Preparation method of quinoa fruit and vegetable composite powder and application thereof

Abstract

The application provides a preparation method and application of quinoa fruit and vegetable compound powder, and the quinoa fruit and vegetable compound powder comprises the following components in parts by weight: 30-50 parts of quinoa, 20-30 parts of yam powder, 10-15 parts of inulin, and 50-80 parts of prune juice. The preparation method comprises the following steps: adding prune juice to sprouted quinoa to beat pulp, then adding yam powder, carrying out alpha-amylase enzymolysis, and then killing the enzyme; after centrifugation, the supernatant is taken; after adding inulin and uniformly mixing, a fermentation base material is prepared; Fe 2+ is added to the fermentation base material, and then plant lactobacillus and bacillus subtilis are inoculated to carry out anaerobic fermentation; Fe 3+ is further added to continue anaerobic fermentation; after sterilization and centrifugation, the supernatant is collected; and the supernatant is subjected to vacuum freeze-drying to obtain the quinoa fruit and vegetable compound powder. The quinoa fruit and vegetable compound powder has the characteristics of significantly increased contents of active ingredients such as glutamic acid, GABA and total flavones, low GI, high content of dietary fiber, and functions of relieving constipation and reducing blood lipids.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a method for preparing quinoa fruit and vegetable compound powder and its application. Background Technology

[0002] With changes in lifestyle and dietary structure, metabolic diseases such as hyperlipidemia and hyperglycemia have become major threats to global public health. Dietary intervention is considered a core strategy for the prevention and control of chronic diseases. Low-GI (glycemic index) diets have been proven to effectively control postprandial blood glucose, improve insulin resistance, and reduce the risk of cardiovascular disease. Therefore, the development of low-GI foods with clearly defined functions and nutritional balance has become a research hotspot in the food processing field. Compound meal replacement powders of whole grains and fruits and vegetables are considered ideal carriers for weight management and metabolic health intervention because they contain dietary fiber, vitamins, minerals, and various phytochemicals. However, the active ingredients such as dietary fiber, polyphenols, and flavonoids in natural food raw materials often exist in bound or macromolecular forms. Traditional processing techniques such as drying and pulverizing result in low bioavailability and functional impairment, limiting the normal exertion of the raw material's biological efficacy. However, existing related products mostly involve simple pretreatment and physical mixing of raw materials, failing to achieve flavor fusion and synergistic effects of nutritional functions between components, resulting in a rough texture, poor palatability, and difficulty in preparation. This fails to meet consumers' sensory needs and cannot fully satisfy the demand for low-GI, high-fiber foods from people with metabolic abnormalities and obesity. Therefore, optimizing raw material formulations and improving processing techniques to simultaneously enhance the sensory quality and nutritional function of products has become a pressing technical challenge for the industry.

[0003] Probiotic fermentation is an effective technology for improving the nutritional quality and bioactivity of plant-based raw materials. Lactic acid bacteria such as *Lactobacillus plantarum* can produce enzyme systems such as cellulase and hemicellulase during fermentation, which enzymatically hydrolyze plant cell walls, promoting the efficient release of active ingredients and converting large molecules in the raw materials into more easily absorbed small molecule metabolites. This may also generate new functional compounds, thereby improving the overall bioactivity and utilization efficiency of the raw materials. *Bacillus subtilis* can utilize its metabolically produced proteases to enzymatically break down proteins in raw materials into peptides, amino acids, and other substances, improving the digestibility and absorption of the raw materials while significantly enhancing the taste and flavor of the final product. However, existing compound microbial fermentation technologies still have the following technical shortcomings:

[0004] First, the metabolic sequence of the strains is difficult to coordinate. In the compound microbial agent fermentation system, the two strains compete for carbon sources and nutrients, and their growth rhythms differ when inoculated simultaneously, resulting in unstable metabolite production.

[0005] Second, existing fermentation control methods lack precise control over the metabolic sequence of strains. Current technologies mostly optimize fermentation by adjusting parameters such as fermentation temperature, time, and inoculation ratio. However, these methods are all static controls and cannot dynamically intervene in the metabolic activities of different strains in stages and selectively during the fermentation process. Therefore, they are insufficient to effectively solve the fundamental problem of asynchronous metabolism among strains in complex systems.

[0006] Therefore, this invention aims to solve the core technical problems in traditional compound microbial agent fermentation, such as disordered metabolic sequence of strains, insufficient release of active ingredients, and poor sensory quality of products, through innovative fermentation processes and formulation design. Summary of the Invention

[0007] Technical Problem to be Solved: This invention addresses the technical problems of uncoordinated metabolic sequences between Bacillus subtilis and Lactobacillus plantarum, resulting in low enrichment efficiency of target functional components in existing compound microbial fermentation technology for fruit and vegetable compound powders. It provides a method for preparing quinoa fruit and vegetable compound powder and its application. By adjusting the iron ion content at different stages of fermentation, the metabolic activities of different strains are activated, significantly improving the enrichment efficiency of target functional components. This effectively solves the metabolic disorder problem caused by strain competition and asynchronous growth in traditional compound microbial fermentation systems, significantly increasing the content of functional components in the fruit and vegetable compound powder. Simultaneously, this invention, supplemented by quinoa germination pretreatment and scientific raw material formulation, combined with prune and compound microbial fermentation, synergistically improves the quality and flavor of the product, significantly increasing the content of functional components such as polyphenols and flavonoids, and enhancing the antioxidant, hypoglycemic, and intestinal function regulating health benefits of low-GI, high-fiber products.

[0008] Technical solution: This invention provides a method for preparing quinoa fruit and vegetable compound powder, comprising the following steps:

[0009] S1. 30-50 portions were subjected to germination treatment to obtain germinated quinoa;

[0010] S2. Sprouted quinoa and 50-80 parts of prune juice are mixed and pulped. The pulp is mixed with 20-30 parts of yam powder, and then α-amylase is added. After enzymatic hydrolysis, the enzyme is inactivated and cooled. The enzymatic hydrolysate is centrifuged and the supernatant is collected. 10-15 parts of inulin are added and mixed evenly to obtain the fermentation substrate.

