Lactobacillus paracasei sq01, its fermentation preparations and uses

Abstract

The application discloses a lactobacillus paracasei SQ01 and a fermentation preparation and application thereof, and belongs to the technical field of microbial preparations. The lactobacillus paracasei SQ01 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2024934. The lactobacillus paracasei SQ01 has the advantages that the lactobacillus paracasei SQ01 has a significant temperature tolerance advantage, can normally multiply at 4-10 DEG C, and can maintain a high number of viable bacteria at 55-60 DEG C, thereby breaking through the limitation of the temperature adaptation range of the lactobacillus paracasei. The lactobacillus paracasei SQ01 has excellent food-grade fermentation function development potential. The fermentation liquor can be used for food preservation, the broken extract can be used as a core raw material of a blood lipid-lowering medicine, and the dried bacterial body can be used in the fermentation process of materials such as fruit juice, vinasse and straw, so that the fermentation efficiency and product quality are improved, and the application range is wide.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparation technology, specifically relating to Lactobacillus paracasei SQ01 and its fermentation preparations and applications. Background Technology

[0002] Lactobacillus paracasei is a commonly used probiotic widely applied in the food fermentation field. Its fermentation products, broken extracts, and bacterial cells can be developed into products with different functions, which have important industrial application value and market prospects.

[0003] Currently available Lactobacillus paracasei strains generally suffer from insufficient heat resistance, with the highest tolerable temperature usually not exceeding 45°C. Above this temperature, bacterial growth is significantly inhibited, making it difficult for the bacteria to survive. They also cannot be adapted to conventional high-temperature processing techniques in the food industry, requiring the addition of additional protective agents or the use of low-temperature processing methods. This not only increases production costs but also limits their large-scale application in functional products.

[0004] Therefore, developing a strain of Lactobacillus paracasei that combines high-temperature resistance with excellent food-grade fermentation potential to address the technical pain points of poor heat resistance and insufficient industrial adaptability of existing strains and meet the needs of large-scale production is an urgent technical problem to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Lactobacillus paracasei that has both high temperature resistance and excellent food-grade fermentation function, as well as its fermentation preparation and applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Lactobacillus paracasei SQ01 is deposited at the China Center for Type Culture Collection (CCTCC) on May 15, 2024, with accession number CCTCC NO: M 2024934, located in Wuhan, China, and classified as Lactobacillus paracasei SQ01.

[0007] The fermentation preparation obtained from Lactobacillus paracasei SQ01 is prepared by the following method: (1) Inoculate Lactobacillus paracasei SQ01 onto MRS solid medium and incubate upside down at 37°C for 48 h; (2) Pick the bacterial colony from the MRS solid medium and transfer it to the MRS liquid medium. Incubate at 37°C with shaking for 24 hours to obtain the seed culture. (3) The seed culture was inoculated into fermentation medium A and cultured at 37℃ for 24 hours to obtain fermentation broth A. The formula of fermentation medium A is as follows: glucose 21.0 g / L, peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 8.0 g / L, dipotassium hydrogen phosphate 3.5 g / L, sodium acetate 6.5 g / L, diammonium hydrogen citrate 2.0 g / L, magnesium sulfate 0.2 g / L, adenine 0.13 g / L, phenylalanine 0.051 g / L, Tween 80 1 mL / L; (4) Transfer fermentation broth A to fermentation medium B. In the early stage of fermentation, stir and culture at 37℃ for 36h. In the middle and late stages of fermentation, seal and anaerobic fermentation at 37℃ for 36h to obtain fermentation broth B. The formula of fermentation medium B is as follows: glucose 16.0g / L, peptone 10.0g / L, beef extract powder 8.0g / L, yeast extract powder 6.0g / L, dipotassium hydrogen phosphate 3.5g / L, sodium acetate 6.5g / L, diammonium hydrogen citrate 2.0g / L, magnesium sulfate 0.5g / L, calcium chloride 1.0g / L, L-glutamic acid 5.0g / L, manganese sulfate 0.1g / L, vitamin C 0.01g / L, adenine 0.15g / L, phenylalanine 0.05g / L, Tween 80 1mL / L; (5) Perform solid-liquid separation on fermentation broth B and collect the fermentation supernatant and bacterial sludge separately; (6) The fermentation supernatant was sequentially separated by microfiltration, ultrafiltration and nanofiltration membranes, and the retentate was collected to obtain fermentation preparation 1; (7) The bacterial sludge is subjected to compound bio-enzyme directional pre-crushing, ultra-high pressure deep crushing, microfiltration, ultrafiltration and nanofiltration in sequence, and the retentate is collected to obtain fermentation preparation 2; or, the bacterial sludge is directly mixed and homogenized and then spray dried to obtain fermentation preparation 3.

[0008] Preferably, in step (4), during the early stage of fermentation, the fermentation process is constant pH fermentation, and the pH of the fermentation liquid is maintained in the range of 5.0-6.0 by adding ammonia water or sodium hydroxide solution; micro-aeration and low-speed stirring are performed once every 12 hours, and this is repeated 3-4 times.

[0009] Preferably, in steps (6) and (7), the pore size of the microfiltration membrane is 0.22 μm and the operating pressure is 0.1 MPa-0.2 MPa; the molecular weight cutoff of the ultrafiltration membrane is 100 kDa and the operating pressure is 0.2 MPa-0.3 MPa; the molecular weight cutoff of the nanofiltration membrane is 10 kDa and the operating pressure is 0.3 MPa-0.4 MPa; and the temperature is controlled at 15℃-25℃ throughout the membrane separation process.

