Probiotic-bacterial cellulose composite membrane as well as preparation method and application thereof

Abstract

The invention relates to a probiotic-bacterial cellulose composite membrane as well as a preparation method and application thereof. The probiotic-bacterial cellulose composite membrane is prepared from lactobacillus plantarum LCC-605 with the preservation number of CCTCC No: M 2016491, a biological membrane, a metabolite and a postbiotic of the metabolite, and the probiotic-bacterial cellulose composite membrane is prepared from lactobacillus plantarum LCC-605 with the preservation number of CCTCC No: M 2016491. The method comprises the following steps: carrying out in-situ co-culture on Acetobacter xylinum and Lactobacillus plantarum LCC-605 to form a Lactobacillus plantarum biofilm based bacterial cellulose membrane and related metabolites of the Lactobacillus plantarum biofilm based bacterial cellulose membrane, and carrying out in-situ co-culture on Acetobacter xylinum and Lactobacillus plantarum LCC-605 to form the Lactobacillus plantarum biofilm based bacterial cellulose membrane. The invention not only provides the bacterial cellulose membrane for encapsulating the probiotics to enhance the survival rate and the stability of the probiotics, but also provides an ideal leavening agent for preparing the fermented black bean milk with excellent texture, high viable count and excellent storage stability.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a probiotic-bacterial cellulose composite membrane, its preparation method, and its application. Background Technology

[0002] Probiotics are susceptible to multiple environmental stresses during processing and storage (such as temperature fluctuations, extreme pH levels, and oxidative stress), leading to a sharp decline in their survival rate and activity (Xu et al., 2022). Therefore, developing a technology that can maintain a high survival rate of probiotics is crucial. Among these technologies, encapsulation, through physical isolation and chemical protection, has been proven to be one of the most effective strategies for ensuring the survival rate of probiotics. Therefore, developing encapsulation delivery technologies for probiotics has significant practical implications for expanding their application scope.

[0003] Currently, various encapsulation strategies are available for protecting probiotics, such as microencapsulation, emulsification, electrospinning, and spray drying (de Oliveira Filho et al., 2025). Among these, microencapsulation is a commonly used and easily implemented method, forming a gel network structure by loading probiotics into a protective matrix. Sodium alginate (SA), a natural anionic polysaccharide, is often combined with Ca2+ due to its safety, low cost, and high solubility. 2+ Cross-linking to form gels is widely used for probiotic encapsulation (Li et al., 2011; Razavi et al., 2021; Torp et al., 2022; Zhang et al., 2021). Furthermore, SA encapsulation enables controlled release of probiotics into the gut (Li et al., 2024). Although microencapsulation improves the survival rate of probiotics in harsh environments, its protective effect can be further optimized by enhancing the stability of the probiotics themselves. In recent years, biofilm encapsulation or biofilm combined with other encapsulation strategies has attracted considerable attention due to its protective effects (Yang et al., 2024; Mgomi et al., 2024). Bacteria often aggregate in nature in the form of biofilms to resist adverse conditions such as extreme pH, high temperature, oxidative stress, and even antibiotics (Cheow & Hadinoto, 2013; Hu et al., 2019; Vega-Sagardía et al., 2018; Flemming & Wuertz, 2019; Motta et al., 2021; Sadiq et al., 2020).

