Lactobacillus paracasei BFS2480 and application thereof in fermented rice protein
By using Lactobacillus casei strain BFS2480 to ferment rice protein, the problem of low rice protein solubility was solved, the quality and taste of fermented beverages were improved, and an efficient and simple fermentation method was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SICHUAN YAXIANGJU FOOD TECHNOLOGY CO LTD
- Filing Date
- 2026-04-03
- Publication Date
- 2026-07-03
AI Technical Summary
Rice protein has high surface hydrophobicity and extremely low solubility under neutral conditions, which makes fermented beverages prone to problems such as layering, clumping and graininess during processing and storage. Existing physical modification methods are energy-intensive and destroy flavor, and there is a lack of research on effective lactic acid bacteria strains to improve solubility.
Rice protein was fermented using a strain of Lactobacillus casei BFS2480. By utilizing its acid-producing characteristics and enzyme activity, the soluble protein content of rice protein was increased, thereby improving solubility and taste.
It significantly increases the soluble protein content and solubility of rice protein, improves the taste and flavor of fermented beverages, simplifies the fermentation process, and enhances the edibility and nutritional value of the product.
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Figure CN122326460A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial fermentation technology. More specifically, it relates to a strain of *Lactobacillus casei* BFS2480 and its application in fermenting rice protein. Background Technology
[0002] Rice protein, as a natural, high-quality plant protein, has an amino acid composition similar to that of milk protein, with a relatively rich content of essential amino acids, closely aligning with the WHO's ideal nutritional model. Furthermore, rice protein offers beneficial health benefits, such as aiding in blood pressure reduction and easy digestion. Due to its low allergenicity, it is often used in infant and toddler beverages to mitigate allergy risks. In recent years, with the rise of the plant-based market, rice protein drinks have received increasing attention.
[0003] Currently, plant-based fermented beverages are experiencing rapid growth in the global market. However, due to the high surface hydrophobicity and extremely low solubility (typically below 5%) of gluten, the main component of rice protein, under neutral conditions, rice protein beverages are prone to problems such as layering, clumping, and graininess during processing and storage, greatly limiting their application in the beverage industry. Current research on lactic acid bacteria strains used for rice protein fermentation mainly focuses on flavor improvement, while combining physical modification methods such as high-pressure homogenization, ultrasound, and microfluidics to enhance solubility. However, these methods are not only energy-intensive and costly, but also easily cause significant damage to the flavor of rice protein, making it difficult for fermented products to achieve the ideal flavor characteristics of rice protein fermentation.
[0004] Currently, while physical modification methods have been studied to address the low solubility of rice protein, there are no reports on improving its solubility using lactic acid bacteria strains. Therefore, to increase the soluble protein content and improve the solubility of rice protein, thereby resolving product quality issues related to rice protein beverages, it is urgent to screen for more microbial agents suitable for rice protein fermentation, especially lactic acid bacteria strains that grow well in rice protein emulsions and can effectively improve the soluble protein content and solubility. This invention application is thus proposed. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to overcome the problem of low soluble protein content in existing rice protein emulsions, and to provide a strain of Lactobacillus casei BFS2480 and its application in increasing the soluble protein content of rice protein and preparing rice protein fermented beverages.
[0006] The first objective of this invention is to provide a strain of *Lactobacillus casei* (… Lacticaseibacillus paracasei ) BFS2480 strain.
[0007] A second objective of this invention is to provide the application of the Lactobacillus casei strain BFS2480.
[0008] A third objective of this invention is to provide a fermentation agent.
[0009] A fourth objective of this invention is to provide a method for fermenting rice protein or increasing the soluble protein content in rice protein.
[0010] The fifth objective of this invention is to provide a rice protein fermented beverage and its preparation method.
[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a strain of Lactobacillus casei ( Lacticaseibacillus paracasei BFS2480 strain was deposited at the China General Microbiological Culture Collection Center on February 5, 2026, with accession number CGMCC No. 37691.
