Lactobacillus rhamnosus LT-1 and application thereof in solid-state fermentation of spirulina
By co-fermenting spirulina with Lactobacillus rhamnosus LT-1 and Bacillus subtilis, the problems of fishy smell and cell wall structure of spirulina were solved, the fermentation efficiency and product quality of spirulina were improved, and it is suitable for industrial application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LANTU BIOTECHNOLOGY (HUZHOU) CO LTD
- Filing Date
- 2026-06-05
- Publication Date
- 2026-07-31
AI Technical Summary
In existing technologies, spirulina has problems such as a strong fishy smell, dense cell wall structure that makes it difficult for nutrients to be absorbed, poor adaptability of conventional lactic acid bacteria and Bacillus to spirulina substrate, and low fermentation efficiency.
Spirulina was co-fermented with Lactobacillus rhamnosus LT-1 and Bacillus subtilis. Bacillus subtilis destroyed the cell wall of Spirulina, while Lactobacillus rhamnosus LT-1 was responsible for the conversion of active ingredients during the fermentation process. Spirulina and oat flour were used as a composite fermentation substrate, and specific fermentation process parameters were set.
It improves the efficiency and product quality of spirulina fermentation, significantly increasing the content of total short-chain fatty acids, β-glucan, and free amino acids. The fermentation process is simple, low-cost, and suitable for industrial production.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial fermentation technology and biological resource utilization, specifically involving a strain of Lactobacillus rhamnosus LT-1 and its application in solid-state fermentation of Spirulina. Background Technology
[0002] Spirulina, a microalga rich in high-quality protein, gamma-linolenic acid, carotenoids, and various trace elements, possesses multiple physiological functions such as antioxidation and lipid reduction, and has broad application prospects in the food and health product fields. However, spirulina itself has problems such as a strong fishy smell, a dense cell wall structure that makes it difficult for humans and animals to absorb nutrients, and low utilization rate of active ingredients, which seriously limit its product development and market promotion.
[0003] Solid-state fermentation technology has become an important technical route for the deep processing of spirulina due to its low cost, simple operation, high product activity, and lack of large amounts of fermentation waste liquid. The performance of the fermentation strain directly determines the efficiency of solid-state fermentation of spirulina, product quality, and conversion efficiency of active ingredients.
[0004] Fresh camel milk is rich in natural microbial communities, whose microorganisms are characterized by tolerance to extreme environments, strong metabolic activity, and adaptability to natural substrates, making it an important natural source for screening high-quality fermentation strains. Currently, the strains used for solid-state fermentation of spirulina are mostly conventional lactic acid bacteria and Bacillus, which have problems such as poor adaptability to spirulina substrates and low fermentation efficiency. Therefore, screening strains adapted to solid-state fermentation of spirulina has significant practical significance and application value. Summary of the Invention
[0005] To address the aforementioned problems, this invention provides a strain of *Lactobacillus rhamnosus* LT-1, which was deposited at the China General Microbiological Culture Collection Center on April 13, 2026, with accession number CGMCC NO.38214. This strain was derived from fresh camel milk and, through morphological analysis and 16S rDNA identification, was identified as belonging to the genus *Lactobacillus* sp., specifically *Lactobacillus rhamnosus*, and named LT-1.
[0006] This invention applies Lactobacillus rhamnosus LT-1 and Bacillus subtilis synergistically to the solid-state fermentation of Spirulina. Bacillus subtilis mainly disrupts the dense cell wall structure of Spirulina, promoting the release of nutrients and active substances within the Spirulina cells, and providing sufficient nutrient substrates for the fermentation metabolism of Lactobacillus rhamnosus LT-1. Lactobacillus rhamnosus LT-1, as the dominant fermentation strain, is responsible for completing the core functions of active ingredient conversion and beneficial metabolite generation during the fermentation process.
[0007] The spirulina solid-state fermentation system uses spirulina and oat flour as a composite fermentation substrate, with a fixed mass ratio of 4:1. Oat flour serves as the sole carbon source, providing carbon support for the growth and metabolism of the fermentation strains. Sterile water is added to adjust the solid-liquid ratio of the fermentation system to 1:0.375. To clarify the fermentation advantages of *Lactobacillus rhamnosus* LT-1 in this invention, three fermentation systems (including a control group) were set up during the fermentation process. The specific groupings are as follows: ① Control group 1: Only *Bacillus subtilis* seed culture was inoculated, allowing only spirulina to break down cell walls; ② Control group 2: *Bacillus subtilis* and *Lactobacillus plantarum* seed cultures were inoculated, with *Lactobacillus plantarum* being a commercially available and commonly used fermentation strain, serving as a control strain; ③ Experimental group: *Bacillus subtilis* and *Lactobacillus rhamnosus* LT-1 seed cultures were inoculated, with *Bacillus subtilis* responsible for breaking down the spirulina cell walls, and *Lactobacillus rhamnosus* LT-1 serving as the dominant fermentation strain.