[0011] S3. After activating Lactobacillus plantarum and Bacillus subtilis, they were inoculated into MRS liquid medium and cultured. After centrifugation, the precipitates were collected, resuspended, and Lactobacillus plantarum suspension and Bacillus subtilis suspension were obtained. The two bacterial suspensions were mixed to obtain a compound bacterial agent.

[0012] S4. Add ferrous ions to the fermentation substrate, inoculate with compound microbial agents for anaerobic fermentation, then add ferric ions to continue anaerobic fermentation. After fermentation, sterilize, collect the supernatant after centrifugation, and freeze-dry the supernatant under vacuum to obtain quinoa fruit and vegetable compound powder.

[0013] Preferably, the amount of amylase added in step S2 is 0.1-0.3% of the total mass of sprouted quinoa, prune juice and yam powder.

[0014] Preferably, in step S3, *Lactobacillus plantarum* is strain BRX-1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38511, and deposited on April 29, 2026; and *Bacillus subtilis* is strain LX-W3, deposited at the same center with accession number CGMCC No. 27046, and deposited on April 10, 2023.

[0015] Preferably, the viable count of the *Lactobacillus plantarum* suspension or *Bacillus subtilis* suspension in step S3 is 1 × 10⁻⁶. 7 ~1×10 8 CFU / mL; the volume ratio of Lactobacillus plantarum suspension to Bacillus subtilis suspension is 1~2:1~2.

[0016] Preferably, the ferrous ions in step S4 are FeSO4, and the final concentration of ferrous ions is 0.2~1 mmol / L.

[0017] Preferably, the ferric ions in step S4 are ferric ammonium citrate, and the final concentration of ferric ions is 0.8~2 mmol / L.

[0018] Preferably, in step S4, the anaerobic fermentation time after adding ferrous ions is 8-12 h, and the anaerobic fermentation time after adding ferric ions is 4-10 h.

[0019] This invention provides a quinoa fruit and vegetable compound powder prepared by the above preparation method.

[0020] This invention provides the application of the above-mentioned quinoa fruit and vegetable compound powder in the preparation of foods, medicines or health products that have antioxidant properties, improve blood lipid health or improve gut health.

[0021] Preferably, the dosage form of the antioxidant, lipid-improving, or gut-health-improving food, medicine, or health product is powder, tablet, capsule, or granule.

[0022] Beneficial effects:

[0023] 1. This invention achieves temporal synergy of bacterial metabolism in a compound microbial agent fermentation system. Addressing the problem of low efficiency in the enrichment of functional components by Bacillus subtilis LX-W3 and Lactobacillus plantarum BRX-1 in a compound fermentation system, this invention achieves this by staged regulation of Fe in the fermentation broth. 2+ and Fe 3+The content of Fe was used to achieve the temporal connection of metabolic activities between the two strains: Fe was added in the early stage of fermentation. 2+ Promotes [4Fe-4S] 2+ Cluster assembly, under anaerobic conditions, Bacillus subtilis Fnr transcriptional regulators and [4Fe-4S] 2+ Clusters assemble into active [4Fe-4S] clusters. 2+ -Fnr dimer activates anaerobic respiration gene transcription, enabling Bacillus subtilis to efficiently metabolize energy and accumulate extracellular proteases, which hydrolyze into amino acids, constructing an amino acid precursor pool rich in glutamate; Fe is added in the middle and late stages of fermentation. 3+ This approach increases the redox potential of the system, guiding the carbon metabolism flow from the synthesis of reducing products to the TCA cycle, promoting the accumulation of precursors such as α-ketoglutarate. These precursors are then converted to glutamate by *Lactobacillus plantarum* glutamate dehydrogenase, and GABA synthesis is efficiently driven by the glutamate decarboxylase (GAD) system. This regulatory strategy achieves temporal connection and functional coupling between the proteolytic precursor production stage dominated by *Bacillus subtilis* and the GABA synthesis and conversion stage dominated by *Lactobacillus plantarum*.

[0024] 2. The quinoa and vegetable compound powder prepared by this invention achieves synergistic and efficient enrichment of glutamic acid, GABA, and various functional components. The content of active ingredients such as glutamic acid, GABA, total phenols, total flavonoids, and dietary fiber, as well as their antioxidant and lipid-lowering abilities, are significantly improved, and their functions in regulating intestinal health and relieving constipation are significantly enhanced. Furthermore, the product's solubility, taste, and flavor are significantly improved, achieving simultaneous optimization of taste, flavor, nutrition, and function, thus solving the technical problem in existing technologies where taste improvement and functional enhancement cannot be simultaneously achieved.

[0025] 3. This invention uses a compound microbial agent composed of *Lactobacillus plantarum* BRX-1 and *Bacillus subtilis* LX-W3 for fermentation. *Lactobacillus plantarum* BRX-1 is isolated from fresh fig fermentation juice, has high biocompatibility, excellent acid and bile acid resistance, and multiple effects such as lowering blood sugar, lowering blood lipids, and antioxidation, and has excellent fermentation performance. *Bacillus subtilis* LX-W3 has a strong extracellular protease secretion capacity. The two strains complement each other in metabolic function: *Bacillus subtilis* LX-W3 decomposes large protein molecules into small peptides and free amino acids, while *Lactobacillus plantarum* BRX-1 converts glutamate into GABA through its glutamate decarboxylase (GAD) system. At the same time, it uses lactic acid bacteria metabolism to convert other large molecules in the raw materials into small functional components. The combination of the two covers the complete metabolic pathway from protein degradation to GABA end product synthesis and synergistically improves the bioavailability and taste of the product.

