A method for producing 1-deoxynojirimycin by fermentation using a soy product by-product and application of a compounded composition

Abstract

The application discloses a method for producing 1-deoxynojirimycin (DNJ) by fermentation of soy product by-products and application of a compound composition. The method uses soybean meal, soybean dregs or yellow pulp water as a fermentation substrate, and uses Bacillus amyloliquefaciens YP2 to perform liquid fermentation to obtain a fermentation liquor rich in DNJ (850-1000 mg / L); then DNJ is extracted from the fermentation liquor, and is compounded with extracellular polysaccharide produced by fermentation of Bacillus amyloliquefaciens YP2 according to a mass ratio of 1: (1000-5000) to prepare a compound composition. The compound composition has a significant synergistic inhibitory effect on alpha-glucosidase. The application realizes comprehensive utilization of soy product processing by-products, and the obtained compound composition can be used for preparing functional food or health products.

Description

Technical Field

[0001] This invention belongs to the fields of microbial fermentation, high-value utilization of agricultural by-products, biomanufacturing and functional food technology, specifically relating to a method for producing 1-deoxynojirimycin by liquid fermentation of soybean by-products and the application of the compound composition. Background Technology

[0002] 1-Deoxynojirimycin (DNJ), a potent α-glucosidase inhibitor, has broad application prospects in hypoglycemic, antiviral, and antitumor fields. Currently, the sources of DNJ are very limited, mainly extracted from mulberry leaves, and its yield is affected by the season. Microbial fermentation, on the other hand, is simple to operate and widely available. However, the yield of DNJ from wild-type strains is generally low, making it difficult to meet industrial demands. For example, the existing technology discloses Bacillus amyloliquefaciens (BAM)... Bacillus amyloliquefaciens Although YP2 has been proven to produce DNJ, its initial yield in conventional culture media is only 93 mg / L (see Chinese Patent CN118064308B), far below the economic threshold for industrial production. Furthermore, existing high-yield fermentation processes largely rely on expensive raw materials such as peptone and yeast extract, leading to high production costs and complex fermentation broth composition, significantly increasing the difficulty of downstream separation and purification. Therefore, developing a low-cost, high-yield, and stable fermentation method is a key technological bottleneck that urgently needs to be addressed in this field.

[0003] Soybean meal, soybean residue, and sour whey are the main byproducts of soybean product processing. my country produces a huge amount of these byproducts annually, which are primarily used as low-value feed or discarded directly, causing serious resource waste and environmental pollution. Although these byproducts are rich in protein, peptides, and dietary fiber, and theoretically excellent substrates for microbial fermentation, their practical application faces significant challenges: soybean residue and sour whey have complex compositions, large batch-to-batch variations, and contain anti-nutritional factors such as trypsin inhibitors. Directly using them as fermentation substrates often leads to inhibited cell growth, low and unstable product conversion rates. How to utilize soybean processing byproducts to replace expensive nitrogen sources and overcome the interference of anti-nutritional factors through process optimization to achieve efficient biosynthesis of DNJ is an important direction for realizing the high-value utilization of soybean byproducts and aligning with the national strategies of "loss reduction and efficiency improvement" and "circular economy."

[0004] On the other hand, microbial extracellular polysaccharides (EPS) possess various biological activities such as antioxidant and immunomodulatory effects. In the development of functional foods, single active ingredients often suffer from low bioavailability or significant side effects, making compounding for synergistic effects a trend. However, there are no reports in the existing technology of combining DNJ obtained from homologous fermentation with extracellular polysaccharides of specific structures to produce a strong synergistic effect. In our research, we discovered that Bacillus amyloliquefaciens YP2 fermentation under specific conditions not only produces high yields of DNJ but also generates a specific extracellular polysaccharide, B-EPS-1. Surprisingly, when DNJ and B-EPS-1 are combined in a specific ratio, they exhibit a very strong synergistic inhibitory effect on α-glucosidase (combination index CI = 0.33), and their inhibitory activity is significantly better than that of either ingredient alone or in combination with other polysaccharides. This discovery not only provides a new strategy for enhancing the efficacy and reducing the dosage of DNJ but also opens up new avenues for the high-value utilization of all components of soybean by-products. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for high-yield 1-deoxynojirimycin (DNJ) through liquid fermentation of soybean product by-products, as well as a composition of DNJ and extracellular polysaccharides and its applications. This invention aims to solve the problems of high cost and low yield in existing DNJ fermentation methods, while simultaneously achieving high-value utilization of soybean product processing by-products.

