Lactobacillus pentosus and application thereof in gossypol detoxification
By using fermentation technology based on Lactiplantibacillus pentosus YL10 and its derivatives, the shortcomings of chemical and solvent extraction methods in gossypol detoxification have been overcome, achieving efficient and safe gossypol detoxification and feed fermentation, thereby improving the detoxification rate and nutritional value.
Patent Information
- Application Number
- CN202511202456.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-26
- Publication Date
- 2025-11-07
AI Technical Summary
Existing methods for detoxifying gossypol suffer from problems such as chemical reagent residues, high solvent recovery costs, environmental pollution, and loss of nutritional value. Furthermore, there are relatively few strains available for large-scale application.
Lactiplantibacillus pentosus YL10 and its derivatives are used to degrade gossypol through anaerobic, facultative anaerobic, or aerobic fermentation. This method utilizes multiple carbon and nitrogen sources to enhance cell viability, inhibit the growth of harmful bacteria, and avoid the drawbacks of chemical and physical methods.
It achieves efficient detoxification of gossypol, avoiding reagent residues from chemical methods and environmental pollution problems from solvent extraction methods, while preserving the nutritional value of feed and providing a safe feed fermentation solution.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of gossypol detoxification, in particular to a pentose Lactobacillus plantarum and its application in gossypol detoxification. BACKGROUND
[0002] Due to the large amount of gossypol contained in cottonseed meal, it is mainly used in ruminant feed as a source of energy and protein. Gossypol is a natural phenolic compound that exists in the form of FG (free gossypol) and bound gossypol in the roots, stems, leaves and seeds of cotton. Excessive intake of FG can cause anemia in animals, damage reproductive function, and organ and intestinal health (Zhuo et al., 2023; Zhang et al., 2024). Since the absorption of bound gossypol in the digestive tract is limited, it exhibits non-toxic properties, while bound gossypol can be hydrolyzed to FG during animal digestion, which is then absorbed by animals, producing toxic side effects (Kadam et al., 2021).
[0003] Currently, gossypol detoxification is divided into chemical method, solvent leaching method, physical method and biological method. The chemical method converts FG into bound gossypol or denatures it by adding ferrous sulfate, urea, alkali and other chemical reagents. However, this method is accompanied by wastewater and waste gas pollution, and the residues of chemical reagents can easily lead to the loss of nutritional components in cottonseed meal. The solvent leaching method has good detoxification effect, but it has problems such as high cost of solvent recovery and reuse, and high risk of environmental pollution; the physical method uses high temperature and high pressure to promote the combination of gossypol and protein to form bound gossypol for detoxification, but this process can easily damage protein and other nutrients, reducing nutritional value; in addition, bound gossypol can be converted to FG after animal digestion; in contrast, the biological method (microbial detoxification method) utilizes the characteristics of microorganisms to degrade toxic substances, and has been widely applied in the degradation of water, soil and feed pollutants, showing good potential in the field of gossypol detoxification.
[0004] Research has found that microorganisms such as Candida tropicalis, Aspergillus niger, Lactobacillus plantarum, Bacillus subtilis and biological enzymes (laccase) can effectively reduce the content of FG (Suntara et al., 2020; Tang et al., 2012; Yusuf et al., 2022). However, fungal and Bacillus subtilis fermentation detoxification requires oxygen, increasing labor costs, and some strains can produce mycotoxins, which are prohibited from use in feed production. Therefore, there are few strains that can be used on a large scale in production.
[0005] In view of this, the present application is proposed. SUMMARY
[0006] The present application aims to provide a Lactiplantibacillus pentosus and its application in gossypol detoxification to solve the above technical problems.
[0007] The present application is implemented as follows:
[0008] In the first aspect, the present application provides a Lactiplantibacillus pentosus, which is preserved in Wuhan University, Wuhan, China, with the preservation number CCTCC NO: M20231547.
[0009] In the second aspect, the present application provides a derivative of the Lactiplantibacillus pentosus, which is a culture of the Lactiplantibacillus pentosus.
