Lactobacillus paracasei Q7-12 with low temperature resistance and oxidation resistance and application of lactobacillus paracasei Q7-12
By screening out Lactobacillus paracasei Q7-12, which has both low-temperature resistance and antioxidant properties, the problem of low fermentation efficiency of silage in the high-altitude and cold region of northwestern Sichuan has been solved. It has achieved rapid fermentation and efficient nutrient retention under low-temperature conditions, thereby improving the quality of silage and the efficiency of livestock production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-11
- Publication Date
- 2026-04-10
AI Technical Summary
In the cold and high-altitude region of northwestern Sichuan, conventional commercial silage fermentation agents are difficult to start fermentation quickly in low-temperature environments, resulting in poor fermentation quality of silage, nutrient loss, and even spoilage, which has become a key technical bottleneck hindering the stable development of animal husbandry.
A strain of Lactobacillus paracasei Q7-12 with both low-temperature resistance and antioxidant properties was screened out for use in the preparation of products with antioxidant, acid-resistant, bile salt-resistant and antibacterial effects, especially for silage and fermented yogurt, to improve fermentation efficiency and quality.
Lactobacillus paracasei Q7-12 exhibited the fastest growth rate at 4℃, with superior antioxidant capacity compared to commercial strains. It significantly improved the fermentation quality and nutrient retention of silage, promoted the growth of Lactobacillus plantarum, and enhanced livestock production efficiency.
Smart Images
Figure CN121825831A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a strain of Lactobacillus paracasei Q7-12 that combines low-temperature resistance and antioxidant properties and its applications. Background Technology
[0002] The high-altitude, cold region of northwestern Sichuan is an important livestock production base in my country, serving as a crucial support for ensuring the supply of livestock products and promoting economic development in ethnic minority areas. However, the region's harsh climate, long and cold winters, and short pasture growth cycle make winter storage difficult, which has become a key technical bottleneck hindering the stable development of local animal husbandry.
[0003] Silage is a key source of nutrition for livestock during the winter and spring seasons, and its fermentation quality directly determines the efficiency of livestock production. Currently, conventional commercial silage fermentation agents are difficult to start fermentation quickly at low temperatures, resulting in poor fermentation quality, nutrient loss, and even spoilage of silage.
[0004] Extreme habitats are natural enrichment sites for microorganisms with special functions. Indigenous lactic acid bacteria, adapted to the extreme conditions of low temperatures, strong ultraviolet radiation, and drastic diurnal temperature variations in the high-altitude and cold regions of northwestern Sichuan, are highly likely to have developed unique physiological and metabolic mechanisms during evolution, possessing the dual potential to resist both low-temperature stress and oxidative damage. Based on this, this project focuses on the systematic screening and functional evaluation of indigenous lactic acid bacteria resources in the northwestern Sichuan plateau. The aim is to obtain superior strains that can efficiently produce acid at low temperatures of 4℃~8℃, while also possessing free radical scavenging capabilities and tolerance to oxidative stress. This will fundamentally solve the technical challenges of low fermentation efficiency and unstable quality of silage in high-altitude and cold regions, effectively ensuring livestock feed supply during the winter and spring seasons. This has significant practical implications and application value for promoting cost reduction and efficiency improvement in local animal husbandry, as well as the coordinated advancement of ecological protection and industrial development. Summary of the Invention
[0005] The purpose of this invention is to provide a lactic acid bacterium with low temperature resistance, antioxidant properties, acid resistance, bile salt resistance, and antibacterial effects; specifically, it relates to a strain of Lactobacillus paracasei Q7-12 that has both low temperature resistance and antioxidant properties and its applications.
[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a strain of *Lactobacillus paracasei* that possesses both low-temperature resistance and antioxidant properties. Lacticaseibacillus paracasei Q7-12 is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on December 11, 2025, with accession number CGMCC No. 37037.