[0008] The core process parameters for the fermentation process are as follows: the amount of each microbial strain added is uniformly 10. 6 -10 7 CFU / g (based on the total mass of the spirulina and oat flour composite substrate); the ratio of the composite bacteria added is 1:1-1:2; the fermentation temperature is 35℃-39℃; and the fermentation time is 12h-56h.
[0009] The present invention has the following beneficial effects:
[0010] 1. The *Lactobacillus rhamnosus* (LT-1) of this invention was screened and isolated from the target spirulina oat flour solid-state fermentation system. Its growth and metabolic characteristics are highly compatible with the fermentation substrate, solving the technical problems of poor compatibility and low fermentation efficiency of existing conventional fermentation strains with spirulina substrate. This invention innovatively adopts a synergistic fermentation mode of "Bacillus subtilis cell wall disruption + LT-1 strain fermentation", combined with specific range of fermentation process parameters, which can achieve precise control of the spirulina solid-state fermentation process and ensure stable quality of fermentation products. This strain is derived from natural fresh camel milk, has high safety, and has good potential for large-scale industrial application.
[0011] 2. The present invention utilizes *Lactobacillus rhamnosus* LT-1 in synergy with *Bacillus subtilis* for solid-state fermentation of *Spirulina*. The fermentation effect was evaluated by measuring indicators such as pH, crude protein, total short-chain fatty acids, β-glucan, and free amino acid content in the fermentation product. Experimental results show that *Lactobacillus rhamnosus* (LT-1) can be well applied in the solid-state fermentation system of *Spirulina*; after 48 hours of fermentation, the total short-chain fatty acid content in the fermentation product increased by more than %, the β-glucan content increased by more than %, and the free amino acid content increased by more than %.
[0012] 3. The spirulina solid-state fermentation process provided by this invention is simple to operate, has low production costs, generates no large amount of fermentation waste liquid, is environmentally friendly, and is easy to achieve industrial-scale production. This process promotes the high-value deep processing of spirulina, provides a new technical path for the upgrading and development of the spirulina industry, and has significant economic, social and environmental benefits. Attached Figure Description
[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0014] Figure 1 The microbial community structure distribution (species level) of the spirulina oat flour solid-state fermentation system (after inoculation with fresh camel milk) at 0h, 24h, 36h and 48h.
[0015] Figure 2 This is a photograph of the colony morphology of Lactobacillus rhamnosus LT-1.
[0016] Figure 3 The phylogenetic tree of the LT-1 strain provided by this invention.
[0017] Figure 4 The effect of the strain LT-1 of this invention combined with Bacillus subtilis on the β-glucan content in a Spirulina solid-state fermentation system. Detailed Implementation
[0018] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.
[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0023] Example 1: Isolation and identification of Lactobacillus rhamnosus (LT-1)
[0024] 1. Preparation of fermentation system: A spirulina-oat flour solid-state fermentation system was prepared, wherein the mass ratio of spirulina to oat flour was 4:1. Oat flour served as a supplementary carbon source to provide carbon support for the growth and metabolism of microorganisms in the system.
[0025] 2. Fresh camel milk inoculation and cultivation: Take fresh, unspoiled raw camel milk and, under aseptic conditions, add it directly to the prepared spirulina oat flour solid fermentation system at an addition rate of 30% (v / w). After thorough mixing, place it in a 37℃ constant temperature incubator and let it stand for 24-48 hours.
[0026] 3. Metagenomic sequencing and screening of dominant strains: Samples of the above fermentation system were collected at 0h, 24h, 36h and 48h after inoculation with fresh camel milk.
[0027] (1) DNA extraction was performed on the sample according to the instructions of DNeasy® PowerSoil® Kit.
[0028] (2) DNA quality testing of samples was performed using the following two methods:
[0029] Agarose gel electrophoresis was used to analyze the degree of DNA degradation and the presence of RNA and protein contamination.
[0030] Qubit 4.0 was used for precise quantification of DNA concentration. The HieffNGS@OnePot Pro DNALibraryPrep Kit V4 One-Step DNA Digestion and Library Construction Kit V4(12972) was used for library construction.