[0026] 4. This invention prepares a quinoa fruit and vegetable compound powder using quinoa, yam powder, inulin, and prune juice as raw materials. First, the quinoa raw material is treated with a germination process to activate endogenous enzyme systems, promoting the release and conversion of bound polyphenols, flavonoids, and other active ingredients. Starch molecules are partially degraded, increasing the content of soluble dietary fiber and enriching GABA. Based on this, the quinoa is mixed with prune juice and compounded with yam powder to effectively improve the taste, flavor, and nutritional properties of the final product. By supplementing with inulin, the dietary fiber content is further increased, and the prebiotic properties of inulin promote the growth of probiotics, rapidly initiate fermentation, shorten the fermentation process, and further enhance the product's gut health benefits. The quinoa fruit and vegetable compound powder provided by this invention has a high dietary fiber content and a low GI value. It can be consumed alone or added as a functional base to diverse product forms such as meal replacement bars, nutritional biscuits, and solid beverages, making it widely applicable to metabolic health management scenarios such as anti-oxidation, weight management, blood lipid improvement, and gut health maintenance. Attached Figure Description

[0027] Figure 1 The inhibition rates of α-amylase and α-glucosidase in Lactobacillus plantarum BRX-1;

[0028] Figure 2 The in vitro lipid-lowering and antioxidant capabilities of Lactobacillus plantarum BRX-1;

[0029] Figure 3 The protease and polypeptide production capabilities of Bacillus subtilis LX-W3;

[0030] Figure 4 The DPPH free radical scavenging rate, ABTS free radical scavenging rate, α-glucosidase activity inhibition rate, α-amylase activity inhibition rate, and sterol removal rate of fermentation broths of Lactobacillus plantarum BRX-1, Bacillus subtilis LX-W3, and compound bacterial agent were measured.

[0031] Figure 5 Sensory evaluation of quinoa fruit and vegetable compound powder;

[0032] Figure 6 The dietary fiber content in quinoa fruit and vegetable compound powder;

[0033] Figure 7 The effect of quinoa fruit and vegetable compound powder on defecation function in mice;

[0034] Figure 8 The effect of quinoa fruit and vegetable compound powder on blood lipid levels in rats. Detailed Implementation

[0035] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the invention, but the invention is not limited to them. Unless otherwise specified, any techniques or conditions not specifically described in the following embodiments shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents, culture media, or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0036] The Lactobacillus plantarum BRX-1 involved in the following examples is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38511 on April 29, 2026.

[0037] Bacillus subtilis LX-W3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27046 on April 10, 2023.

[0038] Lactobacillus rhamnosus ATCC53103 was purchased from the American Type Culture Collection (ATCC).

[0039] Example 1

[0040] This embodiment describes a method for screening and identifying Lactobacillus plantarum BRX-1, including the following steps:

[0041] S1. Initial screening of strains: Select fresh figs, place them in sterilized conical flasks, and ferment naturally at 28℃ for 24 hours. The fermentation broth is then serially diluted (10⁻⁶ ppm). -3 ~10 -7 The colonies were spread on LB medium and incubated at 37°C for 48 h. Colonies of different morphologies were inoculated on MRS solid medium with 2% CaCO3 and incubated at 30°C for 36 h. Colonies with calcium dissolution zones were selected for streak purification.

[0042] S2. Strain identification: The physiological and biochemical characteristics of strain BRX-1 were identified according to the "Classification, Identification and Test Methods of Lactic Acid Bacteria" and the "Handbook of Common Bacterial Identification". Molecular identification was performed using 16S rDNA sequencing. The strain's 16S rDNA was amplified by PCR, and the amplified product was sequenced by Sangon Biotech (Shanghai) Co., Ltd. The results were compared using the BLAST program on the NCBI website. The results showed that the strain had a similarity of more than 99% with Lactobacillus plantarum, and it was named Lactobacillus plantarum BRX-1.

[0043] Example 2

[0044] This example is an evaluation of the beneficial viability of Lactobacillus plantarum BRX-1.

[0045] 1. In vitro hypoglycemic activity analysis: Using the commercial standard probiotic strain Lactobacillus rhamnosus ATCC53103 as a reference and the commonly used clinical hypoglycemic drug acarbose (0.8 mg / L) as a positive control, the in vitro hypoglycemic activity of Lactobacillus plantarum BRX-1 was investigated by detecting the effects of fermentation broth of Lactobacillus plantarum on the inhibition rates of α-amylase and α-glucosidase.

[0046] S1. Sample preparation: Lactobacillus plantarum BRX-1 was inoculated into MRS liquid medium and cultured at 37℃ for 24 h until the bacterial concentration reached 10. 7 The bacterial suspension was inoculated at a rate of 2% (v / v) into MRS liquid medium with a glucose concentration of 100 μg / mL and cultured at 37℃ for 48 h. The fermentation broth was centrifuged at 12000 rpm for 20 min, and the supernatant was collected for analysis.

[0047] S2. α-Amylase Inhibition Rate Determination: 0.25 mL of sample solution and 0.25 mL of α-amylase solution (1 mg / mL) were mixed to prepare a mixture, and reacted at 37℃ for 10 min. Then, 0.25 mL of 1.5% soluble starch solution was added, and the mixture was incubated at 37℃ for 10 min. Next, 1 mL of DNS solution was added, and the mixture was reacted in a boiling water bath for 5 min, then cooled. The OD was measured using a microplate reader. 504 The absorbance at the specified position was measured. Distilled water was used instead of standard distillate for the control and blank groups, and the enzyme solution for the blank and standard groups was replaced with the same volume of PBS buffer. The inhibition rate of α-amylase was calculated for all three groups in parallel using the following formula: R = (1 - (AB) / (CD)) × 100%.

[0048] In the formula: A: contains sample and α-amylase; B: contains sample but no α-amylase; C: no sample but contains α-amylase solution; D: no sample and no α-amylase solution.