[0010] In the third aspect, the present application provides a culture medium of the Lactiplantibacillus pentosus, which comprises the Lactiplantibacillus pentosus or the derivative described above.
[0011] In the fourth aspect, the present application provides a composition, which comprises the Lactiplantibacillus pentosus or the derivative described above.
[0012] In the fifth aspect, the present application provides the application of the Lactiplantibacillus pentosus, the derivative, the culture medium or the composition described above in gossypol detoxification.
[0013] In the sixth aspect, the present application provides a method for gossypol detoxification, which comprises the following steps: mixing a sample to be detoxified with the Lactiplantibacillus pentosus described above, the derivative described above, the culture medium described above or the composition described above for fermentation.
[0014] The present application has the following beneficial effects:
[0015] The present application screens a gossypol detoxification strain from cotton field humus soil, obtains a single strain after multiple streak plate cultures, and then re-screens the strain by increasing the content of gossypol acetate solution in the culture medium to determine a high-efficiency gossypol detoxification strain. After testing, the strain provided by the present application has a high detoxification rate for free gossypol and can be anaerobically, facultatively anaerobically or aerobically fermented. The strain provided by the present application can not only avoid the reagent residue problem existing in the chemical method, but also avoid the solvent recovery cost problem and environmental pollution problem caused by improper recovery of the solvent extraction method, and also avoid the nutrient value loss problem caused by the physical method.
[0016] The strain is identified by molecular biology as Lactiplantibacillus pentosus (L.pentosus) and named as YL10. Except that mannitol cannot be utilized, the strain provided by the application can utilize a plurality of carbon sources and nitrogen sources such as raffinose, cellobiose and sucrose. The biological property results show that the strain L.pentosus YL10 is not hemolytic, sensitive to a plurality of antibiotics, and has high tolerance to intestinal fluid and bile salt, can improve cell viability, and inhibit the growth of Staphylococcus aureus, Salmonella and Escherichia coli. Therefore, the strain provided by the application has high safety and can be used for feed fermentation. BRIEF DESCRIPTION OF DRAWINGS
[0017] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some of the embodiments of the application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.
[0018] Figure 1 Morphological identification of the strain YL10. (A) YL10 colony; (B, C) Gram staining figure (×100);
[0019] Figure 2 Molecular biology identification of the strain YL10. (A) 16S rDNA PCR amplification electrophoresis; (B) 16S rDNA sequencing; (C) 16S rDNA phylogenetic tree;
[0020] Figure 3 Growth curve of the strain YL10;
[0021] Figure 4 Influence result figure of the strain YL10 on IPEC-J2 cell viability;
[0022] Figure 5 Result figure of the strain YL10 on intestinal fluid tolerance and bile salt tolerance (A is the result figure of the strain on intestinal fluid tolerance test, and B is the result figure of the strain on 0.1%, 0.2%, 0.3%, 0.4% bile salt tolerance test);
[0023] Figure 6 Inhibition effect evaluation result figure of the strain YL10. (A) Staphylococcus aureus; (B) Salmonella; (C) Escherichia coli;
[0024] Figure 7Fig. 1 shows the effects of different moisture contents (A, B) and different amounts of bacterial liquid (C, D) and different fermentation times (E, F) on the FG content and detoxification rate of CSM. DETAILED DESCRIPTION
[0025] Reference will now be made in detail to the embodiments of the application, one or more examples of which are set forth below. Each example is provided as an explanation and not as a limitation of the application. Indeed, it will be apparent to one of ordinary skill in the art that numerous modifications and variations of the present application are possible in light of the above teachings. For example, features described or illustrated as part of one embodiment can be used with another embodiment to yield still a further embodiment.
[0026] In a first aspect, the present application provides a Lactiplantibacillus pentosus, which is preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC NO: M 20231547, a preservation date of August 31, 2023, and a classification name of Lactiplantibacillus pentosus YL10. The identification result is survival, and the preservation address is: China. Wuhan. Wuhan University.