[0006] In a first aspect, the present invention provides a method for producing 1-deoxynojirimycin using a soybean product substrate through liquid fermentation, comprising the following steps: (1) Activation of strain: Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Bacillus amyloliquefaciens YP2 was inoculated into LB liquid medium and cultured with shaking at 35–37 °C and 180–220 rpm for 12–16 h to obtain seed culture; (2) Preparation of fermentation medium: Using soybean product substrate as substrate, prepare fermentation medium and adjust the initial pH to 5.5-7.5; the fermentation medium includes 15-30 g / L soybean meal, 15-30 g / L lactose and inorganic salts; (3) Liquid fermentation: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 1% to 5%, and ferment for 48 to 108 h at a fermentation temperature of 30 to 40℃ and a shaking speed of 120 to 220 rpm; (4) Process optimization: The fermentation medium composition and fermentation conditions were optimized by single-factor and response surface methodology to obtain the optimal fermentation process; (5) Application of by-products: The optimized fermentation process in step (4) is directly applied to the liquid fermentation of soybean residue or yellow slurry to obtain a fermentation broth rich in DNJ.

[0007] (6) Component and activity determination: The fermentation broth of soybean product substrate prepared in steps (1)-(5) was used to determine the nattokinase activity, total phenol content, α-glucosidase inhibitory activity, DPPH free radical scavenging ability, ABTS free radical scavenging ability and hydroxyl free radical scavenging ability.

[0008] In a second aspect, the present invention provides a composition of 1-deoxynojirimycin and extracellular polysaccharide and a method for preparing the same, comprising the following steps: (1) Obtaining DNJ components: Take the supernatant of the fermentation broth, extract and separate it to obtain crude DNJ extract or purified DNJ; (2) Obtaining extracellular polysaccharides: The supernatant of the broth fermentation broth of Bacillus amyloliquefaciens YP2 was taken and purified to obtain the extracellular polysaccharide B-EPS-1; (3) Preparation of compound: The DNJ component and the extracellular polysaccharide B-EPS-1 are mixed at a mass ratio of 1:1000 to 1:5000, dissolved in water or buffer solution, stirred evenly and dried to obtain a powder composition.

[0009] Thirdly, the present invention provides the application of the above-mentioned fermentation broth, DNJ and extracellular polysaccharide composition in the preparation of functional foods, health products or dietary supplements with α-glucosidase inhibitory activity.

[0010] Beneficial effects 1. Significantly increases the yield of the target product DNJ and greatly reduces production costs. This invention achieves a DNJ yield of 850–1000 mg / L through response surface methodology, a significant increase compared to the original strain (93 mg / L); and by replacing expensive conventional nitrogen sources with inexpensive byproducts such as soybean meal, it significantly reduces industrial production costs.

[0011] 2. Expanding the sources of fermentation raw materials and realizing the green and high-value utilization of by-products. The fermentation method of this invention has good substrate universality and can be directly applied to the fermentation of soybean residue, yellow whey, and other by-products of soybean product processing. After fermentation, the DNJ yield reached 51 mg / L and 214 mg / L, respectively, while significantly increasing nattokinase activity and total phenol content. This method provides a new approach for the resource utilization of soybean product processing waste, with significant economic benefits and environmental value.

[0012] 3. The fermentation broth is endowed with multiple biological activities, expanding its downstream application prospects. The fermentation broth prepared by the method of this invention exhibits excellent α-glucosidase inhibitory activity, as well as good DPPH, ABTS, and hydroxyl radical scavenging abilities. This fermentation broth has both auxiliary hypoglycemic and antioxidant effects, and can be directly used as an active ingredient in the development of functional foods, dietary supplements, and antioxidant cosmetics.