[0004] Furthermore, probiotics are often developed into functional fermented dairy products. However, probiotics frequently face many problems during fermentation, such as long fermentation time, low density, poor survival ability of probiotics in fermented dairy products, insufficient survival rate of probiotics in fermented dairy products, and the inability to recycle probiotic starter cultures. These issues lead to low production efficiency, high production costs, and unstable product quality (Hu et al., 2019). Bacterial cellulose is a biopolymer produced by some aerobic bacteria (Moon et al., 2011; Ullah et al., 2016), possessing unique physicochemical properties such as natural nanostructure, hydrophilicity, high porosity, high water retention, mechanical strength, crystallinity, and biocompatibility (Li et al., 2022). Moreover, bacterial cellulose has wide applications in dairy products, serving as a stabilizer and emulsifier in dairy production, a fat substitute in products such as cheese, and an additive in functional foods. It can also be used as a carrier for enzymes, bioactive substances, and probiotics to develop dairy products with health benefits, and it also has applications in food packaging (Płoska et al., 2023). Encapsulating probiotics in bacterial cellulose creates a "protective shield" for them, isolating them from the external environment under certain conditions and providing a stable microscopic environment (Frakolaki et al., 2020), allowing them to resist adverse external environments while maintaining their own growth. Furthermore, bacterial cellulose encapsulation of probiotics offers biocompatibility, shortening fermentation time, increasing fermentation density, and enabling the repeated recycling of the starter culture. Therefore, using bacterial cellulose to encapsulate probiotics is an excellent choice.

[0005] Plant-based dairy products (such as soy milk, rice milk, and coconut milk) effectively support the growth of probiotics and maintain high survival rates during fermentation and storage. According to market research data, the global plant-based yogurt market is projected to continue growing at an annual rate of 18.9% (Liu et al., 2023). Soy products are a key product in the plant-based market due to their suitability for vegetarians and lactose-intolerant individuals (Liu et al., 2023; Bock et al., 2024). However, fermented soy yogurt still faces challenges such as poor texture, incomplete fermentation, and low probiotic survival rates, necessitating the screening of novel probiotic strains or the development of engineered probiotic strategies to optimize the fermentation process. Summary of the Invention

[0006] Technical problem solved: This invention provides a probiotic-bacterial cellulose composite membrane, its preparation method and application, to overcome the common problems in existing probiotic encapsulation technology, such as limited protective effect, complex preparation process, low survival rate of probiotics in processing, storage and digestive tract environment, and the inability to recycle traditional fermentation agents.

[0007] Technical solution: A probiotic-bacterial cellulose composite membrane (LP605@BC), which is made from Acetobacter xylinum ( Acetobacter xylinum ) and Lactobacillus plantarum with accession number CCTCC M 2016491 ( Lactobacillus plantarum LCC-605 is formed through in situ co-culture, and sequentially through aerobic and anaerobic culture stages; the composite membrane comprises a matrix composed of bacterial cellulose produced by the Acetobacter xylinum, and the Lactobacillus plantarum LCC-605 loaded and embedded in the matrix and the biofilm formed therein.

[0008] The above-mentioned Acetobacter xylinum ( Acetobacter xylinum The gene sequence of the cellulose-producing strain Komagataeibacter nataicola RZS01 is described in the publicly available literature "Complete genome sequence of the cellulose-producing strain Komagataeibacter nataicola RZS01" (Heng Zhang, Xuran Xu, Xiao Chen, Fanshu Yuan, Bianjing Sun, Yunhua Xu, Jiazhi Yang & Dongping Sun, Scientific Reports, 7, 4431, 2017) and can be obtained from the Microbial Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 10961.

[0009] The above-mentioned Lactobacillus plantarum ( Lactobacillus plantarum LCC-605 is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on September 28, 2016, with accession number CCTCC NO: M2016491.

[0010] The conditions for the aerobic culture stage are as follows: aerobic culture for 2-7 days at 30-37°C in a mixed system containing Acetobacter xylinum fermentation broth and Lactobacillus plantarum LCC-605; the conditions for the anaerobic culture stage are as follows: the membrane material formed after aerobic culture is placed in a liquid culture medium and anaerobic culture for 24-48 hours at 30-37°C.

[0011] In the above-mentioned aerobic culture stage, the volume percentage of Acetobacter xylinum fermentation broth in the mixed system is 5-10%, and the initial OD600 value of Lactobacillus plantarum LCC-605 is 0.01-0.1.

