Composite strain composition for preparing ecological coagulator and application of composite strain composition

Abstract

The invention relates to a compound strain for preparing an ecological coagulator and application of the compound strain, the compound strain is composed of lactobacillus plantarum, lactobacillus casei and saccharomyces cerevisiae, the saccharomyces cerevisiae is subjected to gene introduction and recombination, and high-expression glutamine transaminase is obtained; the method for preparing the ecological coagulator from the compound bacterial strain comprises the following steps: collecting and standardizing bean clear liquid, sterilizing, inoculating and fermenting to prepare the ecological coagulator, applying the ecological coagulator to a bean product, curdling, squeezing and forming. The ecological coagulator has the advantages that a bean product curdled by the ecological coagulator contains GABA, soybean small molecule polypeptide and metabiotics, and is a bean product with nutritional and health-care effects, an exogenous chemical coagulator is not required to be added in the production process, the discharge of bean clear liquid can be effectively reduced by 30%, and clean production of the bean product is realized.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and particularly relates to a composite strain composition for preparing ecological coagulants and its application. Background Technology

[0002] Traditional tofu production primarily uses gypsum, bittern, or glucono-delta-lactone (GDL) as coagulants. While these chemical coagulants are widely used, they present several problems: gypsum tofu tends to have a bitter taste and poor water retention; bittern tofu has a firm texture and its soybean flavor is masked; and GDL tofu tends to develop a sour taste and has a soft texture. Furthermore, these methods fail to effectively utilize the large amount of byproduct generated during tofu production—soybean whey (yellow liquid)—whose direct discharge would cause serious environmental pressure and resource waste.

[0003] In recent years, the preparation of biocoagulants through microbial fermentation has become a research hotspot. This method typically utilizes microorganisms such as lactic acid bacteria to ferment soybean whey to produce acid, which is then used to coagulate the soy milk and produce tofu, providing a direction for the resource utilization of soybean whey. However, existing microbial coagulant technologies still face significant bottlenecks, hindering their large-scale industrial application: Enzyme activity stability and functional singularity: Existing research mainly focuses on the acid-producing capacity of lactic acid bacteria, whose coagulation mechanism is singular (only acid coagulation). The tofu produced from this bacteria cannot compare with traditional coagulants in terms of texture, elasticity, and water retention. Although some studies have attempted to add exogenous transglutaminase (TG enzyme) to improve gel properties, exogenous enzyme preparations are expensive and easily inactivated in the fermentation system, resulting in poor enzyme activity stability. This makes it difficult to maintain their function and precisely control the quality of tofu in the complex fermentation environment of soybean whey.

[0004] Poor synergy in strain combinations: Simple strain combinations often fail to achieve stable and efficient synergistic fermentation due to problems such as growth competition between strains, inhibition of metabolites, or incompatibility of culture conditions. For example, the rapid acid production of lactic acid bacteria leads to a sharp drop in pH, which severely inhibits the growth and metabolic activity of other functional microorganisms (such as enzyme-producing and flavor-producing strains), failing to create a synergistic effect of "1+1>2", and ultimately resulting in limited improvement in product quality.

[0005] Soybean curd liquor has low reuse efficiency and fails to achieve closed-loop production: most technologies treat soybean curd liquor only as a single-use fermentation substrate, and the secondary waste liquid generated after fermentation still needs to be treated. Unlike this invention, it fails to use the soybean curd liquor generated from this coagulation process as a raw material for the next coagulant production, achieving a complete closed-loop cycle of "coagulation-collection-fermentation-coagulation" and truly achieving the goal of reducing emissions. In addition, the nutritional composition of soybean curd liquor is not completely balanced, and single-strain fermentation may be insufficient due to the depletion of carbon sources, vitamins, and other nutrients, resulting in unstable product quality between batches.

[0006] Transglutaminase (TG enzyme) is an enzyme that catalyzes cross-linking between or within protein molecules, significantly improving their gelling properties, water-holding capacity, and stability. Combining high-TG-producing strains with acid-producing strains holds promise for developing a novel eco-friendly coagulant that efficiently produces acid while also improving protein gelling properties. However, naturally occurring high-TG-producing microorganisms (such as *Streptomyces malnourishingus*) are not food-safe strains, and their fermentation conditions differ significantly from those of lactic acid bacteria, making direct combination and application difficult.