[0012] This invention isolated and identified a strain of *Lactobacillus casei* BFS2480 from Guangzhou water kefir. This strain has colonies 1-2 mm in diameter, are milky white with neat edges, slightly raised or flattened, and are semi-opaque with a smooth, moist surface and a distinct lactic acid bacteria odor. Studies show that *Lactobacillus casei* BFS2480 exhibits acid-producing characteristics, with a final fermentation pH of 4.12±0.01 for rice protein, reaching an acidity of 44.67±1.53. It also possesses amylase and protease hydrolytic activity, while exhibiting no hemolytic activity, demonstrating good biocompatibility. Fermenting rice protein using *Lactobacillus casei* BFS2480 can effectively increase the content of soluble rice protein in the fermentation broth, improve the solubility of rice protein, enhance the taste and flavor of the fermented rice protein product, and further improve the edibility and nutritional value of rice protein, enabling the preparation of a wider range of fermented rice products.
[0013] Therefore, the present invention provides the application of Lactobacillus casei strain BFS2480 or its bacterial culture in the preparation of rice soluble protein.
[0014] This invention provides the use of Lactobacillus casei strain BFS2480 or its bacterial culture in improving the solubility of rice protein or in the preparation of products with improved rice protein solubility.
[0015] This invention provides the application of Lactobacillus casei strain BFS2480 or its bacterial culture in the preparation of rice protein fermentation products.
[0016] This invention provides the application of Lactobacillus casei strain BFS2480 or its bacterial culture in fermented rice protein.
[0017] The present invention provides a fermentation preparation containing Lactobacillus casei strain BFS2480 or its bacterial culture.
[0018] This invention provides a method for fermenting rice protein or preparing rice soluble protein, which uses Lactobacillus casei strain BFS2480 or its bacterial culture to treat rice protein raw materials.
[0019] This invention provides a method for preparing a rice protein fermented beverage, which uses Lactobacillus casei strain BFS2480 or its bacterial culture to ferment rice protein products.
[0020] Preferably, the strain of *Lactobacillus casei* BFS2480 or its bacterial culture is inoculated into a raw material containing 1% to 3% rice protein and 5% to 10% sucrose at a weight ratio of 1% to 5% for fermentation preparation.
[0021] Preferably, the Lactobacillus casei strain BFS2480 or its bacterial culture is inoculated into rice protein at a weight ratio of 2-4% for fermentation.
[0022] More preferably, the BFS2480 strain is inoculated into 1%–5% rice protein, 5–10% rice flour and 5%–8% sucrose for fermentation.
[0023] The present invention also provides a rice protein fermented beverage, which is prepared by the above method.
[0024] The present invention has the following beneficial effects: 1. The BFS2480 strain provided by this invention can be used for rice protein fermentation, which can decompose rice protein, increase the content of soluble protein in rice, and improve the solubility of rice protein.
[0025] 2. The BFS2480 strain provided by this invention can improve the rough texture of rice protein fermentation emulsion and enhance the edibility and nutritional value of rice protein.
[0026] 3. The method for fermenting rice protein using microbial strains provided by this invention is simple and quick, can shorten fermentation time, and can produce more fermented products containing rice soluble protein in a targeted and stable manner. Attached Figure Description
[0027] Figure 1 This is a colony diagram of strain BFS2480.
[0028] Figure 2 Phylogenetic tree of strain BFS2480.
[0029] Figure 3 The acidity after fermentation by strain BFS2480.
[0030] Figure 4The images show the protein and amylase activity plates of strain BFS2480 (the left image is the enzymatic digestion diagram of BFS2480 protease; the right image is the enzymatic digestion diagram of BFS2480 amylase).
[0031] Figure 5 This is a plate graph showing the hemolytic activity of strain BFS2480.
[0032] Figure 6 Plate plot showing kanamycin resistance in strain BFS2480.
[0033] Figure 7 This is the Bradford standard curve.
[0034] Figure 8 This represents the soluble protein content after fermentation by strain BFS2480. Detailed Implementation
[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.