[0012] The loading of *Lactobacillus plantarum* LCC-605 in the above composite membrane is not less than 1.0 × 10⁻⁶. 8 CFU / g.

[0013] The method for preparing the above-mentioned probiotic-bacterial cellulose composite membrane includes the following steps: (a) preparing activated fermentation broth of Acetobacter xylinum and activated bacterial suspension of Lactobacillus plantarum LCC-605 respectively; (b) mixing the activated fermentation broth of Acetobacter xylinum obtained in step (a) with the activated bacterial suspension of Lactobacillus plantarum LCC-605, and carrying out a first stage of static or shaking culture under aerobic conditions until a bacterial cellulose membrane is formed; (c) transferring the bacterial cellulose membrane obtained in step (b) to an anaerobic environment, and replacing the original culture medium with a new liquid culture medium for a second stage of anaerobic static culture; (d) taking out the membrane after anaerobic culture in step (c) and washing it to obtain a probiotic-bacterial cellulose composite membrane loaded with Lactobacillus plantarum LCC-605 and its biofilm.

[0014] In step (b), the aerobic culture is carried out at 30-37℃ for 2-7 days; in step (c), the anaerobic culture is carried out at 30-37℃ for 24-48 hours.

[0015] In step (c), the new liquid culture medium is MRS liquid culture medium, and the culture medium is changed 1-2 times during the anaerobic static culture.

[0016] A fermentation agent comprising the probiotic-bacterial cellulose composite membrane.

[0017] The above-mentioned starter culture is used in the preparation of fermented foods by inoculating the starter culture into plant-based milk for fermentation.

[0018] The above-mentioned probiotic-bacterial cellulose composite membrane is used in the preparation of medical devices, pharmaceuticals, skin care products, microecological preparations, feed or pet food.

[0019] Beneficial effects: (1) This strain is biosafe, does not produce toxins, and is environmentally friendly and sustainable; (2) The double encapsulation technology significantly improves the stability of probiotics; through the dual protection mechanism of bacterial cellulose (primary encapsulation) and in-situ biofilm coating (secondary encapsulation), the probiotics maintain high activity in the simulated gastrointestinal environment (GIT) (the number of viable bacteria in the probiotic biofilm bacterial cellulose composite membrane (LP605@BC) reached 8.9 log CFU / g after 3 h of gastric juice digestion, the number of viable bacteria in LP605@BC reached 9.0 log CFU / g after 3 h of intestinal juice digestion, and the number of viable bacteria in fermented black bean yogurt (BSY-LP605@BC) reached 11.2 log CFU / mL). After 30 days of storage at 4 ℃, the survival rate of LP605@BC was as high as 75.8%, and the number of viable bacteria in BSY-LP605@BC remained at 6.1 log CFU / mL, which is far superior to the traditional single-layer encapsulation technology. (3) The fermented dairy products are of excellent quality, combining functionality and stability. BSY-LP605@BC, prepared using LP605@BC as the starter culture, has the following characteristics: LCC-605 can produce extracellular polysaccharides in situ, giving the fermented milk excellent water-holding capacity (WHC), which is superior to traditional starter cultures (such as Lactobacillus bulgaricus + Streptococcus thermophilus mixed strains); long-term storage stability (the viable count reaches 10.7 log CFU / mL after 28 days; the viable count of BSY-LP605@BC remains at 10.1 log CFU / mL after 56 days). The fermented products have good properties, delicate taste, harmonious flavor, and excellent antioxidant properties, cholesterol-lowering ability, and blood sugar-lowering potential. The products meet consumers' demand for plant-based yogurt with high-activity probiotics and simple ingredients. (4) The process is simple and suitable for industrial application. The double encapsulation technology does not require complex equipment, and a high cell load (8.3 log CFU / g) can be achieved through a one-step co-culture method, which is suitable for large-scale production. The encapsulated LP605@BC can be directly used as a high-efficiency starter culture for the production of milk and plant-based yogurts such as soybeans, black beans and oats, reducing the dependence on additives and increasing the added value of the products. (5) The starter culture can be reused and maintains a stable number of live bacteria or even an increased number of live bacteria within a certain number of cycles. Attached Figure Description

[0020] Figure 1 Photos of BC and LP605@BC.