[0031] (3) Document retrieval
[0032] After the library was constructed, it was first quantified using Qubit 4.0, and then fragments were detected by agarose gel electrophoresis / Qsep. After the fragments met the expectations, the effective concentration (3nM) of the library was accurately quantified by qPCR to ensure the quality of the library.
[0033] (4) The sequencing work was completed by Wuhan Benagene Co., Ltd., and the sequencing was performed on the MGIT7 platform PE150.
[0034] By analyzing the microbial community structure of the fermentation system, the dominant bacterial species with the highest relative abundance and most significant growth advantage were screened out. Combined with sequencing results and microbial community analysis data, the dominant bacterial species in this fermentation system was determined to be *Lactobacillus rhamnosus* (see appendix for details). Figure 1 ).
[0035] 4. Isolation and purification of bacterial strains: Take 1g of the above fermentation system sample, add sterile physiological saline under aseptic conditions to dilute to an appropriate multiple, inoculate onto MRS solid medium and incubate at 37℃ for 48h; after the culture is completed, select single colonies that are round, milky white, with neat edges, smooth surface and diameter of 2-5mm on the plate, and purify them three times continuously on MRS solid medium by streak plating to ensure that pure culture strains with the required purity are obtained.
[0036]
[0037] Example 2: Acid and choline resistance of LT-1 strain
[0038] Preparation of simulated gastric juice: Pepsin (1:3000) was added to phosphate buffer solution to a concentration of 3.5 g / L, the pH was adjusted to 2.5, sterilized by membrane filtration (0.22 μm), and stored in a refrigerator at 4℃ for later use.
[0039] Preparation of simulated intestinal fluid: 11 g / L sodium bicarbonate, 2 g / L sodium chloride, 1 g / L trypsin, 10 g / L bile salts, adjust pH to 8.0, sterilize by membrane filtration (0.22 μm), and store at 4℃ for later use.
[0040] A suspension of LT-1 bacteria in good growth condition was collected, centrifuged at room temperature to obtain bacterial cells, washed twice with phosphate buffer (pH 7.4), and 0.5 mL of the bacterial suspension was added to 4.5 mL of simulated gastric fluid. The suspension was then incubated in an anaerobic incubator at 37°C. Samples were taken at 0 h, 1.5 h, and 3 h, and the cells were counted using the MRS agar pour plate method.
[0041] After 3 hours of treatment with simulated gastric fluid, 0.5 mL of the simulated gastric fluid culture was added to 4.5 mL of simulated intestinal fluid. After 2 hours and 4 hours, the cells were counted using the MRS agar pour plate method. The cells were then incubated in an anaerobic incubator at 37°C for 24-48 hours before counting. Each strain was performed in triplicate.
[0042] Survival rate (%) = logα / log β × 100%
[0043] Note: α = number of viable bacteria after treatment with simulated gastrointestinal fluid, β = number of viable bacteria before treatment.
[0044] The experimental results showed that the survival rates of *Lactobacillus rhamnosus* LT-1 in gastric juice were 83.72% and 82.5% at 1.5 h and 3 h, respectively; and in intestinal juice, the survival rates were 78.75% and 72.5% at 2 h and 4 h, respectively. This indicates that LT-1 has high tolerance to simulated gastrointestinal juices and can exert its probiotic effects relatively stably.
[0045] Example 3: Specific application of Lactobacillus rhamnosus LT-1 in solid-state fermentation of Spirulina
[0046] 1. Preparation of fermentation substrate: Take 80g of dried spirulina and 20g of oat flour and mix them evenly. The mass ratio of the two is 4:1. Oat flour serves as the sole carbon source, providing carbon support for the growth and metabolism of the fermentation strain. Add 37.5mL of sterile water to the above composite substrate to adjust the solid-liquid ratio of the fermentation system to 1:0.375 and stir thoroughly.
[0047] 2. Seed Culture Preparation: Seed cultures of *Lactobacillus rhamnosus* LT-1, *Bacillus subtilis*, and *Lactobacillus plantarum* were prepared separately. *Lactobacillus plantarum*, a commonly used fermentation strain, was used as a control strain to compare its fermentation effect with that of the LT-1 strain of this invention. The specific method is as follows: *Lactobacillus rhamnosus* LT-1 and *Lactobacillus plantarum* were inoculated into MRS liquid medium, and *Bacillus subtilis* was inoculated into LB liquid medium. All cultures were placed in a constant temperature shaking incubator at 37℃ and 180 rpm for 24 h. After incubation, the three bacterial cultures were centrifuged at 8000 rpm and 4℃ for 10 min, and the bacterial cells were collected. The cells were resuspended in sterile physiological saline, and the concentration of each seed culture was adjusted to 10. 8 CFU / mL, the amount of each bacterial strain added during inoculation should be controlled at 10. 6 CFU / g (based on the total mass of the spirulina and oat flour composite matrix), for later use.