[0007] Therefore, developing an eco-friendly coagulant and its preparation method that consists of GRAS (Gradually Recycling Assured Strains), exhibits synergistic symbiosis among strains, efficiently utilizes soybean whey to produce an eco-friendly coagulant with the dual advantages of acid and enzyme coagulation, and enables complete wastewater recycling, has significant industrial value and environmental implications. This invention was completed against this backdrop. Summary of the Invention

[0008] This invention provides a composite strain composition for preparing ecological coagulants, which solves the problems of instability and high cost caused by the addition of exogenous enzymes in the prior art.

[0009] To achieve the above objectives, the technical solution of the present invention is: a composite strain composition for preparing an ecological coagulant, the composition comprising Lactobacillus plantarum with accession number CCTCCNO:M2023025, Lactobacillus casei with accession number CCTCCNO:M2023026, and Saccharomyces cerevisiae with accession number CCTCCNO:M2023027, wherein the Saccharomyces cerevisiae is an engineered strain that highly expresses transglutaminase (TG enzyme) obtained through genetic engineering.

[0010] Furthermore, the method for constructing the engineered strain of *Saccharomyces cerevisiae* includes the following steps: (1) Total RNA was extracted from Streptomyces mouyuanensis, cDNA was obtained by reverse transcription, and the TG enzyme-encoded gene was obtained by PCR specific amplification; (2) Artificial optimization and whole-genome synthesis of the above TG enzyme gene sequence were carried out based on the codon preference of Saccharomyces cerevisiae; (3) The optimized TG enzyme gene was cloned into an expression vector suitable for Saccharomyces cerevisiae, which contains a strong promoter (such as TEF1, PGK1 or GAL1 promoter) to drive high-level transcription of exogenous genes. (4) A yeast-efficient secretion signal peptide sequence (such as α-mating factor leader peptide) is attached to the N-terminus of the TG enzyme gene to ensure that the TG enzyme can be secreted extracellularly. (5) The constructed recombinant expression plasmid was introduced into Saccharomyces cerevisiae host cells, and positive transformants were screened using selective culture medium; (6) The successful integration of the TG enzyme gene was verified by PCR, and high-yield strains were screened by enzyme activity detection.

[0011] Furthermore, the method for preparing an ecological coagulant using the co-fermentation of the aforementioned composite strains includes the following steps: (1) Raw material processing: Collect the soy milk produced after curdling tofu and filter it through a 120-mesh sieve; (2) Standardization: Add carbon source to the filtered soybean liquid by adding 1%-2% glucose and 0.2%-0.5% galactose by weight of soybean liquid; (3) Sterilization: Ultra-high temperature instantaneous sterilization (UHT) is adopted at 118°C for 15 seconds; (4) Inoculation: Inoculate the compound strain at 0.5%-1.5% of the mass of the sterilized soybean liquid, wherein the viable count ratio of Lactobacillus plantarum JMF-001, Lactobacillus casei JMF-002 and Saccharomyces cerevisiae JMF-003 is 8:8:1, and the viable count of each strain reaches 1×10⁻⁶. 9 CFU / mL or higher; (5) Fermentation: Ferment at 36-38℃ for 18-22 hours; (6) Termination and post-treatment: When the pH of the fermentation broth drops to 4.0-4.2 and the total enzyme activity reaches 15-20 U / L, the fermentation is terminated. The fermentation broth is first filtered through an 80-mesh filter to remove protein flocculants, and then inactivated by heat treatment at 55-60℃ for 10-15 seconds to obtain the ecological coagulant.

[0012] The ecological coagulant was applied in the preparation of tofu.

[0013] The beneficial effects of this invention are: 1) Synergistic effect of strains: Two kinds of lactic acid bacteria work together to produce acid, which rapidly reduces the pH value; the TG enzyme secreted by the genetically engineered yeast JMF-003 can catalyze the cross-linking of soybean protein. The two work together to form a dual coagulation mechanism of "acid coagulation + enzyme cross-linking", resulting in tofu with high yield, delicate texture, good water retention and elasticity.

[0014] 2) Turning waste into treasure, green and environmentally friendly: Using the waste liquid from soybean product processing—soybean liquid—as raw material to produce high-value-added coagulants, a closed-loop cycle of the production process is realized, solving environmental problems and reducing production costs.