[0021] Figure 2 FESEM image of LP605@BC.

[0022] Figure 3 To simulate the survival rate of probiotics in LP605@BC after gastrointestinal digestion.

[0023] Figure 4The viable count of LP605@BC during storage in sodium chloride and peanut oil at 4°C.

[0024] Figure 5 The viable count of LP605@BC during storage in sodium chloride at 25 °C.

[0025] Figure 6 The viable count of LP605@BC freeze-dried membrane after 50 days of storage at 4 ℃ and 25 ℃.

[0026] Figure 7 Stability of BSY-LP605@BC during storage: A represents the viable bacterial survival rate and pH change in BSY-LP605@BC during storage, and B represents the WHC change in BSY-LP605@BC during storage.

[0027] Figure 8 The antioxidant and cholesterol-lowering properties of black soybean milk and BSY-LP605@BC.

[0028] Figure 9 The α-amylase and α-glucosidase activities of black soybean milk and BSY-LP605@BC were measured.

[0029] Figure 10 The viable cell count and pH changes in BSY-LP605@BC after 5 cycles of fermentation. Detailed Implementation

[0030] The method for obtaining the Lactobacillus plantarum LCC-605 bacterial suspension in the following examples is as follows:

[0031] Lactobacillus plantarum LCC-605 was inoculated into liquid MRS medium at a volume ratio of 3% and incubated statically at 31 °C for 18 h.

[0032] Example 1: Preparation, morphological observation, and viability test of LP605@BC

[0033] Acetobacter xylinum (cultured in seed culture medium for 12-24 h) Acetobacter xylinum Inoculate 7% of the culture medium into the fermentation medium and incubate at 37 ℃ in a shaker for 12 h; then inoculate the activated Acetobacter xylinum ( Acetobacter xylinum ) was inoculated into the fermentation medium at 7 vol% and cultured at 30 ℃ and 150 rpm / min for 12 h; 7 vol% of Acetobacter xylinum ( Acetobacter xylinumThe fermentation broth and LCC-605 were added to 24-well plates to make a total volume of 2 mL. The final OD values ​​of LCC-605 were 0.05, 0.5, and 1, respectively. The plates were cultured under aerobic conditions at 31 °C for 2-7 days until the fermentation medium was completely utilized. The grown membranes were then transferred to an anaerobic chamber, and 2 mL of MRS liquid medium was added to each well to replace the fermentation medium. The plates were cultured at 37 °C for 48 h, with the MRS liquid medium being replaced every 24 h. After the anaerobic period, the membranes were immersed in sterile physiological saline, with frequent replacement of the saline until the membranes turned white. The results are as follows: Figure 1 As shown in Table 1, the probiotic cellulose membranes before and after soaking and decolorization were removed and placed in a sterile mortar. 900 μL of sterile physiological saline was added, and the membranes were ground using a grinding rod. Then, the plate count method was used. The membranes were diluted to a suitable gradient with sterile physiological saline, and 100 μL were spread onto MRS agar plates. After incubation at 31℃ for 24 h, the counts were performed.

