Anti-fatigue leavening and preparation method thereof

Abstract

The preparation method comprises the following steps: S1, raw material pretreatment: mixing ginseng and rhizoma polygonati according to a ratio, crushing and sterilizing to obtain a matrix; s2, inoculation: inoculating lactobacillus plantarum and bifidobacterium longum into the matrix; s3, performing staged solid state fermentation, including primary fermentation: performing fermentation for 10-14 hours under the conditions that the pH is 6.0-7.0, the temperature is 38-42 DEG C and the humidity is 80-90%; secondary fermentation: in an environment with the temperature of 32-37 DEG C and the humidity of 70-80%, regulating the pH to the final pH of 5.0-6.5 in a staged gradient manner, and fermenting for 30-40 hours; and S4, post-treatment: inactivating and drying the fermentation product to obtain a final product.

Description

Technical Field

[0001] This invention relates to the fields of microbial fermentation and food biotechnology, specifically to an anti-fatigue fermented product and its preparation method, a method for preparing an anti-fatigue medicinal and edible fermented product through probiotic targeted transformation technology, and in particular a method for preparing a ginseng-Polygonatum fermented product based on probiotic targeted transformation. Background Technology

[0002] Traditional fermentation processes for food and medicine have several technical limitations in practical applications. Regarding microbial strains, existing processes often rely on single strains or random combinations of microbial communities, lacking scientifically designed formulations. This results in insufficient activity of microbial enzyme systems, making it difficult to target and regulate metabolic pathways. Taking the conversion of ginsenosides as an example, the key active ingredient CK needs to be deglycosylated by β-glucosidase. However, in traditional processes, the expression of this enzyme is unstable, leading to a CK conversion rate generally below 30%, and the easy generation of non-target metabolites.

[0003] In terms of fermentation process control, existing technologies mostly adopt static parameter modes with constant temperature and humidity, which cannot dynamically adapt to the metabolic needs of microbial growth cycles. For example, the temperature and pH requirements of the initial cell proliferation stage and the later product synthesis stage differ significantly. Static control can lead to insufficient cell activity in the early stage or product degradation in the later stage, further reducing the yield of target components. At the same time, current processes lack precise fermentation termination criteria and often rely on experience-based judgment, which can easily lead to incomplete or excessive fermentation, resulting in a loss rate of active ingredients as high as 20%-30%.

[0004] Regarding the retention of functional components, traditional processes suffer from insufficient utilization of synergistic active substances such as polysaccharides and flavonoids. Due to the mismatch between fermentation parameters and the metabolic characteristics of the microbial strains, polysaccharide structures are often damaged or extraction rates are low. For example, in current technologies, the yield of Polygonatum polysaccharides is generally below 15%, making it difficult to realize its prebiotic function. Furthermore, industrial production suffers from poor batch stability, long fermentation cycles, and high energy consumption, hindering the large-scale application of fermented products that are both medicinal and edible. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing an anti-fatigue fermented product and its preparation method. The anti-fatigue medicinal and edible fermented product is prepared using probiotic-directed conversion technology.

[0006] The technical solution provided by this invention is as follows: <First Aspect> A method for preparing an anti-fatigue ferment includes the following steps: S1. Raw material pretreatment: Ginseng and Polygonatum are mixed in proportion, crushed and sterilized to obtain the matrix; S2. Inoculation: Inoculate Lactobacillus plantarum and Bifidobacterium longum into the substrate; S3, staged solid-state fermentation, includes the following steps: Primary fermentation: Ferment for 10-14 hours at pH 6.0-7.0, 38-42℃, and 80-90% humidity; Secondary fermentation: Under an environment of 32-37℃ and 70-80% humidity, the pH is gradually lowered in stages to a final pH of 5.0-6.5, and fermentation is carried out for 30-40 hours. S4. Post-processing: The fermentation product is inactivated and dried to obtain the final product.

[0007] Lactobacillus plantarum is Lactobacillus plantarum CGMCC No. 26622; Bifidobacterium longum is Bifidobacterium longum subspecies BB68S.

[0008] The ratio of viable Lactobacillus plantarum to Bifidobacterium longum is 1.5-3:1; the total amount of Lactobacillus plantarum and Bifidobacterium longum added accounts for 5-7% of the mass of the substrate.

[0009] The bacterial concentration of Lactobacillus plantarum was 1×10⁻⁶. 9 CFU / g-2×10 9 CFU / g.

[0010] The bacterial concentration of *Bifidobacterium longum* subsp. *longum* was 0.5 × 10⁻⁶. 9 CFU / g-1×10 9 CFU / g.