[0049] S3. Assay for α-glucosidase activity inhibition rate: 50 μL of sample solution was added to 50 μL of PNPG solution (1.5 mmol / L), mixed well, and reacted at 37℃ for 10 min. Then, 60 μL of 0.2 U / mL α-glucosidase was added, and the reaction was continued at 37℃ for 30 min. Finally, 50 μL of Na2CO3 solution (0.2 mol / L) was added to terminate the reaction. OD was measured using a microplate reader. 504The absorbance at a certain point was used to calculate the α-glucosidase activity inhibition rate. Distilled water was used instead of standard distillate for the control and blank groups, while the enzyme solution for the blank and standard groups was replaced with the same volume of PBS buffer. All three groups were replicated. *Lactobacillus rhamnosus* ATCC53103 was used as a positive control strain. The formula for calculating the α-glucosidase activity inhibition rate is: R = (1 - (AB) / (CD)) × 100%.

[0050] In the formula: A is the absorbance value of the sample group; B is the absorbance value of the blank sample group; C is the absorbance value of the control group; D is the absorbance value of the blank group.

[0051] like Figure 1 As shown, compared with the positive control strain *Lactobacillus rhamnosus* ATCC53103, the *Lactobacillus plantarum* BRX-1 of this invention showed an increase of 13.17% and 42.05% in the inhibition rates of α-amylase (85.16%) and α-glucosidase (60.27%), respectively; and an increase of 160% compared with the inhibition rate of α-amylase by the commonly used hypoglycemic drug acarbose (0.8 mg / L). This indicates that *Lactobacillus plantarum* BRX-1 of this invention has a significant hypoglycemic function, and its hypoglycemic effect is superior to that of the positive control strain ATCC53103.

[0052] 2. In vitro lipid-lowering activity analysis:

[0053] S1 Cholesterol Removal Rate Determination: Activated *Lactobacillus plantarum* BRX-1 was inoculated into MRS-CHOL medium (cholesterol MRS medium) at an inoculation rate of 5% (v / v) and cultured statically at 37℃ for 24 h. 1 mL of fermentation broth was centrifuged at 6000 rpm for 10 min to obtain the supernatant. The cholesterol content in the supernatant was determined using the o-phthalaldehyde method. The cholesterol removal rate was calculated using the following formula: Cholesterol Removal Rate (%) = [(A0-A1) / A0] × 100%, where A0 is the cholesterol content of the uninoculated supernatant, and A1 is the cholesterol content (mg / mL) of the fermentation supernatant of the strain.

[0054] S2. Determination of triglyceride removal rate: Activated *Lactobacillus plantarum* BRX-1 was inoculated into triglyceride liquid medium at an inoculation rate of 5% (v / v) and cultured statically at 37℃ for 24 h. 1 mL of fermentation broth was centrifuged at 6000 rpm for 10 min, and the supernatant was collected. The triglyceride content in the supernatant was determined according to the triglyceride kit instructions. The triglyceride removal rate was calculated using the following formula: Triglyceride removal rate (%) = [(A0-A1) / A0] × 100%, where A0 is the triglyceride content in the uninoculated supernatant, and A1 is the triglyceride content (mmol / L) in the fermentation supernatant of the strain.

[0055] Most reported plant-based lactic acid bacteria have cholesterol removal rates between 30% and 50% and triglyceride removal rates between 25% and 45%. For example... Figure 2 As shown, the removal rates of cholesterol and triglycerides by *Lactobacillus plantarum* BRX-1 of the present invention were 52.6% and 54.7%, respectively, indicating that *Lactobacillus plantarum* BRX-1 of the present invention has excellent lipid-lowering function.

[0056] 3. Determination of in vitro antioxidant capacity:

[0057] S1. Determination of DPPH free radical scavenging ability: 1.0 mL of BRX-1 bacterial suspension was vortexed with 1.0 mL of DPPH ethanol solution (0.2 mmol / L) and reacted at room temperature in the dark for 30 min. After the reaction, the mixture was centrifuged at 6000 rpm for 3 min, the supernatant was collected, and the absorbance was measured at 517 nm, recorded as A0. At the same time, anhydrous ethanol was used as a control group instead of the sample, and the absorbance was measured and recorded as A1. The DPPH free radical scavenging rate was calculated according to the following formula: DPPH free radical scavenging rate (%) = (A1 - A0) / A1 × 100%, where: A0 is the absorbance of the sample group; A1 is the absorbance of the control group.

[0058] S2. Ferric Reducing Ability Assay (FRAP): Take 0.2 mL of diluted bacterial suspension, add 3.8 mL of FRAP working solution (a mixture of acetate buffer, TPTZ solution, and FeCl3 solution in a 10:1:1 ratio), mix well, and react in a water bath at 37°C for 10 min. Measure the absorbance at 593 nm. Using FeSO4 as a standard, the result is expressed as the FeSO4 equivalent per milliliter of sample (mmol Fe). 2+ / mL).

[0059] like Figure 2 As shown, the *Lactobacillus plantarum* BRX-1 of this invention possesses excellent antioxidant capabilities, with a DPPH free radical scavenging rate of 67.2% and Fe... 3+ The reducing power is 0.81 mmol / mL.

[0060] 4. Acid and bile salt resistance:

[0061] S1. Acid resistance test: Take 10 mL of bacterial culture after 24 h of culture and centrifuge to collect the bacterial cells. Resuspend the bacterial cells in 10 mL of PBS and adjust the pH of the solution to 2.0, 2.5 and 3.0 respectively. Use PBS with pH 7.2 as a control. After standing for 3 h, spread the solution on the culture medium and incubate upside down for 24 h. Calculate the total number of colonies and calculate the survival rate according to the formula: Survival rate of strain (%) = logN1 / logN0 × 100%, where: N1 represents the number of viable bacteria after acid or bile salt treatment / (CFU / mL), and N0 represents the number of viable bacteria in the blank control / (CFU / mL).