[0007] The present invention also provides the aforementioned Lactobacillus paracasei (Lacticaseibacillus paracasei Application of Q7-12 in the preparation of products with antioxidant effects.
[0008] The present invention also provides the aforementioned Lactobacillus paracasei ( Lacticaseibacillus paracasei Application of Q7-12 in the preparation of products with acid resistance.
[0009] The present invention also provides the aforementioned Lactobacillus paracasei ( Lacticaseibacillus paracasei Application of Q7-12 in the preparation of products with bile salt tolerance.
[0010] The present invention also provides the aforementioned Lactobacillus paracasei ( Lacticaseibacillus paracasei Application of Q7-12 in the preparation of products with antibacterial effects.
[0011] Preferably, the product is silage or fermented yogurt.
[0012] The present invention also provides the aforementioned Lactobacillus paracasei ( Lacticaseibacillus paracasei Application of Q7-12 in the preparation of silage with antioxidant, acid-resistant, bile salt-resistant and antibacterial effects.
[0013] The present invention also provides the aforementioned Lactobacillus paracasei ( Lacticaseibacillus paracasei Application of Q7-12 in the preparation of fermented yogurt with antioxidant, acid-resistant, bile salt-resistant and antibacterial effects.
[0014] The present invention also provides a silage feed utilizing the aforementioned Lactobacillus paracasei ( Lacticaseibacillus paracasei Q7-12 was obtained through fermentation.
[0015] The present invention also provides a fermented yogurt, utilizing the aforementioned *Lactobacillus paracasei* (… Lacticaseibacillus paracasei Q7-12 was obtained through fermentation.
[0016] The present invention has the following advantages: This invention provides *Lactobacillus paracasei* Q7-12, which possesses low-temperature resistance and antioxidant properties. At 4°C, this strain exhibits the fastest growth rate. Its antioxidant capacity is superior to the commercial strain XF, with a DPPH radical scavenging rate exceeding 95% and a hydroxyl radical (OH) scavenging rate exceeding 95%. - The clearance rate of Q7-12 was higher than 75%. Furthermore, Q7-12 showed superior performance compared to strain XF in terms of acid resistance, bile salt tolerance, and antibacterial activity. Strain Q7-12 is suitable for silage processing and yogurt fermentation in the western Sichuan plateau region.
[0017] Preservation Instructions
[0018] Lactobacillus paracasei ( Lacticaseibacillus paracaseiQ7-12 is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on December 11, 2025, with accession number CGMCC No. 37037. Attached Figure Description
[0019] Figure 1 The results of morphological observation of strain Q7-12; Figure 2 The results of microscopic morphological observation of strain Q7-12; Figure 3 Phylogenetic tree of strain Q7-12; Figure 4 It is the bacterial community composition of silage grass. Detailed Implementation
[0020] The following detailed description of the solutions provided by the present invention, in conjunction with the embodiments, should not be construed as limiting the scope of protection of the present invention.
[0021] Example 1
[0022] Lactobacillus paracasei ( Lacticaseibacillus paracasei Separation and determination of physicochemical properties of Q7-12
[0023] (a) Method: 1. Sample collection and strain isolation (1) Sample collection Silage and yak yogurt samples were collected from multiple representative locations in Ganzi, Aba, and Liangshan prefectures of the western Sichuan plateau. At least three sampling points were selected in each prefecture to ensure sample diversity and representativeness. Collected samples were placed in sterile sampling bags, stored at low temperatures, and brought back to the laboratory for processing as soon as possible.
[0024] (2) Isolation and purification of strains
[0025] The dilution plating method was used. The collected samples were serially diluted with sterile physiological saline, and the bacterial suspensions of appropriate dilutions were spread on MRS agar plates and incubated at 37°C for 24–72 h. Single colonies with different morphologies were picked and subjected to multiple streak purifications.