[0011] In step S1, the mass ratio of ginseng to polygonatum is (1:1)-(1:3), the particle size is 80-100 mesh, and the sterilization conditions are 110-125℃ for 15-40 min.

[0012] In the secondary fermentation step S3, the pH gradient is achieved by adding a pH adjuster every 6-10 hours, with a single pH reduction of 0.2-0.5.

[0013] The pH adjuster is a citrate-carbonate buffer system. The citrate-carbonate buffer is prepared as follows: citric acid and sodium carbonate are dissolved in water at a molar ratio of (1:0.8)-(1:1.2), the concentration is adjusted to 0.1-0.3 mol / L, and then sterilized.

[0014] The inactivation conditions in step S4 are 70-90℃ for 10-20 min.

[0015] <Second aspect> Fermented products prepared by the method described above are also within the scope of protection of this invention.

[0016] The application of the fermented product in the preparation of anti-fatigue functional foods is also within the scope of protection of this invention.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Through the synergistic fermentation of Lactobacillus plantarum CGMCC No.26622 and Bifidobacterium longum BB68S, combined with staged pH regulation, the bioconversion rate of ginsenoside CK was significantly improved, achieving efficient and targeted conversion of the target active ingredient.

[0018] 2. The fermentation product significantly prolonged the exhaustive swimming time of mice to 709.2±126.1 s, which was 36.8% higher than that of the control group (518.6±84.9 s).

[0019] 3. The staged solid-state fermentation mode of "primary fermentation (cell proliferation) + secondary fermentation (product synthesis)" is adopted, and the polysaccharide yield reaches 19.1% with dynamic pH control of the citrate-carbonate buffer system. Detailed Implementation

[0020] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0021] In this invention: Lactobacillus plantarum CGMCC No. 26622: Huai Zhifang, Fan Wenlei, Wang Binghan, Zhang Jing, Dai Lin, Li Zhixin, Lü Guangxiu, Qi Guofeng, Wang Qiaoli, Lai Xuexiang, Wang Baowei, Zhang Mingai. Effects of Lactobacillus plantarum metabiotics on serum antioxidant indicators, intestinal morphology and flora structure in broiler ducks and its inhibitory effect on harmful bacteria [J / OL]. Journal of Animal Nutrition. Bifidobacterium longum subsp. longum BB68S: Feng Haihong, Wang Ran, Ge Shaoyang, et al. Mechanism of cell damage in Bifidobacterium longum subsp. longum during vacuum freeze-drying [J]. Food and Fermentation Industries, 2025, 51 (13) :62 - 68. FENG Haihong, WANG Ran, GE Shaoyang, et al. Mechanism of cell damage of Bifidobacterium longum subsp. longum during freeze-drying process[ J] . Food and Fermentation Industries, 2025, 51(13) :62 - 68.

[0022] MRS medium: 10.0g tryptone, 10.0g beef extract, 5.0g yeast extract, 20.0g glucose, 2.0g sodium citrate, 2.0g K2HPO4, 0.2g MgSO4, 0.04g MnSO4, 1.0 mL Tween 80, dissolved in deionized water to a final volume of 1L, pH adjusted to 6.2±0.2, sterilized at 121℃ for 15min.

[0023] Bifidobacterium culture medium: 15.0g peptone, 2.5g yeast extract, 15g glucose, 1mL Tween 80, 2.0g K2HPO4, 0.5g MgCl2·6H2O, 0.15g CaCl2, 0.25g ZnSO4·7H2O, 0.01g FeCl3, 0.5g L-Cys·HCl, dissolved in deionized water and brought to a final volume of 1L. The pH was adjusted to 6.5 and sterilized at 121℃ for 15min.

[0024] Example 1 I. Raw Materials and Pretreatment Raw material ratio: 5 parts ginseng, 10 parts polygonatum; all are pulverized through an 80-mesh sieve and sterilized at 121℃ for 20 minutes.

[0025] Strain preparation: Lactobacillus plantarum CGMCC No. 26622: Inoculated into MRS liquid medium and cultured at 37℃ and 150 r / min on a shaker for 12-16 h to achieve a bacterial concentration of 1×10⁻⁶. 9 CFU / g.

[0026] Bifidobacterium longum subsp. longum BB68S: Inoculate with Bifidobacterium liquid culture medium at a 2% inoculum rate and anaerobically culture at 37°C (80% N2, 10% H2, 10% CO2) for 24-36 h until the bacterial concentration reaches 0.5 × 10⁻⁶. 9 CFU / g.