[0027] In the test, different screening media and different concentrations of saturated gossypol acetate solution were first selected to screen gossypol detoxification strains from cotton humus soil, and after multiple streak plate cultures, single strains were obtained. Then, the strains were rescreened by increasing the content of gossypol acetate solution in the culture medium to determine the target strain with ideal detoxification effect. Through testing, the strain provided by the present application has a high detoxification rate for free gossypol and can be anaerobically, facultatively anaerobically or aerobically fermented. The use of the strain provided by the present application can not only avoid the reagent residue problem existing in the chemical method, but also avoid the problems of high solvent recovery cost and environmental pollution caused by improper recovery existing in the solvent extraction method, and also avoid the problem of loss of nutritional value caused by the physical method.
[0028] The strain is identified by molecular biology as Lactiplantibacillus pentosus (L. pentosus) and named as YL10. Morphological observation, biological characteristics and bacteriostatic test of the strain show that the strain provided in the application can utilize raffinose, cellobiose, sucrose and other carbon sources and nitrogen sources except for mannitol. The biological characteristic results show that the strain L. pentosus YL10 is not hemolytic, sensitive to various antibiotics, and has high tolerance to intestinal fluid and bile salt, can improve cell viability and inhibit the growth of Staphylococcus aureus, Salmonella and Escherichia coli. Therefore, the strain provided in the application has high safety and can be used for feed fermentation.
[0029] In a second aspect, the present application provides a derivative of Lactiplantibacillus pentosus, the derivative being a culture of Lactiplantibacillus pentosus.
[0030] The culture includes, but is not limited to, a liquid culture, a solid culture, a fermentation supernatant or a slurry.
[0031] The culture further includes a substance after separation, drying and other treatments of Lactiplantibacillus pentosus, including at least one of a concentrate, a dried substance, a liquid substance and a dilution of Lactiplantibacillus pentosus. The dried substance includes, but is not limited to, a spray-dried substance, a freeze-dried substance, a vacuum-dried substance and a drum-dried substance.
[0032] In a third aspect, the present application provides a culture medium of Lactiplantibacillus pentosus, which includes Lactiplantibacillus pentosus or the derivative described above.
[0033] The culture medium is, for example, a liquid culture medium or a solid culture medium, and the culture medium is, for example, an MRS culture medium.
[0034] In a fourth aspect, the present application provides a composition, which includes Lactiplantibacillus pentosus or the derivative described above.
[0035] In a preferred embodiment of the present application, the composition is a feed composition.
[0036] In a preferred embodiment of the present application, the feed composition includes a feed and Lactiplantibacillus pentosus or the derivative; and the feed is cottonseed meal, cottonseed cake or cottonseed.
[0037] In a fifth aspect, the present application provides application of Lactiplantibacillus pentosus, the derivative, the culture medium or the composition described above in gossypol detoxification.
[0038] Lactiplantibacillus pentosus can reduce the level of free gossypol through the fermentation activity of live bacteria.
[0039] In a preferred embodiment of the application, when gossypol detoxification is performed, the pentosus lactis, derivative or culture medium is mixed with the cottonseed meal to be detoxified, and the fermentation conditions are as follows: the water content of the fermentation broth is controlled at 40-60%, the inoculation amount of pentosus lactis is 4-12%, and the fermentation time is at least 7d. For example, the water content of the fermentation broth is 40%, 45%, 50%, 55% or 60%, the inoculation amount of pentosus lactis is 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% or 12%, and the fermentation time is at least 7d, for example, 7d, 8d, 9d, 10d, 11d, 12d, 13d, 14d, 15d, 16d or 17d.
[0040] Under the above fermentation conditions, the gossypol detoxification effect is higher.
[0041] The results of the effect of different water contents of the fermentation broth on the FG (free gossypol) detoxification rate show that as the water content increases from 35% to 45%, the FG content decreases significantly. When the water content is in the range of 45%-60%, the FG content reaches an inflection point as the water content increases, and the FG detoxification rate gradually decreases. When the water content is 45%, the FG content is the lowest, which is 425.33 mg / kg, and the detoxification rate is 63.77%. The above water content (water content) can also be equivalent to humidity.