[0062] S2. Bile salt tolerance test: Inoculate 10 [units of culture medium] with MRS medium containing 0.1%, 0.2%, and 0.3% taurine salt solutions. 8 The bacterial suspension was prepared at CFU / mL, with the unadded culture medium as a control. After standing for 3 h, the solution was spread onto the culture medium and incubated upside down for 24 h. The total number of colonies was calculated, and the survival rate was calculated.

[0063] Table 1. Results of acid and taurine resistance tests of Lactobacillus plantarum BRX-1

[0064]

[0065] Acid and bile salt tolerance are core prerequisites for probiotics to cross the digestive tract barrier, colonize the intestine, and exert their probiotic functions. Strains must tolerate the acidic environment of gastric juice (pH 1.5–3.5) and the intestinal bile salt environment (0.1%–0.3%) to maintain their activity and continuously exert their regulatory effects on glucose and lipid metabolism. As shown in Table 1, the survival rate of *Lactobacillus plantarum* BRX-1 of this invention is as high as 85% or more in a pH 2–3 environment, and as high as 90% or more in an intestinal bile salt environment of 0.1%–0.3%. This indicates that *Lactobacillus plantarum* BRX-1 of this invention can cross the digestive tract barrier, colonize the intestine, and exert its antioxidant, hypoglycemic, and lipid-lowering effects, demonstrating excellent probiotic viability.

[0066] Example 3

[0067] This embodiment describes the determination of the protease and peptide production capabilities of Bacillus subtilis LX-W3. The specific method includes the following steps:

[0068] S1. Determination of protease activity by the azocasein method: Activated Bacillus subtilis LX-W3 was inoculated into liquid culture medium and cultured at 37℃ with shaking at 120 rpm for 24 h. 1 mL of the fermentation supernatant was taken and centrifuged at 4℃ for 10 min at 10000 rpm. 100 μL of the supernatant was thoroughly mixed with 50 μL of 1% azocasein substrate solution and incubated at 37℃ for 1 h. The reaction was terminated by adding 240 μL of trichloroacetic acid (TCA), and centrifuged at 4℃ for 10 min at 10000 rpm. 100 μL of the supernatant was added to 100 μL of 1M NaOH and allowed to react completely. The absorbance was measured at 450 nm. Physiological saline was used as a blank control. Enzyme activity unit definition: Under constant temperature of 37℃, the amount of enzyme producing 0.001 absorbance in 1 min is defined as 1 unit of enzyme activity. Enzyme activity was calculated according to the following formula:

[0069]

[0070] In the formula: X: enzyme activity (U), A: OD value of the sample at 450 nm, A0: OD value of the blank control at 450 nm, V1: volume of crude enzyme solution (mL), V2: volume of 1% azocasein substrate (mL), V3: volume of 10% TCA (mL), 60: constant temperature for 60 min.

[0071] S2. Determination of peptide production capacity: Take 2 mL of fermentation supernatant, mix with 2.0 mL of 10% trichloroacetic acid, centrifuge at 5000 rpm for 15 min, take 0.5 mL of supernatant, mix with 2 mL of biuret reagent, centrifuge at 3000 rpm for 15 min, and measure the absorbance of the supernatant at 540 nm. Add 0.5 mL of distilled water and 2.0 mL of biuret reagent as blanks.

[0072] like Figure 3 As shown, the fermentation supernatant of Bacillus subtilis LX-W3 exhibited high protease activity (32.24 ± 0.54 U / mL) and a polypeptide content of 12.67 ± 0.25 mg / mL. This indicates that this strain can better break down proteins in raw materials into bioactive peptides and flavor amino acids with health benefits. It has high application potential in improving the health benefits of products, especially in enhancing their flavor.

[0073] Example 4

[0074] This embodiment describes the effect of co-fermentation of *Lactobacillus plantarum* BRX-1 and *Bacillus subtilis* LX-W3 on the function of bacteria. The specific method includes the following steps: *Lactobacillus plantarum* BRX-1 and *Bacillus subtilis* LX-W3 were inoculated separately or mixed at a 1:1 volume ratio in 50 mL of MRS liquid or MRS liquid medium containing 0.20 mg / mL high cholesterol, with an inoculation amount of 5% and 3 replicates per group; after incubation at 30℃ for 36 h, the DPPH free radical scavenging rate, ABTS free radical scavenging rate, α-glucosidase activity inhibition rate, α-amylase activity inhibition rate, and cholesterol removal rate in the culture medium were measured.

[0075] like Figure 4 As shown, the combined fermentation of *Lactobacillus plantarum* BRX-1 and *Bacillus subtilis* LX-W3 exhibits varying degrees of synergistic effects on DPPH free radical scavenging, ABTS free radical scavenging, α-glucosidase activity inhibition, α-amylase activity inhibition, and cholesterol removal. Compared with *Lactobacillus plantarum* BRX-1, the combined bacterial agent increased ABTS free radical scavenging, α-amylase activity inhibition, and cholesterol removal by 30.06%, 27.87%, and 36.98%, respectively; compared with *Bacillus subtilis* LX-W3, the combined probiotic preparation of this invention increased ABTS free radical scavenging, α-amylase activity inhibition, and cholesterol removal by 43.44%, 134.68%, and 41.78%, respectively. These results indicate that the combined probiotic preparation of this invention has good application potential in the preparation of foods, drugs, and health products with antioxidant, hypoglycemic, and hypolipidemic functions, and its effects are significantly better than those of single strains.

[0076] Example 5

[0077] This embodiment describes a method for preparing quinoa fruit and vegetable compound powder, including the following steps:

[0078] S1. Quinoa germination treatment: Select 30 kg of quinoa that is free from pests and mold and has whole and plump grains. Clean it and soak it in deionized water for 4 hours. Spread the quinoa on a gauze and place it in a constant temperature incubator at 25℃ and 85% humidity for 36 hours. Spray deionized water once every 6 hours during the period to obtain germinated quinoa.