[0034] Table 1: Viable bacterial count in the membrane before and after decolorization

[0035]

[0036] The prepared bacterial cellulose membranes and probiotic cellulose membranes were replaced with tert-butanol and then freeze-dried. The membranes were then rapidly frozen in liquid nitrogen and manually broken to obtain cross-sections. The samples were fixed to the sample stage with conductive adhesive and sputtered with gold to improve conductivity. The surface and cross-sectional morphology of the membranes were observed using a cold field scanning electron microscope at an accelerating voltage of 3.0 kV. The results are as follows: Figure 2 As shown, under anaerobic conditions, the probiotic LCC-605, with its typical short rod-like morphology, was distributed on the bottom and top surface of the LP605@BC gel. The short rod-like LCC-605 proliferated extensively and penetrated the entire bacterial cellulose (BC) matrix, with a generally consistent distribution on both sides of the gel. Furthermore, it was found that LCC-605, possessing excellent extracellular polymeric substance (EPS) production capabilities, formed a biofilm inside the BC gel. These results indicate that LCC-605 is uniformly distributed within the BC gel and forms a biofilm, constructing a dual protective layer for the probiotic.

[0037] Example 2: Tolerance to simulated gastrointestinal tract and heat resistance of LP605@BC

[0038] 1. Acid resistance test

[0039] Free LCC-605 cells and one LP605@BC tablet were added to 5 mL of sterile MRS solutions with pH values ​​of 1.5, 2, and 2.5, respectively, and incubated at 37 °C for 3 h. Viable cell counts were determined every h using the agar plate method. The results are shown in Table 2. At pH 2.5, the survival rate of free LCC-605 decreased with increasing reaction time, reaching 84.2% after 3 h. In contrast, the survival rate of LCC-605 in the membrane was significantly higher, decreasing by only 2.1% after 3 h. When the pH was lowered to 2, the survival rate of free LCC-605 decreased to 36.3% after 1 h and died completely after 2 h, while the survival rate of LCC-605 in the membrane remained at 67.9% after 3 h. When the pH continued to decrease to 1.5, all free LCC-605 cells died after 1 hour of reaction, while the survival rate of LCC-605 cells in the membrane remained at 31.5% after 3 hours of reaction. pH 2.0 is the standard condition of extreme acid stress, which is lethal to microorganisms. The presence of the membrane effectively protected the probiotics and greatly improved their survival rate.

[0040] Table 2: Survival rates of LCC-605 and LP605@BC under different pH conditions for different durations

[0041]

[0042] Survival rate (%) calculation formula: (log CFU / mL of bacteria after treatment / log CFU / mL of bacteria before treatment) × 100

[0043] 2. Simulated gastric juice (SGF) tolerance test

[0044] In vitro gastrointestinal digestion of free bacteria and LP605@BC was performed according to a previously reported method (Yuan et al., 2023) with some modifications. Simulated gastric juice (SGF) was prepared by dissolving pepsin and NaCl in sterile water at final concentrations of 0.32% and 0.2% (w / v, g / mL), respectively, and adjusting the pH of the solution to 2.0 with 1 M HCl. Free LCC-605 cells and one LP605@BC tablet were added to 10 mL of SGF and cultured at 31 °C and 150 rpm for 3 h. Viable cell counts were then determined by the agar plate method. Results are as follows: Figure 3 As shown, after 3 hours of simulated gastric digestion, the survival rate of the free bacteria group was 84.4%, while the number of live bacteria in the LP605@BC group remained stable and the survival rate was not significantly affected, both remaining above 99.2%.

[0045] 3. Simulated Intestinal Fluid (SIF) Tolerance Test

[0046] First, 6.8 g / L potassium hydrogen phosphate, 10 g / L trypsin, and 1 g / L bile salts were dissolved in sterile water to prepare SIF (Self-Induced Intestinal Fluid). The pH of the mixture was adjusted to 7.0 with 1 M sodium hydroxide. Free LCC-605 cells and one LP605@BC tablet were added to 10 mL of SIF and incubated at 31 ℃ and 150 rpm for 3 h. Afterward, viable cell counts were determined using the agar plate method. For simulated intestinal digestion, the survival rate of the free bacterial group was 91.9%, while the viable cell count in the LP605@BC group remained relatively stable, with a slight decrease in survival rate with prolonged digestion time, but still as high as 94.1% after 3 h. Figure 3 ).