[0042] The results of the effect of different inoculation amounts of the bacterial solution on the FG detoxification rate when the water content is 45% show that as the inoculation amount increases from 2% to 8%, the FG content decreases significantly. When the inoculation amount is between 8% and 12%, the FG content is close. Therefore, it is preferred to use an inoculation amount of 8% for fermentation of the cottonseed meal to be detoxified, and the FG detoxification rate is 63.90%.
[0043] Under the conditions of water content of 45% and inoculation amount of 8%, as the fermentation time increases, the FG content gradually decreases. On the 11th day and the 13th day of fermentation, the FG content is 274.67 mg / kg and 268.67 mg / kg, respectively, and the FG detoxification rate is 76.73% and 76.54%, respectively. Therefore, the optimal fermentation conditions for the cottonseed meal to be detoxified are water content of 45%, inoculation amount of 8% and fermentation time of 11d.
[0044] In a preferred embodiment of the application, the fermentation conditions are as follows: the water content of the fermentation broth is controlled at 45-55%, the inoculation amount of pentosus lactis is 8-12%, and the fermentation time is 11-13d.
[0045] In a preferred embodiment of the application, the fermentation conditions are anaerobic or aerobic fermentation.
[0046] In a preferred embodiment of the application, the fermentation conditions are anaerobic fermentation. Anaerobic fermentation can better control the growth of mold in the later stage.
[0047] In a sixth aspect, the present application provides a method for detoxifying gossypol, comprising the step of fermenting a sample to be detoxified with gossypol with the Lactobacillus pentosus as described above, the derivative as described above, the culture medium as described above, or the composition as described above.
[0048] In a preferred embodiment of the present application, the sample to be detoxified with gossypol is selected from the group consisting of cottonseed meal.
[0049] In a preferred embodiment of the present application, the fermentation condition is that the water content of the fermentation broth is controlled to be 40-60%, the inoculation amount of the Lactobacillus pentosus is 4-12%, and the fermentation time is at least 7d. For example, the water content of the fermentation broth is 40%, 45%, 50%, 55% or 60%, the inoculation amount of the Lactobacillus pentosus is 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% or 12%, and the fermentation time is at least 7d, for example, 7d, 8d, 9d, 10d, 11d, 12d, 13d, 14d, 15d, 16d or 17d.
[0050] In a preferred embodiment of the present application, the fermentation condition is that the water content of the fermentation broth is controlled to be 45-55%, the inoculation amount of the Lactobacillus pentosus is 8-12%, and the fermentation time is 11-13d. Under the above fermentation condition, the detoxification effect of free gossypol is higher.
[0051] In a preferred embodiment of the present application, the fermentation is anaerobic fermentation, facultative anaerobic fermentation or aerobic fermentation.
[0052] In a preferred embodiment of the present application, the fermentation is anaerobic fermentation.
[0053] In a preferred embodiment of the present application, the culture medium for fermentation is MRS culture medium, and the fermentation temperature is 37℃±0.5℃.
[0054] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the reagents or instruments are not indicated by the manufacturers, they are all conventional products that can be purchased in the market.
[0055] The features and performances of the present application will be further described below in combination with the embodiments.
[0056] Embodiment 1
[0057] In this embodiment, the isolation and identification of the strain are carried out.
[0058] 1. Test materials
[0059] Soil sample collection and processing: Randomly collected soil samples from cotton growing areas in Alar, the seventh division of Xinjiang, Changji Hui Autonomous Prefecture, Kashi and Wujiakuang 105 regiment, and stored in sealed bags. Weigh 100 g of each soil sample, add 5 g of gossypol acetate, stir evenly, add 50% water, and ferment at room temperature for 7 days. Take 1.00 g of unfermented and fermented soil samples into 100 mL triangular flask, add 9 mL of double distilled water, shake and mix well for use.