[0026] 2. Screening of low-temperature resistant strains
[0027] The purified bacterial strain was inoculated into MRS liquid medium and cultured at 4°C. The OD values of the bacterial culture at different time points were measured. 600 Values were used to screen out strains that grew well under low-temperature conditions.
[0028] 3. Screening for antioxidant capacity
[0029] The antioxidant capacity of the selected strains with low-temperature resistance was determined, with commercial strain XF used as a positive control. Commercial strain XF is Pioneer® 1152 silage inoculant, and its main component is Lactobacillus plantarum.
[0030] (1) Determination of DPPH free radical scavenging ability
[0031] Take 1 mL of sample and place it in a test tube. Add 2 mL of DPPH anhydrous ethanol solution (concentration 0.2 mmol / L), mix well, and react at room temperature in the dark for 30 min. Centrifuge at 8000 g for 10 min at 4℃, and take the supernatant. Measure its absorbance at 517 nm, and zero the instrument with deionized water. Each sample has 3 replicates. DPPH scavenging rate (%) = [1 - (A sample - A blank) / A control] × 100% (Blank group: DPPH is replaced with an equal volume of anhydrous ethanol; Control group: Sample solution is replaced with an equal volume of distilled water, and the blank is zeroed with an equal volume of distilled water and anhydrous ethanol mixture).
[0032] (2) OH - Free radical scavenging capacity determination
[0033] 1 mL O - Add 0.5 mL of sample, 1 mL of PBS, 1 mL of 2.5 mmol / L FeSO4, and 1 mL of 20 mmol / L H2O2 to phenanthroline (concentration = 0.1%). After incubation at 37℃ for 1.5 h, the absorbance was measured at 536 nm in triplicate for each sample. Hydroxyl radical scavenging rate (%) = [(Sample A - Control A) / (Blank A - Control A)] × 100% (Blank group: 1 mL of distilled water instead of 1 mL of H2O2; Control group: 0.5 mL of distilled water instead of 0.5 mL of sample).
[0034] (3) Determination of total antioxidant capacity (T-AOC)
[0035] Antioxidant capacity (T-AOC) was determined using a kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., A015-3-1).
[0036] (4) Superoxide dismutase (SOD) activity assay
[0037] The total superoxide dismutase (T-SOD) assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., A001-3-2) was used.
[0038] Based on the antioxidant results, further screening was conducted to identify strains that possess both low-temperature resistance and strong antioxidant capacity.
[0039] 4. Strain identification
[0040] (1) Identification of colony morphology
[0041] The strain was cultured on MRS solid medium using the spread method. After anaerobic culture at 37°C, the macroscopic morphological characteristics of single colonies were observed and recorded, including colony size, shape, edge characteristics, degree of elevation, color and transparency, surface gloss and texture, etc.
[0042] (2) Microscopic morphological identification
[0043] Take a small amount of bacterial suspension, spread it evenly on a glass slide, let it air dry, fix it, and perform Gram staining. The steps include primary staining: crystal violet staining for 1 min, washing with water; mordanting: iodine staining for 1 min, washing with water; destaining: 95% alcohol destaining for 30 s (until the flowing alcohol is colorless), washing with water; counterstaining: safranin / safflower staining for 1 min, washing with water, and air drying. After staining, observe under an oil microscope.
[0044] (3) Identification of 16S rRNA gene
[0045] DNA was extracted from the screened strains using a bacterial genomic DNA extraction kit. Universal primers for 16S rRNA gene amplification (PCR) were: 27F (5'-AGAGTTTGATCMTGGCTCAG-3'SEQ ID NO.1) and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'SEQ ID NO.2). PCR program: pre-denaturation at 94℃ for 5 min; 30 cycles (94℃ 30 s, 55℃ 30 s, 72℃ 90 s); final extension at 72℃ for 10 min. The PCR products were detected by agarose gel electrophoresis, confirming the amplification of a specific band of approximately 1500 bp. The purified PCR products were subjected to bidirectional sequencing. The obtained 16S rRNA gene sequence was compared for homology in the NCBI database using the BLAST program.