[0047] The above results indicate that bacterial cellulose (BC) can protect LCC-605 from some harsh environments and improve its survival rate under severe conditions.

[0048] 4. Heat resistance test

[0049] Free LCC-605 cells and one LP605@BC tablet were added to sterile tubes, respectively. The tubes containing different samples were then heated at 72 °C for 15 s and 3 min, respectively. After different treatments, the viable cell count was determined using the agar plate method.

[0050] Table 3: Viable bacterial counts of LCC-605 in LCC-605 and LP605@BC after different heat treatments.

[0051]

[0052] As shown in Table 3, the thermal stability test results indicate that LP605@BC achieved a 100% survival rate at 72℃ for 15 s, which is higher than that of free bacteria (95.0%). In particular, LP605@BC maintained a 55.9% survival rate at 72℃ for 3 min, breaking through the heat resistance limit of conventional probiotic preparations (< 60℃).

[0053] The above results indicate that the bacterial cellulose membrane encapsulation system can effectively protect probiotics from the gastrointestinal environment and heat treatment, providing key technical support for their application in functional foods.

[0054] Example 3 Storage stability of LP605@BC and freeze-dried LP605@BC

[0055] After soaking in water to remove discoloration, the membranes were placed in sterile physiological saline and stored at room temperature, 4°C, and in peanut oil at 4°C. On days 0, 1, 5, 10, 15, and 30, portions of the culture were taken, thoroughly ground with 900 μL of sterile physiological saline, and diluted to the appropriate bacterial concentration. 100 μL of the diluted bacterial solution was spread onto MRS agar plates and incubated at 31°C for 24 h before colony counting. Each dilution gradient was performed in triplicate.

[0056] The membrane was placed in a 10% skim milk solution, pre-frozen at -80 ℃ for 12 h, and then lyophilized. The lyophilized probiotic cellulose membranes were stored at 4 ℃ and room temperature, respectively. On days 0, 7, 14, 21, and 50, a portion of the culture was taken out, and 900 μL of sterile physiological saline was added to grind it thoroughly and dilute it to the appropriate bacterial concentration. 100 μL of the diluted bacterial solution was spread on MRS agar plates and incubated at 31 ℃ for 24 h before colony counting. Each dilution gradient was performed in triplicate.

[0057] like Figure 4 As shown, after one day of storage in peanut oil and sterile saline at 4 ℃, the viable bacterial count of LCC-605 in LP605@BC showed only a slight decrease, with viable bacterial counts of 8.0 and 7.8 log CFU / g, respectively. After 15 days of storage, the viable bacterial counts in peanut oil and sterile saline at 4 ℃ were 7.9 and 7.7 log CFU / g, respectively. When stored at 4 ℃ for 30 days, the viable bacterial counts in peanut oil and sterile saline were 6.6 and 6.3 log CFU / g, respectively; while the viable bacterial count after 30 days of storage at 25 ℃ was only 3.7 log CFU / g. Figure 5 The above results indicate that storing probiotic cellulose at 25 ℃ for 30 days significantly affects the number of viable bacteria, but it can still maintain a high survival rate of probiotics at 4 ℃.

[0058] Freeze-drying technology is currently the mainstream preservation method for probiotic preparations. Therefore, by adding suitable freeze-drying protectants, probiotic cellulose membranes are freeze-dried to improve membrane stability and convenience. For example... Figure 6 As shown, after adding skim milk, the freeze-dried membranes were stored at 4 ℃ and 25 ℃ respectively. During the first 21 days of storage, the viable count of LP605@BC under different storage conditions was higher than 9.1 log CFU / g. However, as the storage time increased, the viable count of LP605@BC stored at 25 ℃ was 6.7 log CFU / g, while the viable count of LP605@BC stored at 4 ℃ was 8.3 log CFU / g.

[0059] In conclusion, adding skim milk before freeze-drying can significantly improve the survival rate of LP605@BC under storage conditions of 4 ℃ or 25 ℃, which is of key significance for the practical application of LP605@BC.