[0060] CSM sample: Cottonseed meal was purchased from Beijing Zhongcheng Tianli Biological Technology Co., Ltd. After crushing, it was used for CSM fermentation test.
[0061] 2. Preparation of culture medium and reagents
[0062] Lactic acid bacteria culture medium (MRS) was purchased from Qingdao Haibo Biological Company. According to the requirements of the kit, the corresponding weight was weighed, and after adding ultrapure water, it was subjected to high temperature and high pressure sterilization.
[0063] Gossypol medium: add the corresponding saturated gossypol acetate solution (2%, 3%, 4%) to the sterilized medium cooled to about 60±5℃.
[0064] Add 2% agar powder to the liquid medium, which is solid medium; after sterilization, the solid medium is cooled to 60±5℃ and 5% sterile defibrillation sheep blood is added, which is blood plate.
[0065] 3. Screening, isolation and purification of strains
[0066] Take 100 μL of double distilled water diluted sample and spread it on the corresponding medium, and place it in an anaerobic fermentation bag and a natural oxygen fermentation bag, respectively, and then place it in a 37℃ constant temperature incubator for 72h. Observe the appearance and morphology of the colonies, and pick single colonies and four-zone inoculation on the corresponding medium for continuous culture for 48h. Purify multiple times, and obtain single strains with consistent colony morphology on the plate, then store them in a 4℃ refrigerator for standby use.
[0067] 4. Strain verification
[0068] Pick high concentration gossypol culture medium single colony inoculation to 1.5 mL centrifuge tube, put in constant temperature incubator 37℃ static culture overnight; according to 1% inoculation amount (“inoculation amount” refers to the volume of seed liquid of inoculated strain per 100 g raw material is mL / 100 g) transfer to 15 mL centrifuge tube, continue to culture, according to 8% inoculation amount inoculation in crushed cottonseed meal, add 50% moisture (mL / 100 g), mix thoroughly and then put into polyethylene plastic bag (290 mm×230 mm, fermentation feed bag with one-way valve), anaerobic fermentation for 3 d; after drying at 55℃, the free gossypol content of cottonseed meal before and after fermentation is determined by national standard aniline method GB / T 13086-2020 “determination method of free gossypol in feed”, and the detoxification rate of free gossypol is calculated. The strain with the highest free gossypol detoxification rate is selected for further test.
[0069] Free gossypol detoxification rate (%) = (unfermented cottonseed meal free gossypol content-fermented cottonseed meal free gossypol content) / unfermented cottonseed meal free gossypol content x 100.
[0070] 5. Strain identification
[0071] 5.1 Morphological identification
[0072] After the selected strain is activated in MRS liquid medium, the inoculation ring is used to pick the bacterial liquid and spread on MRS solid medium, and then it is placed in a 37℃ incubator for culture. After 2 d of culture, single colonies are picked, stained with Gram and observed for color and shape under an oil microscope.
[0073] According to the CSM fermentation test verification results, a FG detoxification strain YL10 is screened, which grows well on high concentration gossypol MRS medium, round or nearly round, purple after Gram staining (gram-positive bacteria), short rod-shaped ( Figure 1 ). Figure 2 PCR results show that the size of the fragment is 1500 bp ( ).
[0074] 5.2 Molecular biology identification
[0075] The supernatant is removed by centrifugation of the stable period bacterial liquid, and the genomic DNA is extracted. PCR amplification is performed using bacterial 16S rDNA primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1429R: 5'-TTTGATCCTGGCTCAG-3'. The purified PCR product is subjected to DNA sequencing using a sequencer ABI3730-XL. The spliced sequence file is compared with the 16S database in the NCBI Blast program, and a phylogenetic tree is drawn using MEGA 11.0 software. Bootstraps>1000 times are statistically analyzed.