[0013] 4. It exhibits a strong synergistic effect, providing a new strategy for developing highly efficient and low-toxicity products. This invention is the first to discover and verify the strong synergistic effect (synergistic index CI = 0.33) in inhibiting α-glucosidase when combined with homologous DNJ and extracellular polysaccharide (B-EPS-1). Under this combined system, the half-maximal inhibitory concentration (IC50) of B-EPS-1 is... 50 The blood sugar level was significantly reduced by 67%, and its inhibitory effect was significantly better than that of using it alone or in combination with other conventional ingredients. This discovery breaks through the limitations of traditional single blood sugar-lowering ingredients and provides an important theoretical basis and material foundation for the development of new blood sugar-lowering products that are highly effective and low in toxicity.

[0014] 5. High-value utilization across the entire chain: This invention not only achieves efficient biosynthesis of DNJ, but also utilizes polysaccharide components in the fermentation system through compounding technology, perfectly meeting the national strategic needs of "circular economy" and "loss reduction and efficiency improvement" of agricultural products. Attached Figure Description

[0015] Appendix Figure 1 This invention provides a comparison of the DNJ content in soybean meal, soybean residue, and yellow fermentation liquid. Appendix Figure 2 This invention provides a comparison of nattokinase activity in soybean meal, soybean residue, and yellow fermentation liquid. Appendix Figure 3 This invention provides a comparison of the total phenol content in soybean meal, soybean residue, and yellow slurry fermentation products. Appendix Figure 4 The α-glucosidase inhibitory activity and antioxidant activity of soybean meal, soybean residue and yellow whey after fermentation in this invention are (A: α-glucosidase inhibition rate; B: DPPH scavenging rate; C: ABTS scavenging rate; D: hydroxyl radical scavenging rate).

[0016] Appendix Figure 5 Synergistic ability analysis of the compound composition in this invention (A: Fa-dose effect diagram; B: synergistic polygon diagram). Detailed Implementation

[0017] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention is not limited to the following embodiments.

[0018] Example 1: Preparation of a fermentation medium for high-yield DNJ based on soybean meal liquid fermentation Bacillus amyloliquefaciens YP2 (disclosed in Chinese patent CN118064308B) was inoculated at a 2% inoculum into a basal fermentation medium (20 g / L soybean meal, 20 g / L lactose, pH adjusted to 5.0–7.0). Fermentation was carried out at 37 °C for 96 h with a shaking speed of 180 r / min. The DNJ content was determined by FMOC-Cl derivatization HPLC method, and the yield under basal conditions was 123 mg / L.

[0019] Based on the basic fermentation medium, the effects of soybean meal and lactose addition, as well as the types and amounts of inorganic salts, on DNJ yield were investigated through single-factor experiments. Finally, it was determined that the combination of inorganic salts such as ferrous sulfate, ferrous sulfate, tripotassium phosphate, potassium dihydrogen phosphate, trisodium phosphate, manganese sulfate, and dipotassium hydrogen phosphate can improve DNJ yield. Three factors with the most significant impact on DNJ yield (lactose concentration, dipotassium hydrogen phosphate concentration, and manganese sulfate concentration) were selected. A Box-Behnken design was used for response surface methodology optimization, and a quadratic regression model was established to predict and validate the optimal fermentation medium composition when soybean meal was used as the substrate. The optimal composition was: soybean meal 18–22 g / L, lactose 22–24 g / L, manganese sulfate 0.09–0.11 g / L, dipotassium hydrogen phosphate 0.07–0.09 g / L, ferric sulfate 0.07–0.08 g / L, ferrous sulfate 0.02–0.03 g / L, tripotassium phosphate 0.02–0.03 g / L, potassium dihydrogen phosphate 0.04–0.06 g / L, trisodium phosphate 0.02–0.03 g / L, with an initial pH of 6.0–6.5. Under these conditions, the DNJ content was 417 mg / L–444 mg / L.