[0010] Preferably, in step (7), the compound bio-enzyme is composed of lysozyme, β-glucanase and plant-derived pectinase in a mass ratio of 5:4:1, and the amount added is 0.5%-1% of the dry weight of the bacterial mud. The enzymatic hydrolysis conditions are: pH 5.5-6.0, constant temperature shaking at 40℃-45℃ for 120min-150min, and enzyme inactivation at 80℃ for 30min.

[0011] Preferably, in step (7), an ultra-high pressure cell disruptor is used to perform ultra-high pressure deep crushing of the bacterial cells. The nozzle diameter is 50μm, the working pressure is 120MPa-150MPa, the feeding speed is 50mL / min-80mL / min, and the cell is crushed 2-3 times.

[0012] Preferably, in step (7), a composite protective agent is added to the fungal mud at 8%-15% of the dry weight of the fungal mud to adjust the fungal mud. The composite protective agent is composed of trehalose, whey protein and maltodextrin in a mass ratio of 4:2:3. The inlet air temperature of the spray drying is 110℃-130℃, the outlet air temperature is 65℃-75℃, and the feed flow rate is 10L / h-20L / h.

[0013] Application of fermentation preparation 1 in the preparation of food preservatives.

[0014] Application of fermentation preparation 2 in the preparation of lipid-lowering drugs.

[0015] Application of fermentation preparation 3 in the preparation of probiotic fermentation agents for high-temperature processing.

[0016] The advantages of this invention are: (1) The present invention isolates and purifies Lactobacillus paracasei SQ01 from the ground environment of the workshop of Guizhou sauce-flavored liquor factory. Compared with other disclosed Lactobacillus paracasei strains, the Lactobacillus paracasei SQ01 purified by the present invention has a significant temperature tolerance advantage. It can not only reproduce normally under the lower temperature conditions of 4℃-10℃, but also maintain a high number of viable bacteria under the higher temperature conditions of 55℃-60℃, breaking through the limitations of the temperature adaptation range of existing Lactobacillus paracasei.

[0017] (2) The purified Lactobacillus paracasei SQ01 obtained in this invention has excellent potential for food-grade fermentation. Specifically, the fermentation broth (fermentation agent 1) can be used for food preservation, the crushed extract (fermentation agent 2) can be used as a core raw material for lipid-lowering drugs, and the dried bacterial cells (fermentation agent 3) can be used in the fermentation process of materials such as fruit juice, distiller's grains, and straw to improve fermentation efficiency and product quality. The above-mentioned multi-dimensional application scenarios greatly broaden the application scope of Lactobacillus paracasei.

[0018] (3) The fermentation preparations 1, 2 and 3 developed in this invention are highly safe, non-toxic and do not have drug resistance, and can be safely applied in the food field. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of Lactobacillus paracasei SQ01; Figure 2 This is a morphological diagram of Lactobacillus paracasei SQ01. Figure 3 This is a phylogenetic tree diagram of Lactobacillus paracasei SQ01; Figure 4 This is a high- and low-temperature growth curve of Lactobacillus paracasei SQ01; Figure 5 This is a graph showing the results of TC content detection in mouse serum; Figure 6 This is a graph showing the results of TG content detection in mouse serum; Figure 7 This is a graph showing the results of LDL-C content detection in mouse serum; Figure 8 This is a graph showing the results of HDL-C content detection in mouse serum; Figure 9 This is a graph showing the relative expression levels of SREBP-1c in mouse liver. Figure 10 This is a graph showing the results of the relative expression level of PPARα in mouse liver; Figure 11 This is a graph showing the results of relative expression levels of FAS in mouse livers; Figure 12 This is a pathological image of the liver tissue of the control group mice; Figure 13 This is a pathological image of the liver tissue of the model group mice; Figure 14 This is a diagram showing the pathological results of liver tissue in the mice in the treatment intervention group. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0021] I. Culture medium 1. MRS liquid culture medium The specific formula for MRS liquid culture medium is as follows: peptone 10.0 g / L, beef extract powder 5.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, sodium acetate 5.0 g / L, triammonium citrate 2.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, prepared with purified seawater.

[0022] Autoclave at 121℃ for 20 minutes, then cool to room temperature for later use.

[0023] 2. MRS solid culture medium The specific formula for MRS solid culture medium is as follows: peptone 10.0 g / L, beef extract powder 5.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, sodium acetate 5.0 g / L, triammonium citrate 2.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, and agar 15.0 g / L, prepared with purified seawater.

[0024] Sterilize at 121℃ for 20 minutes. After sterilization, cool to 45℃ and pour into sterile plates in a laminar flow hood. Let solidify and set aside.

[0025] 3. Fermentation medium A The specific formula for fermentation medium A is as follows: glucose 21.0 g / L, peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 8.0 g / L, dipotassium hydrogen phosphate 3.5 g / L, sodium acetate 6.5 g / L, diammonium hydrogen citrate 2.0 g / L, magnesium sulfate 0.2 g / L, adenine 0.13 g / L, phenylalanine 0.051 g / L, and Tween 80 1 mL / L. It is prepared with purified seawater and the pH is adjusted to 5.5 ± 0.5.

[0026] Autoclave at 121℃ for 20 minutes, then cool to room temperature for later use.