[0060] Example 4: Preparation and storage stability of BSY-LP605@BC

[0061] Under optimal fermentation conditions, three probiotic cellulose membranes were inoculated into sterile black soybean milk and fermented at 31 °C for 24 h to prepare BSY-LP605@BC. At the end of the fermentation process, the obtained BSY-LP605@BC was stored at 4 °C for 24 h. Subsequently, according to our previously reported method, the viable cell count, bacterial survival rate, pH value, and WHC of BSY-LP605@BC during 56 days of storage were determined (Yang et al., 2025).

[0062] like Figure 7 As shown in Figure A, the viable cell count of BSY-LP605@BC remained relatively stable during the 56-day storage period (reaching 10.1 log CFU / mL after 56 days). Figure 7 As shown in Figure B, the WHC of BSY-LP605@BC after 56 days of storage was 43.8%. This is because no additional thickeners or stabilizers were added to the black soybean yogurt, EPS-605 NPs had limited water retention capacity, and the lactic acid bacteria continued to produce acid. When the pH dropped below 4.0, the casein micelles contracted excessively, squeezing out water, thus the WHC decreased, but it was still much higher than that of other probiotic fermented soybean yogurts reported in the literature (Ziarno et al., 2023).

[0063] The above results indicate that in-situ production of EPS from membrane-fermented black soybean yogurt avoids the need for additional thickeners and stabilizers in the fermentation process of yogurt, which not only simplifies the fermentation process but also reduces production costs, providing a key technological solution for the commercialization of plant-based yogurt.

[0064] Example 5: Functional Characteristic Evaluation of SY-LP605@BC

[0065] Following our previously reported method, we determined the free radical scavenging capacity, cholesterol-lowering capacity, and blood glucose-lowering capacity of SY-LP605@BC (Yang et al., 2025). Figure 8 As shown, fermented black bean yogurt exhibits significantly higher free radical scavenging ability than unfermented black bean milk, especially with an 11-fold increase in hydroxyl radical scavenging ability. Furthermore, the scavenging ability of all three free radicals in black bean yogurt exceeds 95.3%. Figure 8As shown, BSY-LP605@BC also has a strong cholesterol-lowering ability; the cholesterol clearance rate of unfermented black soybean milk is only 16.9%, while the clearance rate increases to 61.4% after fermentation. Figure 9 As shown, the inhibition rates of α-amylase and α-glucosidase were increased in black bean yogurt after membrane fermentation.

[0066] In-situ production of EPS in black bean yogurt via membrane fermentation avoids the need for additional thickeners and stabilizers in the fermented yogurt preparation process, simplifying the fermentation process and reducing production costs. By using probiotic cellulose membranes as a starter culture, the fermented black bean yogurt exhibits better cholesterol-lowering effects, antioxidant activity, and blood sugar-lowering capabilities.

[0067] Example 6: Reusability Test of LP605@BC

[0068] The probiotic cellulose membrane LP605@BC used for fermentation in Example 4 was removed from the fermentation substrate, cleaned, and then used for repeated fermentation. This was used to evaluate the viable cell count of the fermentation product simulating repeated use of the same starter culture.

[0069] like Figure 10 As shown, after five repeated fermentations with the same starter culture, the viable count in BSY-LP605@BC remained at 11.3 log CFU / mL, with a pH of 4.0, remaining stable throughout the cyclic fermentation process. This result may be attributed to the enrichment of LCC-605 in the probiotic cellulose membrane within the black soybean milk during each cycle. The probiotic cellulose membrane slowly releases probiotics into the black soybean milk, with the bacteria multiplying simultaneously on the membrane surface and in the solution, forming a stable microbial community. After each fermentation, some probiotics reattach to the probiotic cellulose membrane, providing sufficient bacterial quantity for the next fermentation. The viable count of the membrane after five cycles remained at 11.0 log CFU / g, higher than the pre-fermentation viable count (8.3 log CFU / g).