[0015] 3) Improve the quality and function of tofu: The tofu prepared by this invention not only has a good taste, but also produces more functional components due to microbial fermentation, such as γ-aminobutyric acid (GABA), with a detected content of up to 16 mg / kg or more, giving the tofu health benefits.

[0016] 4) Stable and controllable preparation process: By optimizing the strain ratio, fermentation conditions and coagulation process, the quality of the coagulant and the consistency of the tofu products are guaranteed.

[0017] Instructions for the Preservation of Biological Materials The strain involved in this invention has been deposited with the China Center for Type Culture Collection (CCTCC), and the information is as follows: Preservation Institution: CCTCC; Preservation Date: January 5, 2023; Preservation Code: M2023025; Classification and Nomenclature: Lactobacillus plantarum strain JMF-001; Viability: Yes; Preservation Institution: CCTCC; Preservation Date: January 5, 2023; Preservation Code: M2023026; Classification and Nomenclature: Lactobacillus casei strain JMF-002; Viability: Yes; Preservation Authority: CCTCC; Preservation Date: January 5, 2023; Preservation Code: M2023027; Classification: Saccharomyces cerevisiae strain JMF-003; Viability: Yes; Address: Wuhan University, Wuhan, China. Attached Figure Description

[0018] Figure 1 : Schematic diagram of strain construction process; Figure 2 Flowchart for the preparation and fermentation of ecological coagulant. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0021] Example 1 A composite strain composition for preparing an ecological coagulant, comprising Lactobacillus plantarum JMF-001 (accession number CCTCCNO:M2023025), Lactobacillus casei JMF-002 (accession number CCTCCNO:M2023026), and Saccharomyces cerevisiae JMF-003 (accession number CCTCCNO:M2023027).

[0022] The construction and validation of the engineered Saccharomyces cerevisiae strain JMF-003 includes the following steps: 1. Gene Cloning and Optimization: Total RNA was extracted from *Streptomyces mobaraensis* and reverse transcribed into cDNA. Primers were designed and PCR was performed to amplify the complete TG enzyme gene (approximately 1.2 kb). The gene sequence was optimized using a codon frequency table based on *Saccharomyces cerevisiae*, and the complete gene synthesis was commissioned to Sangon Biotech (Shanghai) Co., Ltd.

[0023] 2. Vector Construction: The Saccharomyces cerevisiae expression vector pPICZαA (with a strong TEF1 promoter and α-MF secretion signal peptide) was selected. The synthesized TG gene fragment and the pPICZαA empty vector were digested with restriction endonucleases EcoRI and NotI, purified, and ligated using T4 DNA ligase to construct the recombinant plasmid pPICZαA-TG.

[0024] 3. Yeast transformation and screening: The recombinant plasmid pPICZαA-TG was linearized and transformed into Saccharomyces cerevisiae INVSC1 competent cells by electroporation. The cells were then plated on YPD selection plates containing 100 μg / mL Zeocin and incubated at 30°C for 2-3 days.

[0025] 4. Identification of positive clones: Select a single transformant, extract yeast genomic DNA as a template, and perform PCR verification using TG gene-specific primers. Clones that amplify a specific band of approximately 1.2 kb are considered positive recombinant yeast strains.

[0026] 5. Verification of expression level and genetic stability: The verified positive strains were inoculated into YPD liquid medium and cultured at 30°C and 250 rpm for 48 hours with shaking.

[0027] The fermentation supernatant was collected by centrifugation, and its TG enzyme activity was determined by the hydroxylamine trichromatographic method. After multiple rounds of screening, a strain with the highest enzyme activity was obtained and named JMF-003, whose enzyme activity in the fermentation supernatant was stable at 12-16 U / mL.

[0028] The strain was passaged 10 times consecutively, and the enzyme activity of each generation was measured. The results showed that the enzyme activity was stable between 12-16 U / mL, indicating that its genetic traits were stable.

[0029] Physiological and biochemical identification of the strain confirmed it to be Saccharomyces cerevisiae, and it was deposited at CCTCC on January 12, 2023, with accession number CCTCCNO: M2023027.

[0030] The activation and amplification of the complex strains are as follows: 1. The lyophilized powders of preserved Lactobacillus plantarum JMF-001 and Lactobacillus casei JMF-002 were inoculated into MRS liquid medium and activated by static culture at 37℃ for 18h (first generation).