[0079] S2. Preparation of fermentation substrate: Germinated quinoa was mixed with 80 kg of prune juice and pulped. The pulp was mixed with 20 kg of yam powder, and then 0.25% of α-amylase (total mass of germinated quinoa, prune juice and yam powder) was added. The mixture was enzymatically hydrolyzed at 55℃ for 1.5 h, and the enzyme was inactivated at 85℃ for 20 min. After cooling to room temperature, the residue was removed by centrifugation. 10 kg of inulin was added to the supernatant and mixed evenly to obtain the fermentation substrate.

[0080] S3. Preparation of the compound microbial agent: After activation, *Lactobacillus plantarum* BRX-1 and *Bacillus subtilis* LX-W3 were inoculated separately into MRS liquid medium and cultured at 37°C for 24 h. The precipitate was collected by centrifugation and resuspended in physiological saline to a bacterial concentration of 1×10⁻⁶. 8 CFU / mL, to prepare Lactobacillus plantarum BRX-1 bacterial suspension and Bacillus subtilis LX-W3 bacterial suspension, the two bacterial suspensions were mixed at a volume ratio of 1:1 to prepare a compound bacterial agent;

[0081] S4. Inoculation and Fermentation: Add FeSO4·7H2O to the fermentation substrate to a final concentration of 0.6 mmol / L, inoculate with compound microbial agent, and anaerobic ferment at 37℃ for 10 h. Then add ferric ammonium citrate to a final concentration of 1.3 mmol / L and anaerobic ferment at 37℃ for 6 h. After fermentation, sterilize at 90℃ for 20 min to obtain fermentation broth. After centrifugation, collect the supernatant and freeze-dry the supernatant under vacuum to obtain quinoa fruit and vegetable compound powder.

[0082] Example 6

[0083] The difference between this embodiment and Embodiment 5 is that the viable count of the *Lactobacillus plantarum* BRX-1 suspension in this embodiment is 5 × 10⁻⁶. 7 CFU / mL, the remaining steps are the same as in Example 5.

[0084] Example 7

[0085] The difference between this embodiment and Embodiment 5 is that the viable count of the Bacillus subtilis LX-W3 bacterial suspension in this embodiment is 8 × 10⁻⁶. 7 CFU / mL, the remaining steps are the same as in Example 5.

[0086] Example 8

[0087] The difference between this embodiment and embodiment 5 is that the final concentration of FeSO4·7H2O in this embodiment is 0.3 mmol / L, while the other steps are the same as in embodiment 5.

[0088] Example 9

[0089] The difference between this embodiment and embodiment 5 is that the final concentration of ferric ammonium citrate in this embodiment is 1.8 mmol / L, while the other steps are the same as in embodiment 5.

[0090] To further illustrate the technical effects of the present invention, a comparative example is also provided, as follows:

[0091] Comparative Example 1

[0092] The difference between this comparative example and Example 5 is that Lactobacillus plantarum BRX-1 is not added in this comparative example, while the other steps are the same as in Example 5.

[0093] Comparative Example 2

[0094] The difference between this comparative example and Example 5 is that Bacillus subtilis LX-W3 is not added in this comparative example, while the other steps are the same as in Example 5.

[0095] Comparative Example 3

[0096] The difference between this comparative example and Example 5 is that FeSO4·7H2O is not added in this comparative example, while the other steps are the same as in Example 5.

[0097] Comparative Example 4

[0098] The difference between this comparative example and Example 5 is that ferric ammonium citrate is not added in this comparative example, while the other steps are the same as in Example 5.

[0099] Comparative Example 5

[0100] The difference between this comparative example and Example 5 is that in this comparative example, ferric ammonium citrate is added first for fermentation and then FeSO4·7H2O is added; the remaining steps are the same as in Example 5.

[0101] Comparative Example 6

[0102] The difference between this comparative example and Example 5 is that FeSO4·7H2O and ferric ammonium citrate are added simultaneously in this comparative example, while the remaining steps are the same as in Example 5.

[0103] Table 2. Protease activity in fermentation broth and content of glutamic acid, GABA, total phenols, and total flavonoids in fruit and vegetable compound powder.

[0104]

[0105] Note: The contents of glutamic acid, GABA, total phenols, and total flavonoids are all calculated based on the fruit and vegetable compound powder (mg / g); the protease activity is calculated based on the supernatant of the fermentation broth (U / mL).

