculation starter on the firmness, chewiness, and softening properties of Longtian braised pork.

[0054] Table 1 shows the enzyme activity results for single strains and mixed culture ratios. This table lists the lipase activity, protease activity, and overall score for *Lactobacillus plantarum*, *Staphylococcus equi*, and different ratios of LP 17 and SE 55, used to determine suitable compound fermentation starter strains and ratios.

[0055] Table 2 shows the changes in the color characteristics of lean and fat meat in Longtian braised pork under different treatment groups. This table lists the a* and b* values ​​of different treatment groups at different fermentation times. and L The values ​​are used to illustrate the effects of inoculation with the starter culture and frying treatment on the redness, yellowness, and brightness of Longtian braised pork.

[0056] Table 3 shows the changes in the auxiliary textural properties of lean and fatty Longtian braised pork under different treatment groups. This table lists the elasticity, cohesiveness, and adhesiveness indices of different treatment groups at different fermentation times, to supplement the explanation of the influence of the inoculum on the stability and softening degree of the Longtian braised pork tissue structure.

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Claims

1. A dual-strain compound fermentation agent, characterized in that, The dual-strain compound fermentation agent is composed of Lactobacillus plantarum and Staphylococcus equi in a weight ratio of 1:1 to 5:

1.

2. The dual-strain compound fermentation agent as described in claim 1, characterized in that, The dual-strain compound fermentation agent is composed of Lactobacillus plantarum and Staphylococcus equi in a weight ratio of 1:

1.

3. The dual-strain compound fermentation agent as described in claim 1 or 2, characterized in that, The *Lactobacillus plantarum* collection number is CGMCC No. 39555, and it is named *Lactobacillus plantarum* (…). Lactobacillus plantarum LP 17; The *Staphylococcus equi* with accession number CGMCC No. 39554 is named *Staphylococcus equi* (LP 17). Staphylococcus equorum SE 55.

4. The dual-strain compound fermentation agent as described in claim 3, characterized in that, The *Lactobacillus plantarum* and *Staphylococcus equi* were screened from Longtian braised pork.

5. The application of the dual-strain compound fermentation agent as described in claim 4 in the fermentation of Longtian braised pork.

6. A type of *Lactobacillus plantarum* used as a starter culture for Longtian braised pork, characterized in that, The *Lactobacillus plantarum* collection number is CGMCC No. 39555, and it is named *Lactobacillus plantarum* (…). Lactobacillus plantarum )LP 17.

7. A Staphylococcus equine strain used as a starter culture for Longtian braised pork, characterized in that, The *Staphylococcus equi* specimen, with accession number CGMCC No. 39554, was named *Staphylococcus equi* (…). Staphylococcus equorum SE 55.

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