[0048] 3. Fermentation Grouping and Cultivation: Three fermentation systems were set up, with identical fermentation substrate types, amounts, and treatment methods in each group to ensure comparability of experimental results. The specific groupings are as follows: ① Control Group 1: Only Bacillus subtilis seed culture was inoculated. In this group, Bacillus subtilis only played a role in disrupting the cell walls of Spirulina, with no auxiliary fermentation strains. ② Control Group 2: Both Bacillus subtilis seed culture and Lactobacillus plantarum seed culture were inoculated. Bacillus subtilis was responsible for disrupting the cell walls of Spirulina, while Lactobacillus plantarum served as an auxiliary fermentation strain. ③ Experimental Group: Both Bacillus subtilis seed culture and Lactobacillus rhamnosus LT-1 seed culture were inoculated. Bacillus subtilis was responsible for disrupting the cell walls of Spirulina, while Lactobacillus rhamnosus LT-1 served as the dominant fermentation strain.
[0049] 2. Seed culture preparation and inoculation: According to the above grouping scheme, control group 1 was inoculated with Bacillus subtilis seed culture, control group 2 was inoculated with Bacillus subtilis seed culture and Lactobacillus plantarum seed culture, and experimental group 3 was inoculated with Bacillus subtilis seed culture and Lactobacillus rhamnosus LT-1 seed culture. The ratio of the compound strains was 1:1.
[0050] 3. Fermentation culture: All three fermentation systems were placed in a 37℃ constant temperature incubator and fermented in the dark for 48 hours. Samples of the solid-state fermentation system were taken at 0h, 24h, 36h, and 48h for subsequent fermentation effect determination.
[0051] 4. Efficacy Testing: The pH value, crude protein content, short-chain fatty acid content, β-glucan content, and free amino acid content of the three groups of Spirulina ferments were measured at 0h, 24h, 36h, and 48h. The results are as follows:
[0052] (1) pH value determination: accurately weigh 1g of fermentation material, add 10mL of sterile water, vortex mix for 5min, let stand for 10min, centrifuge and take the supernatant, and measure the pH value of the sample at room temperature using a pH meter.
[0053] (2) Determination of crude protein content: After the sample is freeze-dried, it is pulverized through a 40-mesh sieve. The crude protein content is determined according to the Kjeldahl method in GB 5009.5-2016.
[0054] (3) Determination of β-glucan content: After lyophilizing the sample, pulverize it through a 40-mesh sieve, and calculate the content according to the instructions of the β-glucan kit provided by ELISA. Figure 4 )
[0055] (4) Determination of short-chain fatty acids:
[0056] Sample preparation: Weigh 100 mg of the sample to be tested using an analytical balance, add 700 μL of ultrapure water, 3% formic acid and 40 1 mm stainless steel grinding beads, vortex for 10 s, sonicate for 5 min, let stand for 5 min to allow the fatty acids to be fully released, add 700 μL of dichloromethane (chromatographic grade) for extraction, sonicate for 5 min, let stand for 30 min for extraction, take the lower organic phase, filter it through a 0.22 μm microporous membrane and then analyze it.
[0057] Chromatographic conditions: Column: TR-FFAP (30.0 m × 0.25 mm, 0.25 μm); Column temperature: 50°C for 2 min, increased to 120°C at 15°C / min, increased to 160°C at 5°C / min, increased to 210°C at 20°C / min, held for 1 min; Vaporization chamber temperature: 250°C; Carrier gas: He, flow rate: 1.0 mL / min; Splitless injection: injection volume: 1 μL.
[0058] Mass spectrometry conditions: EI source: electron energy 70 Ev; ion source temperature 270℃; scan mode: SIM (m / z 57, m / z 60, m / z 73, m / z 74).
[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A strain of Lactobacillus rhamnosus LT-1, characterized in that, The strain was deposited in China General Microbiological Culture Collection Center (CGMCC) on April 13, 2026; the address is No. 1, Xibaixili, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC NO: 38214.
2. The application of the Ruminococcus lactaris LT-1 in solid-state fermentation of spirulina according to claim 1.