[0027] II. Inoculation and Fermentation Process Mixed inoculation: Mix the two bacterial solutions at a live count ratio of 2:1 (live count ratio of Lactobacillus plantarum CGMCC No. 26622: Bifidobacterium longum subsp. longum BB68S = 2:1), and inoculate (total bacterial solution added as 5% w / w of the matrix mass) into sterilized ginseng-Polygonatum powder, and stir evenly.

[0028] Staged solid-state fermentation: Primary fermentation: Fermentation at an initial pH of 6.5, 40℃, and 85% humidity for 12 hours to promote cell proliferation.

[0029] Secondary fermentation: Cool to 35℃ and humidity 75%, and adjust the pH to 0.3 (final pH 5.5-6.0) every 8 hours with citric acid / sodium carbonate buffer (citric acid and sodium carbonate dissolved in water at a molar ratio of 1:1, adjusted to a concentration of 0.2 mol / L, sterilized and ready for use). Continue fermentation for 36 hours.

[0030] III. Post-processing and testing Inactivation and drying: After fermentation, inactivate at 80℃ for 15 minutes and vacuum dry until the moisture content is ≤8%.

[0031] Conversion rate (%) =

[0032] In the formula ∆ n ck This represents the increase in the molar amount of rare saponin CK before and after fermentation. n 前体 It represents the total molar amount of the precursor saponins (Rb1, Rc, Rb2, Rd) before fermentation.

[0033] Test results: Ginsenoside CK content: 1.25 mg / g (conversion rate 68.3%, detected by HPLC); Polysaccharide yield: 19.1% (determined by phenol-sulfuric acid method); Residual live bacteria count: ≤1×10³ CFU / g (meets food safety standards).

[0034] Example 2 In this embodiment: raw materials include ginseng (4 parts) and polygonatum (9 parts); the remaining steps are the same as in embodiment 1. Test results: Ginsenoside CK content: 1.22 mg / g (conversion rate 66.5%); Polysaccharide yield: 18.7%; Residual viable bacteria count: ≤1×10 3 CFU / g.

[0035] Example 3 In this embodiment: the viable count ratio of *Lactobacillus plantarum* CGMCC No. 26622 to *Bifidobacterium longum* subsp. longum BB68S was 3:1. The remaining steps and conditions are the same as in Example 1.

[0036] Test results: Ginsenoside CK content: 1.14 mg / g (conversion rate 62.5%); Polysaccharide yield: 17.5%; Residual viable bacteria count: ≤1×10 3 CFU / g.

[0037] Comparative Example 1 The difference between this comparative example and Example 1 is that: it does not use staged solid-state fermentation, but uses constant temperature and humidity fermentation at 37°C, 80% humidity, and initial pH 6.5 for 48 hours.

[0038] result: Ginsenoside CK content: 0.77 mg / g (conversion rate 42.6%) Polysaccharide yield: 11.7%; Residual viable bacteria count: ≤1×10 3 CFU / g.

[0039] Comparative Example 2 The difference between this comparative example and Example 1 is that only a single strain of Lactobacillus plantarum CGMCC No. 26622 was used for fermentation, without the addition of Bifidobacterium longum subsp. longum BB68S.

[0040] Specifically as follows: I. Raw Materials and Pretreatment: Raw material ratio: 5 parts ginseng, 10 parts polygonatum; all are pulverized through an 80-mesh sieve and sterilized at 121℃ for 20 minutes.

[0041] Strain preparation: Lactobacillus plantarum CGMCC No. 26622: Inoculated into MRS liquid medium and cultured at 37℃ and 150 r / min on a shaker for 12-16 h to achieve a bacterial concentration of 1×10⁻⁶. 9 CFU / g.

[0042] II. Inoculation and Fermentation Process Mixed inoculation: Inoculate Lactobacillus plantarum CGMCC No.26622 into sterilized ginseng-Polygonatum powder, with an inoculation amount of (5% w / w of total bacterial solution added to the matrix) and mix well.

[0043] Staged solid-state fermentation: Same as Example 1.

[0044] Other conditions (inoculation amount, fermentation process, etc.) are the same as in Example 1.

[0045] Test results: Ginsenoside CK content: 0.68 mg / g (conversion rate 37.2%, detected by HPLC); Polysaccharide yield: 10.2% (determined by phenol-sulfuric acid method); Residual viable bacteria count: 5 × 10 4 CFU / g; Comparative Example 3 The difference between this comparative example and the previous example is that Lactobacillus plantarum CGMCC No. 26622 was replaced with the commercial standard strain Lactobacillus plantarum ATCC 8014 (Thermo Fisher Scientific REMEL), while all other conditions were exactly the same as in Example 1.