[0031] 2. Transfer the activated bacterial culture to fresh MRS medium at an inoculation rate of 2%, and culture under the same conditions for 18 hours (second generation).

[0032] 3. Subculture again (3rd generation). At this point, the viable count of each lactic acid bacteria solution exceeded 1×10⁻⁶. 9 CFU / mL.

[0033] 4. Inoculate Saccharomyces cerevisiae JMF-003 into YPD liquid medium, incubate at 30℃ and 200 rpm for 24 h with shaking, and continue activation for 3 generations until the final viable count exceeds 1×10⁻⁶. 9 CFU / mL.

[0034] Take JMF-001, JMF-002, and JMF-003 bacterial solutions in a live bacteria ratio of 8:8:1, mix them evenly, and prepare a compound bacterial fermentation agent for later use.

[0035] Example 2 The preparation of an ecological coagulant using the bacterial composition of Example 1 includes the following steps: 1. Collect raw materials: Take the soy liquid produced after making tofu using traditional brine.

[0036] 2. Filtration: Filter the soybean liquid through a 120-mesh stainless steel sieve to remove large soybean residue particles.

[0037] 3. Standardization: Based on the total mass of the filtered soybean liquid, add 1.5% glucose and 0.3% galactose, and stir until completely dissolved.

[0038] 4. Sterilization: UHT sterilization unit is used to treat at 118℃ for 15 seconds, and then rapidly cooled to 38℃.

[0039] 5. Inoculation: Under aseptic conditions, 1.0% (by mass) of the compound microbial fermentation agent prepared in Example 2 was added to the cooled soybean liquid base.

[0040] 6. Fermentation: Let it ferment statically in a 37℃ constant temperature fermentation tank for 20 hours.

[0041] 7. Termination and post-treatment: Monitor the fermentation broth. When the pH value drops to 4.1 and the TG enzyme activity is measured to be 18 U / L, terminate the fermentation.

[0042] (1) First, filter the fermentation liquid with an 80-mesh sieve to remove a large amount of protein flocculents produced during the fermentation process.

[0043] (2) Then the filtrate is passed through a plate heat exchanger and heat-treated at 58°C for 12 seconds to inactivate the microorganisms and terminate the fermentation reaction.

[0044] (3) After cooling, a light yellow liquid ecological coagulant with a fermented sour aroma is obtained and stored at 4°C.

[0045] Example 3 describes the preparation of tofu using the eco-friendly coagulant from Example 2, comprising the following steps: 1. Pulping: Select Northeast soybeans with a protein content of 42%, soak them for 10 hours, and then grind them into a pulp. The soy milk is heated in stages: first, raise the temperature to 75℃ and maintain it for 2.5 minutes, then raise the temperature to 93℃ and maintain it for 3.5 minutes, and finally boil for 5 minutes. Filter the soy milk through a 100-mesh filter cloth to obtain soy milk with a concentration of approximately 8.0°Brix, and cool it to 85-88℃ for later use.

[0046] 2. Coagulation: Take 100 kg of soybean milk into a coagulation tank. Prepare 25 kg of the eco-friendly coagulant prepared in Example 3.

[0047] (1) First addition: Turn on the mixer (30 rpm) and slowly add 15 kg of ecological coagulant (60% of the total amount) over 6 minutes. After adding, stop mixing and let stand for 5 minutes.

[0048] (2) Second addition: Start stirring again (30 rpm) and slowly add 7.5 kg of ecological coagulant (30% of the total amount) over 5 minutes. After adding, stop stirring and let stand for 3 minutes.

[0049] (3) Third addition: Start stirring (reducing the speed to 20 rpm), and slowly add the remaining 2.5 kg of ecological coagulant (10% of the total amount) within 3 minutes. After adding, stop stirring and let it stand for 10 minutes.

[0050] 3. Shaping: After the tofu has softened, the tofu curds are smooth and plump. Gently stir the tofu curds at 18 rpm for 2 minutes. Then, divide the tofu curds into tofu molds, cover them with cheesecloth, and press them under 25 kg pressure for 20 minutes.

[0051] 4. Finished Product: After demolding, the finished tofu is obtained. The tofu has a firm yet elastic texture, a smooth and delicate cut surface, a rich bean aroma, and good resilience. Testing revealed that the tofu contains 16.5 mg / kg of γ-aminobutyric acid (GABA), and the tofu yield (based on dry soybeans) is 11.84% higher than that obtained with conventional magnesium chloride.