[0027] 4. Fermentation medium B The specific formula for fermentation medium B is as follows: glucose 16.0 g / L, peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 6.0 g / L, dipotassium hydrogen phosphate 3.5 g / L, sodium acetate 6.5 g / L, diammonium hydrogen citrate 2.0 g / L, magnesium sulfate 0.5 g / L, calcium chloride 1.0 g / L, L-glutamic acid 5.0 g / L, manganese sulfate 0.1 g / L, vitamin C 0.01 g / L, adenine 0.15 g / L, phenylalanine 0.05 g / L, and Tween 80 1 mL / L. It is prepared with purified seawater and the pH is adjusted to 5.5 ± 0.5.

[0028] Autoclave at 121℃ for 20 minutes, then cool to room temperature for later use.

[0029] II. Isolation, purification, and species identification of Lactobacillus paracasei SQ01 1. Isolation and purification of Lactobacillus paracasei SQ01 The Lactobacillus paracasei SQ01 involved in this invention was isolated and purified from the ground environment of a Guizhou-style sauce-flavored liquor factory workshop. The specific isolation and purification process is as follows: (1) Take 10 mL of soil sample and add it to 100 mL of 0.85 wt% NaCl sterile solution to make a turbid solution; (2) The above turbid liquid was serially diluted. 100 μL of the turbid liquid diluted 10,000 times was spread onto MRS solid medium by plate spreading and anaerobic culture was carried out. A total of 3 plates were spread. Then, the 3 plates with the turbid liquid were placed in an incubator at 37°C and inverted for 36 h. After the culture was completed, about 100-150 single colonies grew on each plate. (3) Randomly select 3 single colonies from each plate and anaerobic culture them on a new MRS solid medium for 48 h. Purify the single colonies by streak plating. After streak plating, place 9 plates in an incubator at 37 ℃ and incubate upside down for 24 h. After the culture is completed, about 50 single colonies grow on each plate. Select white colonies with calcium dissolution zones and further isolate and purify them on a new MRS solid medium. Finally, preserve single colonies that are round, milky white, smooth and raised, with neat edges, dense and viscous texture, diameter of 1.0 mm-2.5 mm, and uniform and stable morphology. The strain that forms this colony is the strain finally screened in this invention and is denoted as SQ01.

[0030] 2. Species identification of Lactobacillus paracasei SQ01 (1) Observe the colony morphology and cell shape. Strain SQ01 was plated onto MRS solid medium and incubated anaerobically at 37°C for 48 hours. After incubation, multiple single colonies were isolated from the plates. Figure 1 As shown, these single colonies are round, milky white, with a smooth, raised surface, neat edges, and a dense, viscous texture.

[0031] Take a loopful of the bacterial strain from the above plate and disperse it in MRS liquid medium. Perform Gram staining on strain SQ01 according to Bergey's Manual of Bacteriological Identification, and observe the bacterial morphology under a microscope, such as... Figure 2 As shown, strain SQ01 is a Gram-positive short bacillus with blunt, rounded ends. It is mostly arranged singly, in pairs, or in short chains, and has no spores or branches.

[0032] (2) Gel electrophoresis detection Strain SQ01 was inoculated into MRS liquid medium and cultured at 37°C and 150 rpm for 48 h to obtain bacterial culture.

[0033] Bacterial DNA was extracted from the bacterial culture using a bacterial genomic DNA extraction kit. Then, the extracted DNA was used as a template, with 27F (sequence AGA GTT TGA TCC TGG CTCAG) as the upstream primer and ITS1 (sequence TCCGTAGGTGAACCTGCGG) as the downstream primer for sequence amplification.

[0034] The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min, 56℃ annealing for 1 min, 72℃ extension for 1 min, 31 cycles; 72℃ extension for 5 min, amplification complete.

[0035] The amplification products were detected by 1wt% agarose gel electrophoresis. The results showed that the universal primer PCR amplification was successful.

[0036] (3) Identification of 16S rRNA Strain SQ01 was sent to Beijing Aoke Dingsheng Biotechnology Co., Ltd. for 16S rRNA sequencing. The obtained 16S rRNA sequence (SEQ ID NO: 1) was then searched for homologous sequences in GenBank. The search results showed that strain SQ01 had 100% similarity to Lactobacillus paracasei.

[0037] A phylogenetic tree of the genus *Lactobacillus* constructed based on the 16S rRNA sequence is shown below. Figure 3 As shown. Ultimately, strain SQ01 was identified as Lactobacillus paracasei and denoted as Lactobacillus paracasei SQ01.

[0038] III. Growth of Lactobacillus paracasei SQ01 under high and low temperature conditions The experiment consisted of three groups: a low temperature group (10℃), a high temperature group (60℃), and a control group (37℃).

[0039] The culture and temperature stress test procedure for Lactobacillus paracasei SQ01 is as follows: Lactobacillus paracasei SQ01 was inoculated into MRS liquid medium and continuously cultured at 37℃ and 120 rpm for 24 h to complete activation. Then, it was transferred for fermentation at 37℃ for 12 h. Afterwards, the low-temperature group had its fermentation broth temperature slowly decreased from 37℃ to 10℃ at a rate of 2℃ / h over 12 h and maintained thereafter; the high-temperature group had its fermentation broth temperature slowly increased from 37℃ to 60℃ at a rate of 2℃ / h over 12 h and maintained thereafter; and the control group was maintained at 37℃ throughout the experiment. The entire experiment was conducted continuously for 72 h, with samples taken every 12 h to measure the OD of each treatment group. 600 Values ​​were used to reflect changes in bacterial concentration, and the growth dynamics of *Lactobacillus paracasei* SQ01 under high and low temperature stress were assessed. The results are as follows: Figure 4 As shown.