[0106] As shown in Table 2, the protease activity, glutamic acid, GABA, total phenols, and total flavonoids in the fermentation broths of Examples 5-9 were higher than those in Comparative Examples 1-6. Comparative Example 1 (without *Lactobacillus plantarum* BRX-1) had lower levels of all indicators except glutamic acid compared to Examples. This was because the fermentation system lacked the *Lactobacillus plantarum* glutamate decarboxylase (GAD) system, preventing the glutamate precursor produced by *Bacillus subtilis* through protein hydrolysis from being further converted into GABA. This resulted in an obstructed GABA synthesis pathway, preventing the utilization of glutamic acid and thus leading to a higher glutamic acid content than Examples. Comparative Example 2 (without *Bacillus subtilis* LX-W3) had the *Lactobacillus plantarum* GAD system but lacked *Bacillus subtilis*, preventing protein hydrolysis into amino acids and resulting in a deficiency of glutamate precursors and extremely limited GABA production. The low protease activity also directly led to insufficient cell wall degradation, resulting in reduced total phenol and total flavonoid content. The protease activity, glutamate content, and GABA content of the examples were all higher than those of Comparative Example 2 (without Bacillus subtilis LX-W3) and Comparative Example 3 (without FeSO4·7H2O). This is because Fe 2+ It activated the anaerobic respiratory metabolism of Bacillus subtilis, promoting the efficient secretion of extracellular proteases. Under anaerobic conditions, the energy metabolism of Bacillus subtilis is regulated by Fnr transcription factors, and the activity of Fnr proteins depends on the iron-sulfur cluster ([4Fe-4S)). 2+ The correct assembly of [4Fe-4S] was achieved by adding FeSO4·7H2O during the early stage of fermentation. 2+ The assembly of clusters provides an ample iron source, and under anaerobic conditions, the Fnr of Bacillus subtilis reacts with [4Fe-4S]. 2+ Clusters combine to form active [4Fe-4S] 2+ -Fnr dimer activates anaerobic respiration gene transcription, ensuring energy supply for Bacillus subtilis under anaerobic conditions and accumulating extracellular proteases. These proteases hydrolyze proteins from the raw materials to construct a glutamate-rich precursor pool, thereby increasing GABA content. Comparative Example 2 (without Bacillus subtilis LX-W3) and Comparative Example 3 (without FeSO4·7H2O) lacked Bacillus subtilis and Fe... 2+ Fnr cannot be effectively assembled into its active form, anaerobic respiration metabolism is hindered, extracellular protease secretion is insufficient, and the production of amino acid precursors (including glutamate) from proteolysis is reduced, ultimately limiting GABA synthesis. It is worth noting that Comparative Example 3 still contains trace amounts of endogenous iron ions from the fermentation substrate, therefore the protease activity and GABA content are not completely zero, but they are significantly lower than in the examples. Comparative Example 4 (without added ferric ammonium citrate) showed significantly lower performance in all aspects compared to the examples, due to the addition of only Fe... 2+The system was in a strongly reducing state throughout the process, resulting in a lower redox potential (ORP). This limited the GAD system of *Lactobacillus plantarum*, and even with the provision of some glutamate precursors by *Bacillus subtilis*, the conversion efficiency to GABA remained very low. In contrast, the example added Fe during the later stages of fermentation. 3+ This increases the system's ORP, alters the NAD⁺ / NADH ratio to guide carbon metabolism from the synthesis of reducing products to the TCA cycle, promotes the accumulation of precursors such as α-ketoglutarate, and increases GABA content.

[0107] Comparative Example 5 (Feammonium citrate added first, then FeSO4·7H2O added) added Fe at the beginning of fermentation. 3+ The system's ORP increased, even after Fe was supplemented later. 2+ Bacillus subtilis also missed the optimal window for Fnr assembly under low ORP anaerobic conditions, resulting in insufficient protease secretion and a lack of amino acid precursors; while the Fe supplemented later... 2+ This also lowered the system's ORP, which in turn inhibited the *Lactobacillus plantarum* GAD system. Comparative Example 6 (with simultaneous addition of FeSO4·7H2O and ferric ammonium citrate) had the lowest levels of all indicators among all groups. 2+ and Fe 3+ When present simultaneously in the early stages of fermentation, both strains negatively impact the core metabolic pathways of their respective organisms. For *Bacillus subtilis*, Fnr cannot be effectively activated, anaerobic respiration gene transcription is inhibited, and protease secretion is insufficient. For *Lactobacillus plantarum*, although Fe... 3+ The GAD system can be activated, but due to the lack of a precursor pool constructed in the early stage by Bacillus subtilis, the GAD system cannot achieve GABA conversion.

[0108] Table 3 Sensory Evaluation Criteria for Fruit and Vegetable Compound Powder

[0109]

[0110] Sensory evaluations were performed on the quinoa and vegetable composite powders prepared in Example 5 and Comparative Examples 1-6 of this invention to demonstrate the effect of the method of this invention on improving the taste and flavor of the products. The sensory evaluation results are as follows: Figure 5 As shown, Example 5 had the highest sensory score of 94.2, indicating that the present invention improves the sensory quality of the final product while increasing the active ingredients.

[0111] Table 4. GI value of quinoa fruit and vegetable compound powder

[0112]

[0113] According to Chinese health industry standards, a GI of ≤55 indicates a low-GI food; 55≤GI≤70 indicates a medium-GI food; and GI>70 indicates a high-GI food. As shown in Table 4, the quinoa fruit and vegetable compound powder prepared in Example 5 has a GI value of 28.18, classifying it as a low-GI food. The GI values ​​of Comparative Examples 1-5 are 56.75-68.17, classifying them as medium-GI foods. The GI value of Comparative Example 6 is 72.85, classifying it as a high-GI food. This indicates that the quinoa fruit and vegetable compound powder prepared in this invention has good application potential in the preparation of low-GI foods and health products.

[0114] like Figure 6 As shown, the total dietary fiber (TDF) and soluble dietary fiber (SDF) content of Example 5 were significantly higher than those of Comparative Examples 1-6, indicating that the preparation method of the present invention can significantly increase the dietary fiber content. Dietary fiber has the effects of promoting the proliferation of beneficial intestinal bacteria, delaying the digestion and absorption of carbohydrates, and reducing the glycemic index (GI). Therefore, the quinoa fruit and vegetable compound powder prepared by the present invention is beneficial for maintaining stable blood sugar, reducing GI, increasing satiety, and maintaining intestinal health.

[0115] Example 10

[0116] This embodiment describes the effect of quinoa fruit and vegetable compound powder on the defecation function of mice. The specific method includes the following steps:

[0117] S1. Grouping: C57BL / 6 mice (weighing 18-22 g) were randomly divided into control group, model group, and compound powder group, with 12 mice in each group;

[0118] S2. Model Establishment and Intervention: Mice were administered loperamide hydrochloride solution by gavage at a dose of 0.2 mL / 10 g·bw for 7 consecutive days to establish a mouse constipation model. The control group was administered physiological saline by gavage, while the compound powder group was administered 2.4 mg / kg loperamide hydrochloride solution by gavage, plus 300 mg / mouse of quinoa fruit and vegetable compound powder prepared in Example 5, once a day. Other groups had normal diets. On the 7th day after administration, all mice were fasted but allowed free access to water for 24 hours. Except for the control group which received physiological saline, the model group and the compound powder group were administered loperamide hydrochloride solution (2.4 mg / kg) by gavage at a dose of 20 mL / kg·bw. 30 minutes later, the control group and the model group were given ink (containing 5% activated charcoal), while the compound powder group was given ink containing quinoa fruit and vegetable compound powder prepared in Example 5 (3.0 g / kg·bw). Subsequently, each group of mice was divided into two parts, with 6 mice used to observe the time required for the first black stool; the other 6 mice were used to observe the time required for the first black stool. After a few minutes, the mice were euthanized by cervical dislocation. Blood was collected from the orbital cavity and colon tissue was dissected for later use. The total length of the small intestine and the length of ink propulsion were measured, and the ink propulsion rate of the small intestine was calculated as follows: P (%) = ink propulsion length (cm) / total length of small intestine (cm) × 100%.