[0020] Example 2: A fermentation method for high-yield DNJ based on soybean meal liquid fermentation Under the optimal fermentation medium conditions confirmed in Example 1, the fermentation conditions were further optimized. The effects of different fermentation conditions (pH, temperature, inoculum size, liquid volume, shaking speed, and fermentation time) on DNJ production by Bacillus amyloliquefaciens YP2 were investigated. The three factors with the most significant impact on DNJ yield (pH, liquid volume, and inoculum size) were selected. A Box-Behnken design was used for response surface methodology optimization, and a quadratic regression model was established to predict and confirm the fermentation conditions as follows: inoculum size 2.5%–3.5% (v / v), fermentation temperature 34–37℃, liquid volume 45–55 mL / 250 mL Erlenmeyer flask, shaking speed 180–210 rpm, and fermentation time 72–96 h. Under these conditions, the DNJ yield reached 850–1000 mg / L, which is one of the highest levels of DNJ production reported from soybean meal fermentation.

[0021] Example 3: Application of a high-yield DNJ method based on soybean meal liquid fermentation in soybean milk processing byproducts The fermentation medium and fermentation conditions confirmed in Examples 1 and 2 were applied to the fermentation of soybean residue, a by-product of soybean milk processing. The difference was that the soybean meal in Example 1 was replaced with soybean residue, and the soybean residue fermentation liquid was obtained by fermenting under the optimal fermentation conditions of Example 2.

[0022] Example 4: Application of a high-yield DNJ method based on soybean meal liquid fermentation in yellow whey, a byproduct of tofu processing. The fermentation medium and fermentation conditions confirmed in Examples 1 and 2 were applied to the fermentation of yellow slurry, a byproduct of tofu processing. The difference was that the 18-22 g / L soybean meal in Example 1 was removed, and the 45-55 mL pure water in Example 2 was replaced with an equal amount of yellow slurry. The yellow slurry fermentation liquid was obtained by fermentation under the optimal fermentation conditions of Example 2.

[0023] Example 5: Preparation and synergistic activity evaluation of the DNJ and B-EPS-1 compound composition. The supernatant from soybean meal fermentation was precipitated with ethanol and concentrated by rotary evaporation, then freeze-dried to obtain crude DNJ extract, with a DNJ content of 5%–15%; or purified by cation exchange resin, macroporous resin, anion exchange resin, or silica gel column chromatography, concentrated, and freeze-dried to obtain purified DNJ (purity ≥90%). The supernatant from meat broth fermentation was purified by ethanol precipitation, trichloroacetic acid precipitation, and DEAE 52 cellulose column chromatography, eluted with pure water, and freeze-dried to obtain purified B-EPS-1. The DNJ component and B-EPS-1 were further dissolved separately in appropriate amounts of pure water or phosphate buffer (50 mM, pH 6.8), and compounded at a DNJ to B-EPS-1 mass ratio (actual mass of DNJ: mass of B-EPS-1) of 1:1000–1:5000. After stirring evenly, the mixture was dried to obtain a powdered composition.

[0024] Comparative Example 1: Preparation of Unoptimized Soybean Meal Fermentation Broth Unoptimized soybean meal fermentation broth was prepared according to the basic fermentation medium and fermentation conditions in Example 1.

[0025] Comparative Example 2: Preparation of Unoptimized Soybean Residue Fermentation Broth Unoptimized soybean residue fermentation broth was prepared according to the basic fermentation culture medium and fermentation conditions in Example 1, except that the 18-22 g / L soybean meal in Example 1 was replaced with 18-22 g / L soybean residue.

[0026] Comparative Example 3: Preparation of Unoptimized Yellow Slurry Fermentation Broth Unoptimized yellow slurry fermentation broth was prepared according to the basic fermentation medium and fermentation conditions in Example 1, the difference being that the 18-22 g / L soybean meal in Example 1 was removed and pure water was replaced with yellow slurry.