[0040] Depend on Figure 4 It can be seen that all three groups (control group, high temperature group, and low temperature group) exhibited typical growth kinetic characteristics, but temperature had a significant regulatory effect on the growth, stabilization, and death phases of the strains. Specifically: (1) Control group (optimal temperature, 37℃): The strain exhibited a complete growth cycle, with the logarithmic growth phase lasting from 0 to 12 hours, and OD... 600 The value rose rapidly to approximately 1.8, then entered a stable period, OD 600 The value continued to increase, reaching a peak at 48 hours (approximately 2.55). Afterward, as fermentation time prolonged, the cells gradually entered a death phase, and the OD value... 600 The value decreased slowly, but remained at around 2.1 after 72 hours, indicating that the strain has strong proliferation ability and later stability under optimal conditions. (2) High temperature group (60℃ stress): The growth rate of the strain in 0-12h was similar to that of the control group, OD 600 The value reached approximately 1.7 at 12 hours, and after 24 hours, the OD value... 600 The value rose to approximately 1.95, indicating that the strain could still maintain a certain level of growth activity under high-temperature stress of 60℃. This contrasts sharply with the characteristic that conventional Lactobacillus paracasei cannot survive at 60℃, demonstrating the high-temperature tolerance advantage of Lactobacillus paracasei SQ01. However, under continuous high-temperature stress, the bacterial cell death process was significantly accelerated, and the OD value increased significantly after 24 hours. 600 The value gradually decreased, dropping to about 1.8 at 48h and 1.1 at 72h, showing the characteristics of rapid proliferation and premature death. This is consistent with the growth pattern of disordered cell metabolism and easy autolysis under high temperature conditions. (3) Low temperature group (10℃ stress): The growth kinetics of the strain showed obvious inhibition characteristics, and it still maintained a certain degree of proliferation from 0 to 12 h, OD 600 The value rose to about 1.7, but growth was significantly inhibited after 12 hours, and OD at 24 hours... 600 Its value was only 1.3, after which it entered a slow decay phase, OD 600 The value continued to decrease with the extension of fermentation time, dropping to about 0.5 at 72h. This result indicates that although the low temperature of 10℃ cannot support the continuous proliferation of the strain, the cells can still maintain a certain survival state for a relatively long time. The decline process is gradual and slow, which is consistent with the typical characteristics of microbial metabolic rate reduction and cell activity decrease at low temperature, but they are not easy to die quickly.

[0041] In summary, Lactobacillus paracasei SQ01 exhibits good adaptability across a wide temperature range of 10℃-60℃, maintaining high early growth activity under high temperature (60℃) stress and achieving long-term stable survival in low temperature (10℃) environments. This lays the foundation for its application in special scenarios such as low-temperature fermentation of food and high-temperature fermentation of feed and fertilizer.

[0042] Lactobacillus paracasei SQ01 was continuously cultured on MRS liquid medium at 4℃ and 120 rpm for 72 h, with samples taken every 6 h to determine the OD of Lactobacillus paracasei SQ01. 600 The value is used to determine the growth status of the strain under low temperature conditions.

[0043] The growth curve of Lactobacillus paracasei SQ01 is as follows: Figure 3 As shown in the figure, the results indicate that the strain grows rapidly at a low temperature of 4°C.

[0044] IV. Carbon source fermentation of Lactobacillus paracasei SQ01 The sugar fermentation characteristics of Lactobacillus paracasei SQ01 were detected using 16 carbon source substrates, namely: glucose, lactose, sucrose, maltose, fructose, mannose, sorbitol, mannitol, xylose, arabinose, trehalose, melibiose, rhamnose, mesotriose, erythritol, and starch. The fermentation results are detailed in Table 1.

[0045] Table 1. Identification results of sugar fermentation characteristics of Lactobacillus paracasei SQ01

[0046] Note: + indicates that the strain can utilize the carbon source substrate, and - indicates that the strain cannot utilize the carbon source substrate.

[0047] The results showed that Lactobacillus paracasei SQ01 can utilize glucose, lactose, sucrose, maltose, fructose, mannose, sorbitol, mannitol, xylose, arabinose, trehalose, and melibiose, but cannot utilize rhamnose, pine triose, erythritol, and starch.

[0048] V. Cultivation and fermentation of Lactobacillus paracasei SQ01 and preparation of fermentation reagents 1. Culture of Lactobacillus paracasei SQ01 (1) Planar culture of Lactobacillus paracasei SQ01 The purified Lactobacillus paracasei SQ01 was inoculated onto MRS solid medium and cultured anaerobically at 37°C for 48 hours.

[0049] (2) Seed culture of Lactobacillus paracasei SQ01 One loop of independent bacterial colony was picked from the MRS solid medium obtained after 48 hours of culture and transferred to 200 mL of MRS liquid medium. The culture was sealed with plastic wrap and cultured continuously at 37°C and 120 rpm for 24 hours to obtain the seed culture.

[0050] 2. Fermentation of Lactobacillus paracasei SQ01 (1) Fermentation of Lactobacillus paracasei SQ01 The seed culture obtained after 24 hours of cultivation was inoculated into 3000 mL of fermentation medium A at a 5% inoculation rate. The medium was sealed with plastic wrap and incubated at 37°C for 24 hours to obtain fermentation broth A.