[0046] The 16S rRNA gene sequence is shown in SEQ ID NO.3; SEQ ID NO.3:
[0047] (4) Determination of physicochemical properties
[0048] The selected functional strains underwent a series of physicochemical property tests, including Gram staining, catalase test, and sugar fermentation test.
[0049] Acid production capacity: The strain was inoculated into MRS liquid medium and cultured at 37°C for 24 h. After centrifugation at 12,000 rpm for 3 min, the supernatant was collected and the pH was measured.
[0050] Carbohydrate fermentation: Referring to Bergey's Manual of Bacterial Identification, acid production (yellowing of color) and gas production were observed using phenol red carbohydrate broth medium.
[0051] Catalase test: Pick up a colony from the solid culture medium with an inoculation loop, place it in a clean test tube, add 2 mL of 3% hydrogen peroxide solution, and observe the results; if bubbles appear within half a minute, it is positive, and if no bubbles appear, it is negative.
[0052] Acid resistance test: The strain was inoculated into MRS liquid medium at pH 2.5 and pH 3.0 and incubated at 37°C for 24 h. The culture was serially diluted, spread on MRS plates, and anaerobically incubated at 37°C for 36 h. The viable count was then calculated.
[0053] Bile salt tolerance test: The strain was inoculated into MRS liquid medium containing 0.3% bovine bile salts and incubated at 37°C for 24 h. The culture was then serially diluted and spread onto MRS plates, and anaerobically incubated at 37°C for 36 h. The viable count was then calculated.
[0054] Antibacterial test: The inhibitory effect of the strain's metabolites on Escherichia coli and Clostridium was tested using the agar diffusion method (perforation method). The fermentation broth was added dropwise to Oxford cups containing indicator bacteria on agar plates and incubated at 37°C for 24 h. The diameter of the inhibition zone was then measured.
[0055] (II) Results
[0056] 1. Isolation and screening of strains
[0057] Twenty-eight bacterial strains were isolated from silage and yak yogurt samples, and 21 strains that could reproduce normally at 4℃ were successfully screened from these 280 strains. The OD values of the 21 strains at different time points at 4℃ were also analyzed. 600 The values are shown in Table 1. Q7-12 had the highest OD value at 24 h, which was 0.32, indicating that it had the fastest growth rate.
[0058] Table 1. OD values of 21 bacterial strains at different time points at 4℃ 600 value
[0059] 2. Screening of strains with antioxidant capacity
[0060] Of the 21 strains mentioned above, Q7-12 showed the best antioxidant capacity, as shown in Table 2. The T-AOC and OH values of Q7-12 were both higher than those of the commercial strain XF. - The free radical scavenging rate was higher than 79.00%, and the DPPH scavenging rate was above 97%.
[0061] Table 2 Antioxidant capacity of strain Q7-12
[0062] 3. Colony morphology identification
[0063] Strain Q7-12 differed significantly from the commercial strain XF in colony size, shape, edge, and color (Table 3), suggesting that Q7-12 may differ from strain XF at the genus level. Morphological observations of strain Q7-12 are as follows: Figure 1 As shown.
[0064] Table 3. Colony morphology of strain Q7-12
[0065] 4. Microscopic morphological identification
[0066] Microscopic morphological results of strain Q7-12 are shown below. Figure 2 The Q7-12 strain showed a positive Gram staining result, with uniform staining and a clear blue-purple color in the microscopic field of view. The Q7-12 strain is rod-shaped, slender, with most cells slightly curved, exhibiting numerous single arrangements, as well as paired or short chain arrangements.
[0067] 5. 16S rDNA gene identification
[0068] The 16S rDNA sequence of strain Q7-12 was amplified to 1427 bp (SEQ ID NO.3). BLAST comparison revealed that the strain with 100% similarity to it was *Lactobacillus paracasei*. Lacticaseibacillus paracasei The phylogenetic tree analysis results are as follows: Figure 3 As shown.