[0046] Test results: Ginsenoside CK content: 0.64 mg / g (conversion rate 35.3%, detected by HPLC); Polysaccharide yield: 9.8% (determined by phenol-sulfuric acid method); Residual viable bacteria count: 5 × 10 4 CFU / g.

[0047] Comparative Example 4 The difference between this comparative example and the embodiment is that Bifidobacterium longum subsp. BB68S is replaced with Saccharomyces cerevisiae, numbered BNCC 336054, sourced from Beina Chuanglian Biotechnology Co., Ltd., and the rest is the same as in embodiment 1.

[0048] Test results: Ginsenoside CK content: 0.95 mg / g (conversion rate 52.8%, detected by HPLC); Polysaccharide yield: 14.5% (determined by phenol-sulfuric acid method); Residual viable bacteria count: 5 × 10 4 CFU / g.

[0049] Comparative Example 5 The difference between this comparative example and the embodiment is that *Bifidobacterium longum subsp. BB68S* is replaced with *Bifidobacterium pseudomicrobium*. (Bifidobacterium pseudocatenulatum) with the number BMZ014603 is from Ningbo Mingzhou Biotechnology Co., Ltd.

[0050] Test results: Ginsenoside CK content: 0.90 mg / g (conversion rate 49.1%, detected by HPLC); Polysaccharide yield: 13.7% (determined by phenol-sulfuric acid method); Residual viable bacteria count: 5 × 10 4 CFU / g.

[0051] Comparative Example 6 The difference between this comparative example and the specific example is that the viable count ratio of *Lactobacillus plantarum* CGMCC No. 26622 to *Bifidobacterium longum* subsp. longum BB68S is 1:2.

[0052] Test results: Ginsenoside CK content: 1.04 mg / g (conversion rate 57.2%, detected by HPLC); Polysaccharide yield: 15.9% (determined by phenol-sulfuric acid method); Residual viable bacteria count: 5 × 10 4 CFU / g.

[0053] Performance testing: Animal fatigue resistance experiments Laboratory animals: 21 C animals aged 6-8 weeks 57 BL / 6J mice (18 ± 2 g) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. They were fed SPF environmental acclimatization diet for 7 days. Starting on day 8, they were administered the drug via gavage at a dose of 200 mg / kg / day, once daily. On day 14, 30 minutes after gavage, a swimming test was conducted to exhaustion. Each mouse had a lead bar (5% of its body weight) secured to its tail, and was placed in a swimming tank with a water depth of 30 cm and a water temperature controlled at (30 ± 2) ℃. Swimming time was recorded until the mouse could no longer surface through its nostrils within 10 seconds; this time was considered the exhaustion swimming time. The experimental results are shown in Table 1.

[0054] Table 1. Swimming time to exhaustion

[0055] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing an anti-fatigue ferment, characterized in that, Includes the following steps: S1. Raw material pretreatment: Ginseng and Polygonatum are mixed in proportion, crushed and sterilized to obtain the matrix; S2. Inoculation: Inoculate Lactobacillus plantarum and Bifidobacterium longum into the substrate; S3, staged solid-state fermentation, includes the following steps: Primary fermentation: Ferment for 10-14 hours at pH 6.0-7.0, 38-42℃, and 80-90% humidity; Secondary fermentation: Under an environment of 32-37℃ and 70-80% humidity, the pH is gradually lowered in stages to a final pH of 5.0-6.5, and fermentation is carried out for 30-40 hours. S4. Post-processing: The fermentation product is inactivated and dried to obtain the final product.

2. The preparation method according to claim 1, characterized in that, Lactobacillus plantarum is Lactobacillus plantarum CGMCC No. 26622; Bifidobacterium longum is Bifidobacterium longum subspecies BB68S.

3. The preparation method according to claim 1, characterized in that, The ratio of viable Lactobacillus plantarum to Bifidobacterium longum is 1.5-3:1; inoculate the substrate at an inoculum size of 3-7% of the substrate mass.

4. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of ginseng to polygonatum is 1:1 to 1:3, the particle size is 80-100 mesh, and the sterilization conditions are 110-125℃ for 15-40 min.

5. The preparation method according to claim 1, characterized in that, In the secondary fermentation step S3, the pH gradient is achieved by adding a pH adjuster every 6-10 hours, with a single pH reduction of 0.2-0.

5.

6. The preparation method according to claim 5, characterized in that, The pH adjuster is a citrate-carbonate buffer system.

7. The preparation method according to claim 1, characterized in that: The inactivation conditions in step S4 are 70-90℃ for 10-20 min.

8. A fermentation product prepared by any one of claims 1-7.

9. The use of the fermentation product as described in claim 8 in the preparation of anti-fatigue functional foods.

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