[0076] The PCR-amplified fragment was sequenced, and the sequence was compared with the NCBI database. The results showed that the strain had the highest similarity with Lactiplantibacillus pentosus, and the strain was preliminarily considered to be Lactiplantibacillus pentosus (L.pentosus), named YL10 (L.pentosus YL10). Figure 2
[0077] 5.3. Physiological and biochemical identification
[0078] According to the method described in the "Bacterial System Identification Manual" and the micro-biochemical identification tube, the strain was preliminarily identified and screened. After the bacteria were inoculated on blood plates and placed in a 37°C incubator, whether there was a hemolytic ring around the colonies was observed.
[0079] The physiological and biochemical results of the strain YL10 are shown in Table 1. In addition to mannitol, the strain YL10 can utilize serum glucose, cellobiose, sucrose, salicin, maltose, raffinose, lactose, 1% hippuric acid and sorbitol. At the same time, the strain is not hemolytic and does not produce gas.
[0080] Table 1 Biochemical characteristics of strain YL10
[0081]
[0082] Note: "-" indicates negative, "+" indicates positive.
[0083] Example 2
[0084] In this example, the biological characteristics of the strain were analyzed. The data were arranged using Excel 2021 software. Single factor analysis of variance in SPSS 26.0 software was used for multiple comparisons, and Duncan method was used for multiple comparisons between groups. Two-group comparison was analyzed by t-test. Data mapping was performed using GraphPad Prism 9.3.0 software. Each feed sample was set up with three replicates, and the data results were set up with parallel samples, expressed as mean ± standard error, and P<0.05 indicated significant difference.
[0085] 1. Growth curve
[0086] Activated bacteria were inoculated in MRS medium at 1% inoculation amount, and incubated at 37°C. The absorbance of the fermentation broth at OD 600 was measured every 2h, and the absorbance value was used as the vertical coordinate and the culture time was used as the horizontal coordinate to draw the growth curve of the strain.
[0087] Growth curve reference Figure 3 As shown in the reference, at 0-4h, the strain YL10 was in lag phase. At 4-12h, the strain was in logarithmic growth phase and showed geometric growth. At 12-38h, the strain grew slowly and entered the stationary phase.
[0088] 2. Safety
[0089] Drug sensitivity: Take the bacteria in the stable phase with 1% inoculation amount in MRS medium, evenly coat with a coating rod, and then place the drug sensitivity paper containing 12 kinds of antibiotics on the medium. After 48h, observe whether there is an inhibition zone.
[0090] The drug sensitivity test results are shown in Table 2. The strain is sensitive to chloramphenicol, streptomycin, gentamicin, ampicillin, erythromycin, tetracycline and clindamycin, which can inhibit the growth of the strain YL10.
[0091] Table 2 Analysis of drug sensitivity of strain YL10
[0092]
[0093] Note: "S" represents sensitivity, and "R" represents insensitivity.
[0094] Cell viability: 100μL of IPEC-J2 cell (porcine small intestinal epithelial cell) suspension was inoculated in a 96-well plate. After the cells grew to 80%-85%, the bacteria suspension after centrifugation was added to the cell culture medium. After 4h of incubation in an incubator, 10μL of CCK-8 solution was added to each well. After 2-4h of incubation in an incubator, the absorbance at OD 450 was measured by an enzyme marker instrument, and the cell survival rate was calculated.
[0095] As Figure 4 shown, the strain can improve the IPEC-J2 cell viability (P<0.05).
[0096] 3. Tolerance
[0097] Take the bacteria in the early stable phase after culture activation with 1% inoculation amount, inoculate the bacteria in the liquid medium, centrifuge at 4℃, 6000xg for 3min after 12h of culture, collect the bacteria; rinse the bacteria with sterile normal saline for 2 times to remove the culture medium, resuspend in an equal volume of sterile water; mix thoroughly, and then test the tolerance of the strain to intestinal juice and bile salt.
[0098] Artificial intestinal juice tolerance: Preheat the artificial intestinal juice to 37℃, inoculate the 1% bacteria suspension, and then place it in an incubator. Sample and measure the strain survival rate at 0h, 1h, 3h and 4h, respectively.