[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.
[0037] MRS medium: Add 10.8 g of MRS broth to a 200 mL Erlenmeyer flask containing grade III water, add 2 g of light calcium carbonate powder (1%) and 4 g (2%) of agar, stir with a glass rod to dissolve, and prepare a solid medium. Sterilize the solid medium at 121℃ for 20 min. (Without agar, it is the MRS liquid medium.) Blood agar medium formula: 10.0g casein trypsin digest, 3.0g cardiac trypsin digest, 1.0g corn starch, 5.0g venipuncture enzyme digest, 5.0g yeast extract, 5.0g sodium chloride, 15.0g agar, 50-100mL sterile defibrinated sheep blood, 1000mL distilled water. Final pH 7.3±0.2 Antibiotics: Weigh 12 mg of amphotericin B, dissolve it in 10 mL of grade III water, and then dissolve it in 100 mL of grade III water. After dissolving in the solution, use a syringe to draw the solution into a sterile 5 mL centrifuge tube through a 0.22 μm filter membrane. When using, add 1 mL of antibiotics to 200 mL of culture medium.
[0038] Method for preparing amylase screening plates: 10g soluble starch, 10g tryptone, 5g yeast extract, 10g sodium chloride, 17g agar, pH 7.2, add water to a final volume of 1L.
[0039] Method for preparing protease screening plates: Add 50g of skim milk powder and 20g of agar to a final volume of 1L with water.
[0040] Example 1 Isolation and Identification of Strains 1. Isolation of strains Guangzhou water kefir granules cultured in the laboratory were washed with sterile physiological saline and inoculated into a mixture of 2% (w / v) rice protein and 5% brown sugar for fermentation. The mixture was then incubated at 37°C for 24 h. The granules were then filtered out using a sterile sieve and transferred to a fresh fermentation nutrient solution for further incubation for 24 h. This process was repeated 3–5 times.
[0041] Take 0.5 mL of the activated water kefir fermentation broth, add 4.5 mL of sterile physiological saline, shake to mix, and adjust the concentration to 10. -1 This process is repeated sequentially to a gradient dilution of 10. -7 .
[0042] Take 10 respectively -5 ~10 -7 Place 1 mL of each diluent into a sterile Petri dish. Melt the MRS solid medium and cool it to below 50°C. Then pour approximately 20 mL into the Petri dish, quickly and gently agitate to mix the bacterial culture. After cooling and solidification, seal the dish with plastic wrap and incubate at 37°C and 30°C upside down for 24–48 h. Pick single colonies of different morphologies (round / irregular, transparent / milky, raised / flat), label them, and then streak them onto solid agar plates for purification. After culturing, pick the last single colony and streak it again, purifying twice. Using an inoculation loop, pick a single colony from the last section of the plate that has been purified at least twice, and insert it into 10 mL of the corresponding liquid MRS medium and stir. When inoculating, be careful not to let the inoculation loop touch the mouth of the test tube. After inoculation, sterilize the mouth of the tube before plugging it with a rubber stopper, and finally seal it with plastic wrap. Inoculate the purified strain into the corresponding liquid medium and incubate at 37°C and 30°C for 24–48 h, until the test tube becomes noticeably turbid. Name the obtained strains accordingly.
[0043] Long-term preservation: Prepare 200 mL of 60% glycerol (add 120 mL of glycerol and 80 mL of grade III water, sterilize at 121℃ for 20 min), add 900 μL of bacterial solution and 300 μL of 60% glycerol to each glycerol tube, mix well, and freeze at -80℃.
[0044] 2. Identification of strains (1) Morphological identification: Take the strains isolated and purified above, and record their size, color, edge, ridge condition, transparency, odor, and calcification zone. Finally, number and photograph them for preservation. At the same time, perform microscopic examination and Gram staining.
[0045] The results of BFS2480 are as follows: Figure 1 As shown, the colonies of this strain are 1-2 mm in diameter, milky white in color, with neat edges. The colonies may be slightly raised or flat, semi-opaque, smooth and moist, and have a distinct lactic acid bacteria odor.