[0027] Test Example 1: Component Determination Soybean meal fermentation broth, soybean residue fermentation broth, and yellow whey fermentation broth were obtained using the fermentation methods of Examples 1-4 and Comparative Examples 1-3. The DNJ content, nattokinase activity, and total phenol content in the fermentation broth were determined, and the results are shown in the appendix. Figure 1 As shown in the figure, under the optimized fermentation method, the DNJ content in soybean meal fermentation broth, soybean residue fermentation broth, and yellow slurry fermentation broth were 910 mg / L, 51 mg / L, and 214.3 mg / L, respectively, which were 7.4 times, 2.5 times, and 2.2 times higher than those in the unoptimized soybean meal fermentation broth, soybean residue fermentation broth, and yellow slurry fermentation broth, respectively. The above experiments demonstrate that the method for high-yield DNJ based on liquid fermentation of soybean meal provided by this invention can not only utilize soybean meal but also synthesize DNJ from other soybean processing byproducts such as yellow slurry and soybean residue. When soybean meal is used as raw material, the raw material cost per ton of fermentation broth is approximately 425 yuan, which translates to a raw material cost of approximately 0.5 yuan / g for DNJ in the fermentation broth, far lower than that of mulberry leaf extraction (approximately 200 yuan / g) and chemical synthesis (approximately 50 yuan / g).

[0028] In addition, Figure 2 and attached Figure 3 The results showed that the total phenol content and nattokinase activity increased to varying degrees. Nattokinase activity increased by 2.4 times, 2.4 times, and 1.6 times, respectively, with the soybean meal fermentation broth showing the strongest nattokinase activity, followed by the yellow whey fermentation broth. The total phenol content increased by 3.2 times, 4.3 times, and 2.27 times, respectively, with the yellow whey fermentation broth showing the highest total phenol content, followed by the soybean meal fermentation broth. These experiments demonstrate that the method for high-yield DNJ based on soybean meal liquid fermentation provided by this invention can not only increase the DNJ content but also improve the total phenol content and nattokinase activity.

[0029] Test Example 2: Activity Assay Soybean meal fermentation broth, soybean residue fermentation broth, and yellow whey fermentation broth were obtained using the fermentation methods of Examples 1-4 and Comparative Examples 1-3. The α-glucosidase inhibitory activity and antioxidant activity of the fermentation broths were determined. (See attached...) Figure 4 As shown in Figure A, the α-glucosidase inhibitory activity assay revealed that even after diluting the fermentation broth 100-fold, the optimized broth still exhibited strong inhibitory activity. (See attached figure.) Figure 4 B~Attachment Figure 4 D represents the DPPH radical scavenging capacity, ABTS radical scavenging capacity, and hydroxyl radical scavenging capacity of the three fermentation broths before and after optimization. The antioxidant activities of the optimized soybean meal fermentation broth, soybean residue fermentation broth, and yellow pulp fermentation broth were all improved to varying degrees. Even after diluting the fermentation broth 8 times, it still had more than 50% scavenging capacity, and all of them were significantly higher than the unoptimized control. In particular, the scavenging rate of hydroxyl radicals of the optimized soybean meal fermentation broth was 68%, which was 2.2 times higher than that of the unoptimized broth.

[0030] Test Example 3: Evaluation of the synergistic activity of the DNJ and B-EPS-1 compound composition The inhibitory rate of the compound composition prepared in Example 5 against α-glucosidase was determined, and the IC50 of the compound composition was calculated. 50 The values ​​were calculated, and the dose-effect ratio and combination index (CI) were calculated using CompuSyn software. (See attached...) Figure 5 As shown in the Fa-dose-response curve of A, the dosage of the compound composition at different inhibition rates was significantly lower than that of the single composition. When DNJ and B-EPS-1 were compounded at a ratio of 1:4000, the IC50 value was significantly lower. 50 Compared to B-EPS-1 alone, it reduces costs by 67% (Table 1); Appendix Figure 5 The cooperative polygon diagram of B further visually shows that there is a strong cooperative relationship between DNJ and B-EPS-1 (CI=0.33). Other ratios also show cooperative relationships, but the CI values ​​are all higher than 0.33.