[0099] Bile salt tolerance: Preheat the bile salt to 37℃, inoculate the 1% bacteria suspension in the bile salt solution with a concentration of 0.1%, 0.2%, 0.3% and 0.5%, and then place it in an incubator. Sample and measure the strain survival rate at 0h, 1h, 3h and 4h, respectively.
[0100] As Figure 5As shown in (A), the strain exhibits high tolerance to intestinal fluid, with a survival rate of 86.33% after 4 hours of culture. Figure 5 As shown in Figure (B), the survival rates after 1 h, 3 h, and 4 h of culture were 97%, 90%, and 85.33% for a bile salt concentration of 0.1%; 85%, 70%, and 66.33% for a bile salt concentration of 0.2%; 64%, 59.67%, and 56.65% for a bile salt concentration of 0.3%; and 28.33%, 25.33%, and 23.00% for a bile salt concentration of 0.5%.
[0101] 4. Antibacterial properties
[0102] Take the activated bacterial solution and inoculate it into MRS medium at a rate of 1%. Incubate at 37°C. After the initial stage of the stationary phase, spread the bacterial solution onto a culture plate using a medical cotton swab. Add the activated Staphylococcus aureus (30 μL), Salmonella (30 μL), and Escherichia coli (10 μL) to a pre-laid blank antimicrobial susceptibility test disc. Incubate in an incubator for 48 hours and then measure the inhibition zone using calipers.
[0103] Strain YL10 inhibited the growth of Staphylococcus aureus, Salmonella, and Escherichia coli. The size of the inhibition zone was in the order of Salmonella > Staphylococcus aureus > Escherichia coli, with diameters of 12.47 mm, 11.24 mm, and 11.13 mm, respectively (Table 3). Figure 6 ).
[0104] Table 3 Evaluation of the antibacterial effect of strain YL10
[0105]
[0106] Example 3
[0107] This embodiment optimizes the fermentation conditions of the strain.
[0108] The solid-state fermentation process was optimized using a single-factor analysis method. While optimizing one parameter, other parameters were kept constant. Following the fermentation procedure described in Example 1, the strain was harvested during the stabilization period to prepare a seed solution. This seed solution was inoculated into CSM at ratios of 2%, 4%, 6%, 8%, and 10% (v / w), maintaining a fixed moisture content of 50%, and anaerobic fermented at 37°C for 3 days. Using unfermented CSM as a control, the FG content in FCSM was measured, and the FG detoxification rate was calculated to determine the optimal inoculum rate. At the optimal inoculum rate, the moisture content of CSM was adjusted to 40%, 45%, 50%, 55%, and 60%, respectively, and anaerobic fermented at 37°C for 3 days. Again, using unfermented CSM as a control, the FG content in FCSM was measured, and the FG detoxification rate was calculated to determine the optimal fermentation moisture content. After determining the optimal inoculum and moisture content, CSM was continuously anaerobic fermented at 37°C for 13 days, with samples taken on days 3, 5, 7, 9, 11, and 13. Using unfermented CSM as a control, the FG content in FCSM was determined, and the FG detoxification rate was calculated to determine the optimal fermentation time. Each treatment was repeated in triplicate, and the final conditions were used as fermentation process parameters in subsequent experiments.
[0109] The effect of different moisture contents on the detoxification rate of foetida (FG) was investigated. The results showed that as the moisture content increased from 35% to 45%, the FG content decreased significantly. When the moisture content was in the range of 45%–60%, the FG content tended to stabilize with the addition of water, while the detoxification rate gradually decreased with further water addition. The lowest FG content (425.33 mg / kg) was observed at a moisture content of 45%, with a detoxification rate of 63.77%. Figure 7 (A, B)
[0110] The effect of different inoculum sizes on the FG detoxification rate at a moisture content of 45% showed that the FG content decreased significantly as the inoculum size increased from 2% to 8%. When the inoculum size was between 8% and 12%, the FG content remained relatively consistent. Therefore, an inoculum size of 8% was selected for CSM fermentation, resulting in a detoxification rate of 63.90%. Figure 7 (C, D)
[0111] Under conditions of 45% moisture content and 8% inoculum, the FG content gradually decreased with increasing fermentation time. On days 11 and 13 of fermentation, the FG content was 274.67 mg / kg and 268.67 mg / kg, respectively, with detoxification rates of 76.73% and 76.54%, respectively. Figure 7 (E, F). Therefore, the optimal fermentation conditions for CSM were determined to be a moisture content of 45%, an inoculum size of 8%, and a fermentation time of 11 days.