[0046] (2) Molecular biological identification: Genomic DNA was extracted from the selected target strains, and the 16S rRNA gene was amplified by PCR and sequenced. The sequence was compared with the NCBI database to determine the species of the strain.
[0047] BLAST alignment analysis was performed in the NCBI database, with the results for BFS2480 as follows: Figure 2 As shown, the comparison results indicate that this strain is similar to *Lactobacillus casei* (…). Lacticaseibacillus paracasei Based on the high homology and morphological characteristics, this strain was classified as *Lactobacillus casei*. Lacticaseibacillus The bacteria, belonging to the genus *Lactobacillus* (sp.), were identified as *Lactobacillus Lacticaseibacillus paracasei The strain, named BFS2480, was deposited on February 5, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37691, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Simultaneously, the *Lactobacillus casei* (…) provided in this application… Lacticaseibacillus paracasei Based on the updated list of microbial strains that can be used in food published in Announcement No. 4 of 2022, the strain has been renamed: *Lactobacillus paracasei*. Therefore, the BFS2480 strain provided in this invention is named *Lactobacillus paracasei* according to the latest name change. Lacticaseibacillus paracasei ) BFS2480 strain.
[0048] Similar to the isolation and identification process described above, this embodiment isolated multiple strains from different sample sources, and obtained corresponding strains after isolation, purification and identification. The specific strain sources and information are shown in Table 1. Other strains were identified and preserved in the strain resource bank of the Fermentation Engineering Team of the College of Food Science and Technology, South China Agricultural University.
[0049] Table 1 Sources of strain screening
[0050] Example 2: Preparation of Rice Protein Fermentation Broth (1) Activation of bacterial strain: Using a sterile pipette tip, 200 μL of bacterial solution was taken from the above-mentioned bacterial strain glycerol preservation tube and inoculated into 5 mL of MRS liquid medium. After mixing evenly, the culture was incubated at 37℃ for 24 h. Then, 200 μL of bacterial solution was taken from the MRS culture medium and inoculated into 5 mL of MRS liquid medium. Activation was carried out once more under the same conditions. (2) Preparation of bacterial solution: Take 1 mL of activated bacterial solution, centrifuge at 6000 r / min and 4℃ for 5 min, discard the supernatant, resuspend the bacterial sludge in physiological saline (0.9% NaCl), centrifuge again, and then resuspend in 1 mL of physiological saline to obtain bacterial solution; (3) Inoculation and fermentation: Inoculate the bacterial solution into a culture medium containing rice protein (3% rice protein, 5% rice flour, 7% sucrose (v / v)) at 2% (v / v), and let it ferment at 30℃ for 16 hours to obtain rice protein fermentation liquid.
[0051] Example 3: Performance determination of the strain 1. pH and acidity According to GB 5009.239-2016 standard, the titratable acidity of rice protein fermentation broth prepared by fermentation from different strains was determined by acid-base titration. 1 mL of the prepared fermentation broth was diluted 5 times by volume with deionized water, and 2 drops (approximately 0.1 mL) of 10 g / L phenolphthalein indicator were added. The acidity was then determined by neutralization titration using 0.1 M sodium hydroxide (NaOH) standard solution. The endpoint was defined as the solution turning a stable rose-red color (lasting ≥30 s). Uninoculated liquid culture medium was used as a blank control.
[0052] The formula for calculating acidity is as follows: ; Where TA represents titratable acidity (°T); V2 represents the volume of NaOH standard solution consumed in the sample titration (mL); V1 represents the volume of NaOH standard solution consumed in the blank control titration (mL); and m represents the sample volume (mL).
[0053] The results, shown in Table 2, reveal significant differences in acid-producing performance among different strains. Compared to other strains, strain BFS2480 exhibited a significantly lower pH and a significantly higher acidity after fermenting rice protein, demonstrating superior acid-producing characteristics. Figure 3 As shown, this facilitates fermentation preparation.