[0069] 6. Physiological and biochemical characteristics of the strain
[0070] The physiological and biochemical characteristics of strain Q7-12 are shown in Table 4. Strain Q7-12 exhibits good acid production ability, positive for sugar fermentation, and negative for catalase; its acid tolerance and bile salt tolerance are better than those of the commercial strain XF.
[0071] Table 4 Physiological and biochemical characteristics of strain Q7-12
[0072] The antibacterial properties of strain Q7-12 are shown in Table 5. *Escherichia coli* is the most common Gram-negative bacterium, and *Bacillus cereus* is also a Gram-negative bacterium, commonly found in poorly fermented silage, thus representing undesirable bacteria in silage fermentation. Therefore, *Escherichia coli* and *Bacillus cereus* were selected as indicator bacteria. The supernatant of Q7-12 showed strong antibacterial activity against both indicator bacteria, with inhibition diameters of 16.2 mm and 13.0 mm against *Escherichia coli* and *Bacillus cereus*, respectively, both higher than that of the commercial strain XF.
[0073] Table 5. Antibacterial activity of strain Q7-12 (inhibition zone diameter, mm)
[0074] Example 2
[0075] Using *Gnaphalium affine* at different growth stages (heading stage and booting stage) in the high-altitude cold region of northwestern Sichuan as silage material, three treatment groups were set up for silage: the control group (Control) received no treatment agent; the treatment group (S) received silage with commercial lactic acid bacteria XF; and the treatment group (Q7-12) received silage with strain Q7-12 screened in this invention. The fermentation characteristics and chemical composition of the *Gnaphalium affine* silage were determined after 90 days of silage; the bacterial community composition after 90 days of silage fermentation was analyzed using 16S third-generation sequencing technology. The results are shown in Tables 6 and 7. Figure 4 As shown.
[0076] The fermentation characteristics of *Gynostemma pentaphyllum* silage are shown in Table 6. The pH value, lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) showed interactive effects in each treatment group (treatment group × growth period). P < 0.001). During both reproductive periods, compared to the control group, the levels of LA and AA in the Q7-12 treatment group were significantly increased ( P < 0.05), and its content was also significantly higher than that of the S treatment group ( P < 0.05). Meanwhile, the pH, PA, and BA content in the Q7-12 treatment group were significantly lower than those in the control group ( P < 0.05), and also significantly lower than the S treatment group ( P < 0.05). The above results indicate that the Q7-12 treatment group can rapidly reduce the pH of silage, promote LA fermentation, and improve the fermentation quality of silage.
[0077] Table 6. Fermentation characteristics of silage forage at different growth stages.
[0078] Note: Y represents heading stage, W represents booting stage, S represents commercial lactic acid bacteria treatment group, Q7-12 represents screening strain treatment group; LA represents lactic acid, AA represents acetic acid, PA represents propionic acid, BA represents butyric acid; A represents treatment group, G represents growth stage (heading stage, booting stage), A × G represents treatment group × growth stage, different lowercase letters indicate significant differences between groups ( P < 0.05).
[0079] The chemical composition of silage after 90 days of fermentation is shown in Table 7. Dry matter (DM), soluble carbohydrates (WSC), and acid detergent fiber (ADF) showed interactions in each treatment group (treatment group × growth period). P <0.001). During both reproductive periods, compared to the control group, the Q7-12 treatment group significantly increased the levels of DM, WSC, and CP ( ). P < 0.05), significantly reduced NPN (non-protein nitrogen) and NH3-H (ammonia nitrogen) content ( P < 0.05); and compared with the S treatment group, the DM and CP (crude protein) contents in the Q7-12 treatment group were significantly higher than those in the S treatment group ( P < 0.05), NPN content was significantly lower than that of the S treatment group ( P < 0.05); During the heading stage, compared with the control group and the S treatment group, the Q7-12 treatment group significantly increased the content of aNDF and ADF ( P < 0.05). The above results indicate that the Q7-12 treatment group retained the nutrients in the forage silage.