[0112] In conclusion, the application successfully screens and separates a high-concentration gossypol-tolerant strain from cotton humus soil, which can effectively remove the content of FG, and is preliminarily identified as Lactiplantibacillus pentosus, named YL10.
[0113] The physiological and biochemical characteristics research results show that, except for mannitol, the strain provided by the application can utilize a variety of carbon sources and nitrogen sources such as raffinose, cellobiose and sucrose. The biological characteristics results show that the strain L.pentosus YL10 does not lyse blood, is sensitive to a variety of antibiotics, has high tolerance to intestinal fluid and bile salt, can improve cell viability, and inhibit the growth of Staphylococcus aureus, Salmonella and Escherichia coli. Therefore, the strain has high safety and can be used for feed fermentation.
[0114] The application is further obtained by single-factor strain fermentation condition optimization, and the optimal fermentation conditions of CSM are moisture content 45%, inoculation amount 8%, and fermentation time 11d, and the highest FG detoxification rate is 76.73%.
[0115] The above only describes the preferred embodiments of the application and is not used to limit the application, and the application can have various changes and variations for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.
Claims
1. A Lactiplantibacillus pentosus, characterized in that, It is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20231547.
2. A derivative of Lactobacillus pentosus, characterized in that, The derivative is a culture of the pentosus lactis of claim 1.
3. A culture medium for Lactobacillus pentosus, characterized in that, It comprises the pentosus lactis of claim 1 or the derivative of claim 2.
4. A composition characterized in that, The composition comprises the pentosus lactis of claim 1 or the derivative of claim 2; Preferably, the composition is a feed composition; Preferably, the feed composition comprises feed and the pentosus lactis or the derivative; the feed is cottonseed meal.
5. The pentosus lactis of claim 1, the derivative of claim 2, the culture of claim 3 or the composition of claim 4 is used in gossypol detoxification.
6. Use according to claim 5, characterized in that, In the gossypol detoxification, the pentosus lactis, the derivative or the culture is mixed with cottonseed meal to be detoxified for fermentation, and the fermentation conditions are as follows: the water content of the fermentation broth is controlled at 40-60%, the inoculation amount of the pentosus lactis is 4-12%, and the fermentation time is at least 7d; Preferably, the fermentation conditions are as follows: the water content of the fermentation broth is controlled at 45-55%, the inoculation amount of the pentosus lactis is 8-12%, and the fermentation time is 11-13d; Preferably, the fermentation conditions are anaerobic or aerobic fermentation. Preferably, the fermentation conditions are anaerobic fermentation.
7. A method of gossypol detoxification, characterized in that, It comprises the following steps: mixing a sample to be detoxified with the pentosus lactis of claim 1, the derivative of claim 2, the culture of claim 3 or the composition of claim 4 for fermentation.
8. The method of gossypol detoxification according to claim 7, wherein, The sample to be detoxified is selected from cottonseed meal; Preferably, the fermentation conditions are as follows: the water content of the fermentation broth is controlled at 40-60%, the inoculation amount of the pentosus lactis is 4-12%, and the fermentation time is at least 7d; Preferably, the fermentation conditions are as follows: the water content of the fermentation broth is controlled at 45-55%, the inoculation amount of the pentosus lactis is 8-12%, and the fermentation time is 11-13d.
9. The method of gossypol detoxification according to claim 8, wherein, The fermentation is anaerobic, facultative anaerobic or aerobic fermentation; Preferably, the fermentation is anaerobic fermentation.
10. The method of gossypol detoxification according to claim 8, wherein, The fermentation medium is MRS medium, and the fermentation temperature is 37℃±0.5℃.