[0070] This is likely because during fermentation, the black soybean milk permeates the membrane's network structure, protecting the probiotics within. This probiotic cellulose membrane offers many practical advantages, such as reducing the cost of repeatedly purchasing starter cultures in traditional yogurt preparation; high storage stability, tolerance to various adverse environments, and ease of transportation and processing; reusability and resistance to degradation; and ease of cleaning, removing residual yogurt without damaging the immobilized bacteria and preventing contamination by other microorganisms.

[0071] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A probiotic-bacterial cellulose composite membrane, characterized in that it is Acetobacter xylinum ( Acetobacter xylinum ) and Lactobacillus plantarum with accession number CCTCC M 2016491 ( Lactobacillus plantarum LCC-605 is formed through in situ co-culture, and sequentially through aerobic and anaerobic culture stages; the composite membrane comprises a matrix composed of bacterial cellulose produced by the Acetobacter xylinum, and the Lactobacillus plantarum LCC-605 loaded and embedded in the matrix and the biofilm formed therein.

2. The probiotic-bacterial cellulose composite membrane according to claim 1, characterized in that, The conditions for the aerobic culture stage are as follows: aerobic culture for 2-7 days at 30-37°C in a mixed system containing Acetobacter xylinum fermentation broth and Lactobacillus plantarum LCC-605; the conditions for the anaerobic culture stage are as follows: the membrane material formed after aerobic culture is placed in a liquid culture medium and anaerobic culture for 24-48 hours at 30-37°C.

3. The probiotic-bacterial cellulose composite membrane according to claim 2, characterized in that, During the aerobic culture phase, the volume percentage of Acetobacter xylinum fermentation broth in the mixed system is 5-10%, and the initial OD600 value of Lactobacillus plantarum LCC-605 is 0.01-0.

1.

4. The probiotic-bacterial cellulose composite membrane according to claim 1, characterized in that, The loading of *Lactobacillus plantarum* LCC-605 in the composite membrane is not less than 1.0 × 10⁻⁶. 8 CFU / g.

5. A method for preparing the probiotic-bacterial cellulose composite membrane according to any one of claims 1-4, characterized in that, Includes the following steps: (a) Prepare activated fermentation broth of Acetobacter xylinum and activated bacterial suspension of Lactobacillus plantarum LCC-605 respectively; (b) Mix the activated fermentation broth of Acetobacter xylinum obtained in step (a) with the activated bacterial suspension of Lactobacillus plantarum LCC-605 and carry out the first stage of static or shaking culture under aerobic conditions until a bacterial cellulose membrane is formed; (c) Transfer the bacterial cellulose membrane obtained in step (b) to an anaerobic environment and replace the original culture medium with a new liquid culture medium for the second stage of anaerobic static culture; (d) Take out the membrane after anaerobic culture in step (c) and wash it to obtain a probiotic-bacterial cellulose composite membrane loaded with Lactobacillus plantarum LCC-605 and its biofilm.

6. The method according to claim 5, characterized in that, In step (b), the aerobic culture is carried out at 30-37℃ for 2-7 days; in step (c), the anaerobic culture is carried out at 30-37℃ for 24-48 hours.

7. The method according to claim 5 or 6, characterized in that, In step (c), the new liquid culture medium is MRS liquid culture medium, and the culture medium is changed 1-2 times during the anaerobic static culture.

8. A fermenting agent, characterized in that, The probiotic-bacterial cellulose composite membrane comprising any one of claims 1-4.

9. The application of the starter culture of claim 8 in the preparation of fermented foods, characterized in that, The fermenting agent is inoculated into plant-based milk for fermentation.

10. The use of the probiotic-bacterial cellulose composite membrane according to any one of claims 1-4 in the preparation of medical devices, pharmaceuticals, skin care products, microecological preparations, feed or pet food.

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