[0054] Table 2. Acidity determination of rice protein fermentation broth
[0055] 2. Enzyme activity assay Crude enzyme solution preparation: Different bacterial solutions prepared in Example 2 were inoculated into MRS fermentation medium at an inoculation rate of 2% (v / v) and cultured at 37℃ for 48 h. The fermentation supernatant was obtained by centrifugation at 10000 r / min for 10 min and stored at 4℃ for later use.
[0056] Protease activity measurement: A 1 cm diameter punch was used to create wells on a rice protein screening plate. 100 μL of supernatant was injected into each well, with sterile water as a blank control. After incubation at 37 ℃ for 48 h, the diameter of the hydrolysis zone was measured.
[0057] Amylase activity measurement: Use a 1 cm diameter punch to make holes in the amylase screening plate, inject 200 μL of the prepared crude enzyme solution into the holes, treat with enzyme at 28 ℃ for 48 h, and observe and measure the diameter of the transparent zone.
[0058] The results are shown in Table 3, indicating significant differences in the size of the lysis zones among different strains. BFS2480 exhibited better enzyme production performance than BFS1543, BFS1779 (of the same species), and BFS272 (from a different genus). The plate assay results are shown below. Figure 4 As shown.
[0059] Table 3. Enzyme activity assay results of the strains
[0060] 3. Hemolytic test BFS2480 strain was inoculated and streaked onto MRS solid medium and cultured for 48 h until a single colony grew. Then, a single colony was picked and streaked onto blood agar medium and cultured for 48 h. The growth of the colony on the blood agar plate was observed to determine whether hemolysis was present.
[0061] The results are as follows Figure 5 As shown, no hemolysis was observed around the colony, indicating that strain BFS2480 is non-hemolytic and has good biosafety.
[0062] 4. Antibiotic susceptibility testing The antibiotic resistance of 10 commonly used antibiotics, including chloramphenicol, cefotaxime, penicillin, carbenicillin, polymyxin B, vancomycin, kanamycin, tetracycline, rifampin, and amoxicillin, was tested. The bacterial strains were activated twice in MRS medium, and microscopic examination was performed after each generation to confirm the absence of contamination. An appropriate amount of bacterial suspension was diluted to a concentration of 1×10⁻⁶ in a clean bench. 8 CFU / mL, pipette 1.0 μL and add it to 15 mL of sterile, thawed solid MRS medium. Mix well and slowly pour into sterile plates, being careful to avoid air bubbles. Wait approximately 20 minutes for the liquid to solidify, then evenly place three antimicrobial susceptibility test strips of the same antibiotic onto the plate. Incubate at 37°C for 48 hours. Measure the diameter of the inhibition zone using calipers and calculate the average value. Evaluate the antibiotic susceptibility of the strain according to the "Standards for Antimicrobial Susceptibility Testing".
[0063] The results are shown in Table 4. Based on the susceptibility testing of strain BFS2480 to the drug susceptibility test strips, it was found that strain BFS2480 was sensitive to tetracycline, amoxicillin, chloramphenicol, penicillin, and rifampin. The results for kanamycin plate testing are as follows: Figure 6 As shown, its good safety performance is confirmed.
[0064] Table 4 Antibiotic susceptibility results
[0065] Note: S- indicates sensitive; I- indicates moderately sensitive; R- indicates drug resistance.
[0066] Example 4: Detection of soluble protein content At room temperature, the rice protein fermentation broth of the strain in Example 2 was centrifuged at 1000 r / min for 10 min using a high-speed centrifuge. The supernatant was then collected, and a standard curve was constructed using the Bradford method with BSA as the standard to determine the soluble protein content of the rice protein fermentation broth. The specific steps are as follows: (1) Reagent preparation: 0.01% Coomassie Brilliant Blue G-250 staining solution: Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 mL of ethanol. Slowly add 100 mL of phosphoric acid while stirring. Make up to 1000 mL with grade III water. Filter through filter paper and store in a brown bottle at room temperature.