[0080] Table 7 Effects of different strains on the chemical composition of silage grass
[0081] Note: Y represents heading stage, W represents booting stage, S represents commercial lactic acid bacteria treatment group, Q7-12 represents screening strain treatment group; DM represents dry matter, WSC represents soluble carbohydrates, CP represents crude protein, NPN represents non-protein nitrogen, NH3-H represents ammonia nitrogen, aNDF represents neutral detergent fiber, ADF represents acid detergent fiber; A represents treatment group, G represents growth stage (heading stage, booting stage), A × G represents treatment group × growth stage, different lowercase letters indicate significant differences between groups (…). P < 0.05).
[0082] The bacterial community composition of silage hay is as follows Figure 4 As shown. Figure 4 The results showed that, during both growth stages, compared with the control group and the S treatment group, the Q7-12 treatment group had significantly higher levels of *Lactobacillus plantarum*. Lactobacillus plantarum The relative abundance increased. These results indicate that the Q7-12 treatment group promoted the growth of *Lactobacillus plantarum* in *Gnaphalium affine* silage. Lactobacillus plantarumThe growth of.
[0083] As can be seen from the above embodiments, the present invention provides a *Lactobacillus paracasei* Q7-12 strain that possesses both low-temperature resistance and strong antioxidant properties. At 4°C, this strain exhibits the fastest growth rate. Its antioxidant capacity is superior to the commercial strain XF, with a DPPH free radical scavenging rate exceeding 95% and a hydroxyl free radical (OH) scavenging rate exceeding 95%. - The clearance rate of the strain was higher than 75%. Furthermore, Q7-12 showed superior performance compared to strain XF in terms of acid resistance, bile salt tolerance, and antibacterial activity. This strain also demonstrated excellent efficacy in silage processing in the western Sichuan plateau region, providing a basis for the processing of silage in this area.
[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A strain of *Lactobacillus paracasei* possessing both low-temperature resistance and antioxidant properties (… Lacticaseibacillus paracasei Q7-12, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on December 11, 2025, with accession number CGMCC No. 37037.
2. The *Lactobacillus paracasei* as described in claim 1 ( Lacticaseibacillus paracasei Application of Q7-12 in the preparation of products with antioxidant effects.
3. The *Lactobacillus paracasei* as described in claim 1 ( Lacticaseibacillus paracasei Application of Q7-12 in the preparation of products with acid resistance.
4. The *Lactobacillus paracasei* as described in claim 1 ( Lacticaseibacillus paracasei Application of Q7-12 in the preparation of products with bile salt tolerance.
5. The *Lactobacillus paracasei* as described in claim 1 ( Lacticaseibacillus paracasei Application of Q7-12 in the preparation of products with antibacterial effects.
6. The application according to any one of claims 2 to 5, characterized in that, The product is either silage or fermented yogurt.
7. The *Lactobacillus paracasei* as described in claim 1 ( Lacticaseibacillus paracasei Application of Q7-12 in the preparation of silage with antioxidant, acid-resistant, bile salt-resistant and antibacterial effects.
8. The *Lactobacillus paracasei* as described in claim 1 ( Lacticaseibacillus paracasei Application of Q7-12 in the preparation of fermented yogurt with antioxidant, acid-resistant, bile salt-resistant and antibacterial effects.
9. A type of silage, characterized in that, Using the Lactobacillus paracasei (as described in claim 1) Lacticaseibacillus paracasei Q7-12 was obtained through fermentation.
10. A fermented yogurt, characterized in that, Using the Lactobacillus paracasei (as described in claim 1) Lacticaseibacillus paracasei Q7-12 was obtained through fermentation.