[0052] Comparative Example: Preparation of Tofu Using a Single Lactic Acid Bacteria Fermentation Coagulant Except for the use of only Lactobacillus plantarum JMF-001 and Lactobacillus casei JMF-002 (ratio 1:1, total inoculum 1.0%) as the starter culture without the addition of JMF-003, the remaining steps are exactly the same as in Examples 3 and 4.

[0053] Texture characteristics of tofu products with different coagulants:

[0054] Results: The prepared coagulant had a distinctly sour taste but no enzyme activity. The tofu made from the coagulant was soft, brittle, had poor water retention, a sour taste, and a low GABA content (only 3.2 mg / kg). The tofu yield was approximately 13.2% lower than that of the method described in this invention. Based on the above data, the tofu made with the ecological coagulant showed significantly better texture analysis results than tofu made with other coagulants.

[0055] The above results indicate that the composite strain and its ecological coagulant preparation method provided by this invention can effectively utilize soybean whey waste liquid through the synergistic effect between strains and the "acid-enzyme complex" coagulation mechanism to produce high-quality, high-yield tofu rich in functional components, thus achieving the dual goals of resource recycling and product quality improvement.

[0056] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A composite bacterial strain composition for preparing an ecological coagulant, characterized in that, The composition comprises Lactobacillus plantarum, Lactobacillus casei, and Saccharomyces cerevisiae, wherein the Saccharomyces cerevisiae is a genetically engineered recombinant strain capable of efficiently expressing transglutaminase.

2. The composite bacterial strain composition for preparing an ecological coagulant according to claim 1, characterized in that, The construction method of Saccharomyces cerevisiae includes the following steps: (1) extracting total RNA from Streptomyces mouyuanensis, reverse transcribing to obtain cDNA, and amplifying the TG enzyme gene by PCR; (2) The TG enzyme gene sequence was optimized based on the codon preference of Saccharomyces cerevisiae, and the whole gene was synthesized; (3) The optimized TG enzyme gene was cloned into an expression vector suitable for Saccharomyces cerevisiae, the vector containing a strong promoter; (4) Link the yeast secretion signal peptide sequence to the N-terminus of the TG enzyme gene; (5) The recombinant plasmid was introduced into the host cell of Saccharomyces cerevisiae and transformants were obtained by resistance screening; (6) PCR and enzyme activity detection were used to verify the high efficiency of TG enzyme expression.

3. The composite strain composition for preparing an ecological coagulant according to claim 2, characterized in that, The strong promoter is one of TEF1, PGK1 or GAL1 promoter; the secretion signal peptide sequence is an α-mating factor leader peptide.

4. The composite strain composition for preparing an ecological coagulant according to claim 3, characterized in that, The TG enzyme activity of the brewing yeast in the fermentation supernatant was 12-16 U / mL, and the enzyme activity remained stable after 10 generations of subculturing.

5. The composite bacterial strain composition for preparing an ecological coagulant according to claim 1, characterized in that, The application of the composite strain composition in the preparation of ecological coagulant includes the following steps: (1) after filtering soybean liquid, add carbon source, add 1%-2% glucose and 0.2%-0.5% galactose; (2) The soybean liquid after feeding was sterilized by UHT at 118°C for 15 seconds; (3) Inoculate with the compound bacterial strain composition at an inoculation amount of 0.5%-1.5%, wherein the ratio of viable Lactobacillus plantarum, Lactobacillus casei and Saccharomyces cerevisiae is 8:8:1; (4) The inoculated soybean extract was fermented at 36-38℃ for 18-22 hours; (5) Terminate fermentation when the pH of the fermentation broth reaches 4.0-4.2 and the enzyme activity reaches 15-20 U / L; (6) After the fermentation broth is filtered, it is heat-treated at 55-60℃ for 10-15 seconds to inactivate it, thereby obtaining the ecological coagulant.

6. The composite strain composition for preparing an ecological coagulant according to claim 5, characterized in that, The soy milk comes from the yellow liquid produced during the curdling process of tofu.

7. The composite strain composition for preparing an ecological coagulant according to claim 6, characterized in that, The ecological coagulant was applied in the preparation of tofu.

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