[0031] Table 1 IC of DNJ and B-EPS-1 at different mixing ratios 50 Value and CI value In summary, the method for high-yield DNJ based on liquid fermentation of soybean meal provided by this invention can also be applied to the fermentation of soybean residue and yellow liquid, byproducts of other soybean processing. While achieving high DNJ yield, it also promotes the synthesis of nattokinase and phenolic substances. The resulting fermented soybean meal, soybean residue, and yellow liquid exhibit α-glucosidase inhibitory activity and antioxidant capacity. The application examples provided by this invention demonstrate that the method for high-yield DNJ and the compound composition of DNJ and extracellular polysaccharide B-EPS-1 can greenly and efficiently solve the problem of resource waste from soybean processing byproducts, providing added value and producing functional factors with multiple functional activities. These factors can be used as components of dietary supplements or as functional factors in functional products (such as functional foods, cosmetics, and pharmaceuticals), playing a role in regulating blood sugar and lipids and delaying aging, showing good application prospects and economic benefits.

[0032] The preferred embodiments of the present invention have been described above, but the present invention is not limited to the specific embodiments described above, and the present invention can be applied in various ways. Any modifications, substitutions, and improvements made within the spirit and principles of the present invention are within the protection scope of the technical solutions in this aspect.

Claims

1. A method for producing 1-deoxynojirimycin using a soybean product substrate through liquid fermentation, characterized in that, Includes the following steps: Using soybean product substrate as the base, a fermentation medium was prepared, and the initial pH was adjusted to 5.5–7.5; Bacillus amyloliquefaciens (BAM) was inoculated. Bacillus amyloliquefaciens YP2 was fermented for 48–108 h at a fermentation temperature of 30–40℃, an inoculum size of 1%–5%, a liquid volume of 30–90 mL / 250 mL Erlenmeyer flask, and a shaking speed of 120–220 rpm. The fermentation medium includes soy products, lactose, and inorganic salts; wherein the content of soy products is 15-30 g / L, the content of lactose is 15-30 g / L, and the inorganic salts include ferric sulfate, ferrous sulfate, tripotassium phosphate, potassium dihydrogen phosphate, trisodium phosphate, manganese sulfate, and dipotassium hydrogen phosphate.

2. The method according to claim 1, characterized in that, The fermentation medium consists of: soybean meal 18–22 g / L, lactose 22–24 g / L, manganese sulfate 0.09–0.11 g / L, dipotassium hydrogen phosphate 0.07–0.09 g / L, ferric sulfate 0.07–0.08 g / L, ferrous sulfate 0.02–0.03 g / L, tripotassium phosphate 0.02–0.03 g / L, potassium dihydrogen phosphate 0.04–0.06 g / L, trisodium phosphate 0.02–0.03 g / L, and an initial pH of 6.0–6.

5.

3. The method according to claim 1, characterized in that, The fermentation conditions were as follows: inoculum size 2.5%–3.5% (v / v), fermentation temperature 34–37°C, liquid volume 45–55 mL / 250 mL Erlenmeyer flask, shaking speed 180–210 rpm, and fermentation time 72–96 h.

4. The method according to claim 1, characterized in that, The soybean product substrate is at least one of soybean meal, soybean residue, or yellow liquid.

5. A fermentation broth rich in 1-deoxynojirimycin, characterized in that, Prepared by the method described in any one of claims 1 to 4.

6. A composition of 1-deoxynojirimycin and extracellular polysaccharide, characterized in that, It comprises 1-deoxynojirimycin or its crude extract, and the extracellular polysaccharide B-EPS-1; the mass ratio of 1-deoxynojirimycin or its crude extract to the extracellular polysaccharide B-EPS-1 is 1:1000 to 1:5000; the 1-deoxynojirimycin or its crude extract is prepared by the method according to any one of claims 1 to 4, and the extracellular polysaccharide B-EPS-1 is prepared from the broth fermentation broth of Bacillus amyloliquefaciens YP2.

7. The composition according to claim 6, characterized in that, The mass ratio of 1-deoxynojirimycin or its crude extract to the extracellular polysaccharide B-EPS-1 is 1:4000.

8. A method for preparing the composition according to claim 6 or 7, characterized in that, include: 1-Deoxynojirimycin or its crude extract was mixed with extracellular polysaccharide B-EPS-1 at the stated mass ratio, dissolved in water or buffer solution, stirred evenly, and then dried to obtain a powdered composition.

9. The use of the composition according to claim 6 or 7 in the preparation of functional foods, health products or dietary supplements having α-glucosidase inhibitory activity.

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