[0051] (2) Optimized fermentation of Lactobacillus paracasei SQ01 The fermentation broth A obtained after 24 hours of cultivation was transferred to 30L of fermentation medium B (contained in a 50L fermenter) and fermented continuously at 37℃ and 120rpm for 36 hours (early stage of fermentation). During the fermentation process, ammonia (or sodium hydroxide solution) was added to maintain the pH of the fermentation broth within the range of 5.0-6.0. Micro-aeration (0.1VVM) and low-speed stirring (50rpm) were performed every 12 hours, for a total of 3 times (to promote cell proliferation). Subsequently (mid-to-late stage of fermentation), the mixture was sealed and anaerobic for 36 hours (to avoid high dissolved oxygen inhibiting cell growth and reducing viable cell count), yielding fermentation broth B. The viable cell count of fermentation broth B reached 9.7 × 10⁻⁶. 9 CFU / mL.

[0052] 3. Preparation of Lactobacillus paracasei SQ01 microbial preparation After optimized fermentation, Lactobacillus paracasei SQ01 was used to separate the solid and liquid components of the resulting fermentation broth B using a tubular centrifuge. The broth was centrifuged at 16,000 rpm for 3 minutes, and the fermentation supernatant and bacterial sludge were collected separately. The collected bacterial sludge was then divided into two equal portions.

[0053] The collected fermentation supernatant was sequentially treated with microfiltration, ultrafiltration, and nanofiltration membranes. The microfiltration membrane had a pore size of 0.22 μm and operated at a pressure of 0.1 MPa. The collected filtrate was then subjected to ultrafiltration, using a membrane with a molecular weight cutoff of 100 kDa and an operating pressure of 0.2 MPa. The collected retentate was then subjected to nanofiltration, using a membrane with a molecular weight cutoff of 10 kDa and an operating pressure of 0.3 MPa. The retentate was collected to obtain fermentation preparation 1. The temperature was controlled throughout the process at 15℃-25℃. The solids content of fermentation preparation 1 was determined to be 30 wt%.

[0054] Experiments showed that when the operating pressure of microfiltration membrane, ultrafiltration membrane, and nanofiltration membrane was increased to 0.2 MPa, 0.3 MPa, and 0.4 MPa, respectively, the solid content of the fermentation preparation could reach 35 wt%.

[0055] Half of the collected bacterial sludge was suspended. First, a compound bio-enzyme (composed of lysozyme, β-glucanase, and plant-derived pectinase in a mass ratio of 5:4:1) was added to the bacterial sludge suspension at 0.5% (up to 1%) of the dry weight of the bacterial sludge. The pH of the system was adjusted to 5.5-6.0, and the mixture was placed in a constant temperature shaker at 40℃ (up to 45℃) and shaken at 120 rpm (up to 150 rpm) for 120 min (up to 150 min). After enzymatic hydrolysis, the temperature was raised to 80℃ and maintained for 30 min to inactivate the enzyme, completing the directional pre-disruption of the compound bio-enzyme. Subsequently, the bacterial cells were subjected to ultra-high pressure deep crushing using an ultra-high pressure cell disruptor. The nozzle diameter of the ultra-high pressure cell disruptor was 50 μm, the working pressure was controlled at 120 MPa (up to 150 MPa), the feed rate was 50 mL / min (up to 80 mL / min), and the crushing was cyclically repeated 2 times (up to 3 times). Finally, membrane separation was performed using the same method as before to obtain fermentation preparation 2.

[0056] The other half of the collected mycelium sludge was mixed and homogenized. First, a composite protective agent (composed of trehalose, whey protein, and maltodextrin in a mass ratio of 4:2:3) was added at 8% (up to 15%) of the dry weight of the mycelium sludge to form a glassy protective film. Then, spray drying was performed, controlling the inlet air temperature at 110℃ (up to 130℃), the outlet air temperature at 65℃ (up to 75℃), and the feed flow rate at 10L / h (up to 20L / h), to obtain fermentation preparation 3. Testing showed that the droplet size after atomization was in the range of 20μm-50μm, the moisture content of fermentation preparation 3 was ≤4.0%, and the viable cell retention rate was ≥80%.

[0057] VI. Validation of the antibacterial activity of Lactobacillus paracasei SQ01 Lactobacillus paracasei can inhibit the growth and reproduction of pathogenic and putrefactive bacteria by producing active substances such as bacteriocins, organic acids (lactic acid, acetic acid, etc.), and short-chain fatty acids through metabolism, or by adhering to itself, occupying space, and competing for nutrients.

[0058] Indicator bacteria: Staphylococcus aureus ( Staphylococcus aureus ATCC 6538), Salmonella ( Salmonella enterica ATCC 14028), Escherichia coli ( Escherichia coli ATCC 25922), Listeria monocytogenes ( Listeria monocytogenes ATCC 19115), Bacillus subtilis ( Bacillus subtilis ATCC6633), Clostridium perfringens ( Clostridium perfringens (ATCC 13124).

[0059] Detection method: Oxford cup plate diffusion method.

[0060] Antibacterial plates: Staphylococcus aureus, Salmonella, Escherichia coli, Listeria monocytogenes, Bacillus subtilis, and Clostridium perfringens were added to molten MRS solid medium at a concentration of 1% (v / v). The mixture was then poured into different petri dishes and cooled to obtain the corresponding antibacterial plates.

[0061] Fermentation agent 1, fermentation agent 2 and fermentation agent 3 were each prepared into a solution with a concentration of 1 mg / mL using ultrapure water and were ready for use.