[0067] Protein standard solution: bovine serum albumin (BSA).
[0068] Test sample solution: Take rice protein fermentation broth and centrifuge at 10000 r / min for 10 min to obtain fermentation supernatant. Dilute it twice with tertiary water so that the protein concentration falls within the linear range of the standard curve.
[0069] (2) Experimental method: Take 7 1.5mL centrifuge tubes and number them. Prepare the solution according to the formula shown in Table 5 below, and accurately add the solvent (also matching sample grade III water) and 1.0mg / mL BSA stock solution using a pipette. At the same time, set up a CK group that does not use the strain for fermentation (i.e., the rice protein content before dissolution is measured).
[0070] Table 5 Experimental Sample Preparation System
[0071] Colorimetric reaction: Add 5.0 mL of Coomassie Brilliant Blue staining solution to each tube and immediately vortex thoroughly to mix. Let stand at room temperature for 5-10 minutes. The color stabilizes within 2-5 minutes and can be maintained for about 1 hour. Avoid prolonged standing.
[0072] Absorbance measurement: A spectrophotometer was used, with the instrument zeroed using a blank tube. The absorbance values (OD) of each standard tube and sample tube were measured sequentially at a wavelength of 595 nm. 595 ).
[0073] Plotting and Calculating the Standard Curve: The standard curve is plotted with the mass (μg) of the standard protein on the x-axis (X), and its corresponding OD value... 595 The value is the ordinate (Y). Using linear regression, the standard curve equation is obtained: Y = aX + b, and the correlation coefficient R² (usually required to be >0.99).
[0074] Calculate the protein concentration in the sample: Calculate the average OD of the sample tube. 595 Substitute the values into the standard curve equation: X (sample protein amount, μg) = (Yb) / a, and calculate the stock solution concentration. Sample protein concentration (μg / mL or mg / mL) = X (μg) / loading volume (mL), and finally multiply by the dilution factor 2.
[0075] The standard curve is drawn as follows Figure 7 As shown, the soluble protein content after fermentation by different strains is statistically combined as follows: Figure 8 As shown, the soluble protein content obtained by the BFS2480 strain from rice protein decomposition reached 147 μg / mL, which is 200%~300% higher than the 50 μg / mL of the unfermented CK group. The soluble protein content was also significantly increased compared to other strains.
[0076] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A strain of Lactobacillus casei ( Lacticaseibacillus paracasei BFS2480 strain, characterized in that, This strain was deposited at the China General Microbiological Culture Collection Center on February 5, 2026, with accession number CGMCC No. 37691.
2. The use of the Lactobacillus casei BFS2480 strain or its bacterial culture as described in claim 1 in improving the solubility of rice protein or in the preparation of products with improved rice protein solubility.
3. The application of the Lactobacillus casei BFS2480 strain or its bacterial culture as described in claim 1 in the preparation of soluble rice protein.
4. The use of the Lactobacillus casei BFS2480 strain or its bacterial culture as described in claim 1 in the preparation of rice protein fermentation products.
5. The use of the Lactobacillus casei BFS2480 strain or its bacterial culture as described in claim 1 in fermented rice protein.
6. A fermentation preparation, characterized in that, Contains the Lactobacillus casei BFS2480 strain or its bacterial culture as described in claim 1.
7. A method for decomposing rice protein or preparing rice soluble protein, characterized in that, Rice protein raw materials were treated using the Lactobacillus casei BFS2480 strain or its bacterial culture as described in claim 1.
8. A method for preparing a rice protein fermented beverage, characterized in that, Rice protein products are prepared by fermentation using the Lactobacillus casei BFS2480 strain or its bacterial culture as described in claim 1.
9. The method according to claim 8, characterized in that, The bacteria are prepared by inoculating a raw material containing 1%–3% rice protein and 5–10% sucrose with Lactobacillus casei strain BFS2480 or its bacterial culture at a weight ratio of 1–5%.
10. A rice protein fermented beverage, characterized in that, Prepared by the method described in claim 8 or 9.