[0119] like Figure 7 As shown, compared with the control group, the model group mice had an increased time to expel the first black feces and a decreased rate of ink propulsion in the small intestine, indicating that the model was successfully established. After intervention with the quinoa fruit and vegetable compound powder of this invention, compared with the model group, the time required for the first black feces to be expelled in mice was significantly shortened and the rate of ink propulsion in the small intestine was increased, indicating that the quinoa fruit and vegetable compound powder of this invention has a positive effect on intestinal peristalsis in mice, has significant laxative activity, and improves intestinal function in constipated mice.

[0120] Example 11

[0121] This embodiment describes the effect of quinoa fruit and vegetable compound powder on blood lipid levels in rats. The specific method includes the following steps:

[0122] S1. Five-week-old healthy male SD rats were randomly divided into three groups after one week of acclimatization: control group, model group, and compound powder group. The control group was fed a regular diet, the model group was fed a high-fat diet, and the compound powder group was fed a high-fat diet and administered quinoa fruit and vegetable compound powder (300 mg / rat) prepared in Example 5 by gavage daily. The control group and the model group were administered an equal volume of physiological saline by gavage for four consecutive weeks.

[0123] S2. Fasting for 16 hours before the end of the experiment, blood was collected by enucleation to measure serum total cholesterol (TC), serum triglycerides (TG), serum high-density lipoprotein cholesterol (HDL), and serum low-density lipoprotein cholesterol (LDL).

[0124] like Figure 8 As shown, compared with the control group, the model group rats had significantly increased serum total cholesterol (TC), serum triglycerides (TG), and serum low-density lipoprotein cholesterol (LDL) levels, and decreased serum high-density lipoprotein cholesterol (HDL) levels, indicating that the hyperlipidemia model was successfully established. After intervention with the quinoa fruit and vegetable compound powder of this invention, compared with the model group, the levels of TC, TG, and LDL decreased, while the level of HDL increased, indicating that the quinoa fruit and vegetable compound powder has a significant lipid-lowering effect.

[0125] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing quinoa fruit and vegetable compound powder, characterized in that, Includes the following steps: S1. 30-50 parts of quinoa are germinated to obtain germinated quinoa; S2. Sprouted quinoa and 50-80 parts of prune juice are mixed and pulped. The pulp is mixed with 20-30 parts of yam powder, and then α-amylase is added. After enzymatic hydrolysis, the enzyme is inactivated and cooled. The enzymatic hydrolysate is centrifuged and the supernatant is collected. 10-15 parts of inulin are added and mixed evenly to obtain the fermentation substrate. S3. After activating Lactobacillus plantarum and Bacillus subtilis, they were inoculated into MRS liquid medium and cultured. After centrifugation, the precipitates were collected, resuspended, and Lactobacillus plantarum suspension and Bacillus subtilis suspension were obtained. The two bacterial suspensions were mixed to obtain a compound bacterial agent. S4. Add ferrous ions to the fermentation substrate, inoculate with compound microbial agents for anaerobic fermentation, then add ferric ions to continue anaerobic fermentation. After fermentation, sterilize, collect the supernatant after centrifugation, and freeze-dry the supernatant under vacuum to obtain quinoa fruit and vegetable compound powder.

2. The method for preparing quinoa fruit and vegetable compound powder according to claim 1, characterized in that: In step S2, the amount of α-amylase added is 0.1-0.3% of the total mass of sprouted quinoa, prune juice, and yam powder.

3. The method for preparing quinoa fruit and vegetable compound powder according to claim 1, characterized in that: In step S3, *Lactobacillus plantarum* strain BRX-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38511, on April 29, 2026; and *Bacillus subtilis* strain LX-W3 is deposited at the same center with accession number CGMCC No. 27046, on April 10, 2023.

4. The method for preparing quinoa fruit and vegetable compound powder according to claim 1, characterized in that: In step S3, the viable count of the *Lactobacillus plantarum* suspension or *Bacillus subtilis* suspension is 1 × 10⁻⁶. 7 ~1×10 8 CFU / mL; the volume ratio of Lactobacillus plantarum suspension to Bacillus subtilis suspension is 1~2:1~2.

5. The method for preparing quinoa fruit and vegetable compound powder according to claim 1, characterized in that: In step S4, the ferrous ions are FeSO4, and the final concentration of ferrous ions is 0.2~1 mmol / L.

6. The method for preparing quinoa fruit and vegetable compound powder according to claim 1, characterized in that: In step S4, the ferric ions are ferric ammonium citrate, and the final concentration of ferric ions is 0.8~2 mmol / L.

7. The method for preparing quinoa fruit and vegetable compound powder according to claim 1, characterized in that: In step S4, the anaerobic fermentation time after adding ferrous ions is 8-12 hours, and the anaerobic fermentation time after adding ferric ions is 4-10 hours.

8. Quinoa fruit and vegetable compound powder prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the quinoa fruit and vegetable compound powder according to claim 8 in the preparation of foods, medicines or health products that have antioxidant properties, improve blood lipid health or improve intestinal health.

10. The application according to claim 9, characterized in that: The dosage forms of the antioxidant, lipid-lowering, or gut-health-improving foods, medicines, or health products mentioned are powders, tablets, capsules, or granules.

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