[0062] Four Oxford cups (6 mm inner diameter) were placed on each inhibition plate, with one Oxford cup serving as the control group and the other three as parallel experimental groups. 100 μL of sterile water was added to the Oxford cup in the control group, and 100 μL of solution was added to each Oxford cup in the parallel experimental groups. The plates were then anaerobically incubated at 37°C for 24 h. The diameter of the inhibition zone was observed and measured (accurate to 0.1 mm), and the average of the three replicates was taken as the final result. The antibacterial activity intensity of fermentation preparations 1, 2, and 3 was determined based on the size of the inhibition zone diameter.

[0063] The criteria for classifying antibacterial activity levels are shown in Table 2.

[0064] Table 2 Classification Criteria for Antibacterial Activity Levels

[0065] The antibacterial activity test results of fermentation preparation 1, fermentation preparation 2 and fermentation preparation 3 are shown in Tables 3, 4 and 5, respectively.

[0066] Table 3 Antibacterial effect of fermentation preparation 1

[0067] Table 4 Antibacterial effect of fermentation preparation 2

[0068] Table 5. Antibacterial effect of fermentation preparation 3

[0069] As shown in Tables 3, 4, and 5, fermentation preparation 1 exhibits the best antibacterial activity, showing strong inhibition against Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis (inhibition zone diameter 15.3-17.3 mm), and moderate inhibition against Salmonella, Escherichia coli, and Clostridium perfringens (10.6-11.6 mm). Fermentation preparation 2 is the second best, mainly showing moderate inhibition, with only good inhibitory effect against Gram-positive bacteria. Fermentation preparation 3 has moderate antibacterial activity, mainly showing moderate and weak inhibition.

[0070] The above results indicate that the antibacterial activity of Lactobacillus paracasei SQ01 not only comes from live bacterial colonization, but also from the bacterial cells themselves and their metabolites.

[0071] Antibacterial spectrum analysis: Lactobacillus paracasei SQ01 showed significantly better inhibitory effects on Gram-positive bacteria (Staphylococcus aureus, Listeria monocytogenes, Bacillus subtilis) than on Gram-negative bacteria (Salmonella, Escherichia coli). This is related to the barrier effect of the cell wall structure (including the outer membrane) of Gram-negative bacteria on antibacterial substances.

[0072] In summary, Lactobacillus paracasei SQ01 has good antibacterial application potential in food preservation, feed antibiotic alternatives and other fields.

[0073] VII. Verification of the lipid-lowering activity of Lactobacillus paracasei SQ01 Model construction: Nine male C57 mice (6-8 weeks old) were randomly divided into three groups of three mice each, as follows: Control group: fed regular feed for 30 consecutive days, and administered physiological saline by gavage daily.

[0074] Model group: fed a high-fat diet for 30 consecutive days and administered physiological saline by gavage daily.

[0075] Preparation intervention group: fed high-fat diet for 30 consecutive days, and administered fermented preparation 2 times daily by gavage at a dose of 100mg / kg.

[0076] After the administration was completed, serum samples were collected to detect the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Liver samples were collected to detect the relative expression levels of key liver lipid metabolism genes SREBP-1c, PPARα, and FAS. The pathological characteristics of liver tissue were also analyzed.

[0077] The results of detecting the levels of TC, TG, LDL-C, and HDL-C in the serum of the three groups of mice are shown below. Figure 5 , Figure 6 , Figure 7 and Figure 8 .

[0078] Depend on Figures 5 to 8 The results showed that, compared with the model group, the serum HDL-C level in the treatment group was significantly increased (p<0.001), indicating that fermentation preparation 2 can effectively remove excess lipids in blood vessels and has good lipid-regulating and vascular-protective effects. Meanwhile, the serum TC, TG, and LDL-C levels in the treatment group were significantly lower than those in the model group (p<0.001), indicating that fermentation preparation 2 can regulate cholesterol metabolism, reduce TC and TG levels, effectively prevent hyperlipidemia and fatty liver, and also downregulate LDL-C levels, reducing lipid deposition in the vascular wall and thus inhibiting the occurrence of atherosclerotic lesions.

[0079] The relative expression levels of the key genes SREBP-1c, PPARα, and FAS in the liver of the three groups of mice were detected as follows: Figure 9 , Figure 10 and Figure 11 .

[0080] Depend on Figures 9 to 11 The results showed that the relative expression levels of genes SREBP-1c and FAS were significantly increased and the relative expression level of gene PPARα was significantly decreased in the model group mice, indicating that lipid metabolism disorder induced by high-fat diet is closely related to the imbalance of lipid synthesis / decomposition pathways. After intervention with fermentation agent 2 (treatment group), the relative expression levels of genes SREBP-1c and FAS were significantly downregulated and the relative expression level of gene PPARα was significantly upregulated. Therefore, it is indicated that fermentation agent 2 may regulate lipid metabolism balance bidirectionally by inhibiting lipid synthesis pathway and activating fatty acid oxidation pathway, thereby improving lipid accumulation induced by high-fat diet.

[0081] The histopathological results of the liver tissues of the three groups of mice are shown below. Figure 12 , Figure 13 and Figure 14 .

[0082] Depend on Figure 12 It can be seen that the liver tissue structure of the control group mice was intact, the lobule boundaries were not obvious (which is a normal histological feature of mouse liver), and the central vein, peripheral hepatic cords and hepatic sinusoids of the lobules were arranged regularly. Only a very small number of hepatocytes showed mild edema, which was characterized by loose and pale cytoplasm (black arrow). No obvious fatty degeneration, necrosis and portal inflammatory infiltration were observed, indicating that the liver of the control group was in a normal physiological state and had no pathological damage.

[0083] Depend on Figure 13 It can be seen that: the liver tissue of the model group mice showed typical hyperlipidemic pathological damage, and the central vein of the liver lobules showed varying degrees of congestion (black arrows), indicating obstruction of hepatic circulation; hepatocytes showed significant fatty degeneration, and a large number of tiny round vacuoles were visible in the cytoplasm (a characteristic feature of lipid droplets dissolved in HE staining, yellow arrows), indicating a large amount of lipid deposition in hepatocytes; no obvious abnormalities were seen in the portal area, and there was no severe inflammation or necrosis, indicating that the high-fat diet mainly induced liver damage with fatty degeneration as the core, and the hyperlipidemic animal model was successfully established.

[0084] Depend on Figure 14It can be seen that the pathological damage to the liver tissue of mice in the intervention group was significantly improved, steatosis was significantly reduced, only a small number of hepatocytes showed mild steatosis (yellow arrow), and the number of lipid droplet vacuoles was much less than that in the model group, indicating that fermentation agent 2 effectively alleviated liver lipid deposition, partially alleviated congestion, reduced central venous congestion in some samples (green arrow), improved liver circulation, and slightly increased hepatocyte edema compared with the control group (black arrow), but there was no serious necrosis or inflammatory infiltration. The overall pathological condition was significantly better than that of the model group.

[0085] Histopathological results of liver tissue showed that a high-fat diet successfully induced hyperlipidemic liver damage in mice, characterized by extensive hepatocyte steatosis and central venous congestion, indicating the effectiveness of the hyperlipidemia model. After intervention with fermentation agent 2, the degree of hepatic steatosis in mice was significantly reduced, lipid deposition was markedly decreased, and some hepatic circulatory disturbances were alleviated, indicating that fermentation agent 2 can effectively improve liver pathological damage induced by a high-fat diet and has a certain protective effect against liver lesions caused by hyperlipidemia.

[0086] VIII. Research on the fermentation of distiller's grains, straw, etc. by Lactobacillus paracasei SQ01 To verify the fermentation effect of Lactobacillus paracasei SQ01 on a mixture of distiller's grains and straw, changes in pH, crude protein, and crude fiber before and after fermentation were measured to determine whether Lactobacillus paracasei SQ01 can improve the fermentation quality and nutritional value of the raw materials, thereby enabling the application of Lactobacillus paracasei SQ01 in feed fermentation fields such as antibiotic alternatives and roughage improvement.

[0087] Raw material pretreatment: The distiller's grains and corn stalks are crushed separately and mixed evenly at a mass ratio of 7:3 to obtain a mixed raw material of distiller's grains and corn stalks for later use.

[0088] Inoculation and sealing: Add fermentation agent 3 to the experimental group mixed raw materials at a dosage of 5% (v / v), and add purified water to the control group mixed raw materials at a dosage of 5% (v / v). After stirring evenly, compact the mixture and cover it with plastic film.

[0089] Fermentation culture: Ferment continuously for 15 days, and record the fermentation temperature.

[0090] Index testing: After fermentation, three replicate samples were taken from each group, and the pH value, crude protein and crude fiber content were tested respectively, and sensory evaluation was carried out.

[0091] The results of temperature, pH value, crude protein and crude fiber content before and after fermentation in the experimental group and the control group are shown in Table 6.

[0092] Table 6. Results of temperature, pH value, crude protein and crude fiber content before and after fermentation.

[0093] Table 6 shows that fermentation agent 3 can effectively promote the fermentation of the mixed raw material of distillers' grains and straw. The temperature of the experimental group increased significantly after fermentation, with the highest internal temperature of the mixed raw material of distillers' grains and straw reaching 64.5℃. It significantly reduced the fermentation pH value (pH dropped to 4.7, the suitable pH range for fermented feed is 4.5-5.5), inhibited the growth of putrefactive bacteria, and increased the crude protein content (the crude protein content of the experimental group increased from 8.8% to 11.7%, an increase of 32.9%) and decreased the crude fiber content (the crude fiber content of the experimental group decreased from 32.5% to 26.3%, a decrease of 19.1%). This indicates that Lactobacillus paracasei SQ01 can degrade the crude fiber in straw, convert the nitrogen source in the raw material, increase the crude protein content, and improve the palatability and nutritional value of fermented feed. It provides basic experimental data for the promotion and application of Lactobacillus paracasei SQ01 in the field of roughage fermentation.

[0094] The sensory evaluation results of the mixed raw materials after fermentation are shown in Table 7.

[0095] Table 7 Sensory evaluation of the mixed raw materials after fermentation

[0096] As shown in Table 7, the experimental group produced a strong sweet and sour taste after fermentation and did not spoil, while the control group produced a light sour taste and showed slight spoilage. This indicates that Lactobacillus paracasei SQ01 can inhibit the growth of spoilage bacteria and promote fermentation and maturation by producing acid.

[0097] IX. Preservation of Lactobacillus paracasei SQ01 Based on the effects of fermentation preparations 1, 2, and 3, it is evident that the *Lactobacillus paracasei* SQ01 screened in this invention has high practical application value in fields such as food preservation, lipid-lowering drug development, and high-temperature processing-specific probiotic fermentation agents. Therefore, the *Lactobacillus paracasei* SQ01 strain was preserved, and the preservation information is as follows: Lactobacillus paracasei SQ01 is deposited at the China Center for Type Culture Collection (CCTCC) on May 15, 2024, with accession number CCTCC NO: M 2024934, located in Wuhan, China, and classified as Lactobacillus paracasei SQ01.

[0098] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. Lactobacillus paracasei SQ01, characterized in that, It is deposited at the China Center for Type Culture Collection (CCTCC) on May 15, 2024, with accession number CCTCC NO: M 2024934, located in Wuhan, China, and classified as Lactobacillus paracasei SQ01.

2. The fermentation preparation obtained by fermentation of *Lactobacillus paracasei* SQ01 according to claim 1, characterized in that, The preparation method is as follows: (1) Inoculate Lactobacillus paracasei SQ01 onto MRS solid medium and incubate upside down at 37°C for 48 h; (2) Pick the bacterial colony from the MRS solid medium and transfer it to the MRS liquid medium. Incubate at 37°C with shaking for 24 hours to obtain the seed culture. (3) The seed culture was inoculated into fermentation medium A and cultured at 37℃ for 24h to obtain fermentation broth A. The formula of fermentation medium A is as follows: glucose 21.0g / L, peptone 10.0g / L, beef extract powder 8.0g / L, yeast extract powder 8.0g / L, dipotassium hydrogen phosphate 3.5g / L, sodium acetate 6.5g / L, diammonium hydrogen citrate 2.0g / L, magnesium sulfate 0.2g / L, adenine 0.13g / L, phenylalanine 0.051g / L, Tween 80 1mL / L, pH 5.5±0.5; (4) Transfer fermentation broth A to fermentation medium B. In the early stage of fermentation, stir and culture at 37℃ for 36h. In the middle and late stages of fermentation, seal and anaerobic fermentation at 37℃ for 36h to obtain fermentation broth B. The formula of fermentation medium B is as follows: glucose 16.0g / L, peptone 10.0g / L, beef extract powder 8.0g / L, yeast extract powder 6.0g / L, dipotassium hydrogen phosphate 3.5g / L, sodium acetate 6.5g / L, diammonium hydrogen citrate 2.0g / L, magnesium sulfate 0.5g / L, calcium chloride 1.0g / L, L-glutamic acid 5.0g / L, manganese sulfate 0.1g / L, vitamin C 0.01g / L, adenine 0.15g / L, phenylalanine 0.05g / L, Tween 80 1mL / L, pH 5.5±0.5; (5) Perform solid-liquid separation on fermentation broth B and collect the fermentation supernatant and bacterial sludge separately; (6) The fermentation supernatant was sequentially separated by microfiltration, ultrafiltration and nanofiltration membranes, and the retentate was collected to obtain the fermentation preparation, which was designated as SQ01 microbial preparation 1; (7) The bacterial sludge is subjected to compound bio-enzyme directional pre-crushing, ultra-high pressure deep crushing, microfiltration membrane filtration, ultrafiltration membrane filtration, and nanofiltration membrane filtration in sequence. The retentate is collected to obtain the fermentation preparation, which is denoted as SQ01 microbial preparation 2; or, the bacterial sludge is directly mixed and homogenized, and then spray-dried to obtain the fermentation preparation, which is denoted as SQ01 microbial preparation 3.

3. The fermentation preparation according to claim 2, characterized in that, In step (4), during the early stage of fermentation, the fermentation process is constant pH fermentation. The pH of the fermentation liquid is maintained in the range of 5.0-6.0 by adding ammonia or sodium hydroxide solution. Micro-aeration and low-speed stirring are performed once every 12 hours, and this is repeated 3-4 times.

4. The fermentation preparation according to claim 2, characterized in that, In steps (6) and (7), the pore size of the microfiltration membrane is 0.22 μm and the operating pressure is 0.1 MPa-0.2 MPa; the molecular weight cutoff of the ultrafiltration membrane is 100 kDa and the operating pressure is 0.2 MPa-0.3 MPa; the molecular weight cutoff of the nanofiltration membrane is 10 kDa and the operating pressure is 0.3 MPa-0.4 MPa; the temperature is controlled at 15℃-25℃ throughout the membrane separation process.

5. The fermentation preparation according to claim 2, characterized in that, In step (7), the compound bio-enzyme is composed of lysozyme, β-glucanase and plant-derived pectinase in a mass ratio of 5:4:

1. The amount added is 0.5%-1% of the dry weight of the bacterial mud. The enzymatic hydrolysis conditions are: pH 5.5-6.0, constant temperature shaking at 40℃-45℃ for 120min-150min, and enzyme inactivation at 80℃ for 30min.

6. The fermentation preparation according to claim 2, characterized in that, In step (7), an ultra-high pressure cell disruptor is used to perform ultra-high pressure deep crushing of the bacteria. The nozzle diameter is 50μm, the working pressure is 120MPa-150MPa, the feeding speed is 50mL / min-80mL / min, and the crushing is repeated 2-3 times.

7. The fermentation preparation according to claim 2, characterized in that, In step (7), a composite protective agent is added to the mushroom mud at 8%-15% of the dry weight of the mushroom mud. The composite protective agent is composed of trehalose, whey protein and maltodextrin in a mass ratio of 4:2:

3. The inlet air temperature of the spray dryer is 110℃-130℃, the outlet air temperature is 65℃-75℃, and the feed flow rate is 10L / h-20L / h.

8. Application of SQ01 microbial preparation 1 in the preparation of food preservatives.

9. Application of SQ01 microbial preparation 2 in the preparation of lipid-lowering drugs.

10. Application of SQ01 microbial preparation 3 in the preparation of high-temperature resistant probiotic starter.

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