Fermentation type beet root pigment as well as preparation method and application thereof

Abstract

The invention relates to the technical field of food processing, in particular to a fermentation type beet root pigment and a preparation method and application thereof. The method comprises the following steps: slicing and crushing beet roots, inoculating a proper amount of specific strains, fermenting for a certain time in an intermittent multi-frequency ultrasonic mode, collecting fermentation liquor, adding a proper amount of maltodextrin and acid substances, and obtaining the fermented beet root pigment powder with dual functions of reducing blood sugar and uric acid by adopting a spray drying mode. Through the synergistic effect of a specific strain combination and an ultrasonic fermentation process, the content of betacyanin in a fermentation product is remarkably increased, and the beet root pigment powder has good inhibitory activity of alpha-amylase, alpha-glucosidase and xanthine oxidase. The prepared pigment powder not only has good stability, can be used as a natural pigment for baked foods such as bread, but also can effectively reduce the starch digestibility of the bread, and has a wide application prospect in preparation of functional products for reducing blood sugar and uric acid.

Description

Technical Field

[0001] This application relates to the field of food processing technology, and in particular to a fermented beetroot pigment, its preparation method, and its application. Background Technology

[0002] Hyperglycemia, or diabetes mellitus (DM), is the seventh leading cause of death worldwide and is projected to affect approximately 591.9 million people by 2035. This vulnerable group is at higher risk of debilitating complications such as cardiovascular disease, peripheral vascular disease, kidney disease, retinal changes, blindness, and even premature death. Diabetes is a metabolic disease caused by insufficient insulin secretion from pancreatic β-cells or decreased insulin sensitivity in peripheral tissue cells, and its prevalence is multifactorial. Long-term excessive consumption of highly digestible foods leading to elevated postprandial blood glucose levels is a significant contributing factor to its high incidence. Current management strategies primarily focus on low glycemic index (GI) diets to control postprandial blood glucose spikes. Various studies have been conducted in this direction. Since the human body possesses α-amylase / α-glucosidase that degrades starch in food, the time-dependent digestion pattern characterizes the glucose response based on carbohydrate source and categorizes it into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS). Therefore, inhibiting α-amylase / α-glucosidase activity can reduce the rate of glucose release and play a role in the prevention and treatment of DM.

[0003] Hyperuricemia (HUA) is a systemic metabolic disorder caused by abnormal purine metabolism, characterized by fasting serum uric acid levels exceeding 420 μmol / L in adults after two consecutive days of a normal purine diet. The increased prevalence of HUA is attributed to a variety of factors, such as changes in dietary patterns and lifestyle, rising incidence of chronic diseases, genetic susceptibility, and environmental influences. In recent years, the prevalence of HUA has been rising globally, with significant differences in prevalence among different populations. HUA not only leads to gout but is also closely associated with the development of hypertension, kidney disease, atherosclerosis, coronary artery disease, and insulin resistance. Currently, clinical treatment mainly focuses on symptom relief and recurrence prevention. Current medications, such as xanthine oxidase (XOD) inhibitors (e.g., allopurinol) and uricosuric agents (e.g., benzbromarone), can effectively lower uric acid levels, but long-term use often produces side effects including severe skin reactions, muscle pain, gastrointestinal discomfort, diarrhea, polyarthritis, nephrotoxicity, anaphylactic shock, methemoglobinemia, hepatic steatosis, and elevated liver enzyme levels. Some patients even experience intolerance or poor response to these medications. These factors contribute to the continued prevalence of hypersensitivity to airway obstruction (HUA) and its complications, imposing a significant economic burden on society and families. Therefore, there is an urgent need to develop safe and effective alternatives or supplements to address or alleviate this growing health problem.

[0004] Microbial therapy, including the use of probiotics with specific functions, has proven to be an effective means of controlling diabetes and has been well-tolerated in most randomized clinical trials. Meanwhile, anthocyanin extracts, or purified flavonoids or polyphenols, have been shown to alleviate hyperuricemia, such as by inhibiting enzyme activity in purine metabolism and altering the gut microbiota.

[0005] With increasing global health awareness and improved quality of life, consumers are increasingly demanding natural, organic, and functional foods. Beetroot, with its rich nutritional value and numerous health benefits, including nitrates, betaine, betaine, carotenoids, phenolic compounds, and ascorbic acid, has shown promise in the supplement and food markets due to its ability to enhance athletic performance, promote cardiovascular health, provide antioxidant and anti-inflammatory effects, and improve cognitive function. However, existing research has not yet reported on how to efficiently and conveniently prepare fermented beetroot pigment powder with dual functions of lowering blood sugar and uric acid.

[0006] Therefore, how to effectively synergize the beneficial effects of probiotics and beet juice to obtain food ingredients with multiple functions is a problem that this industry needs to address. Summary of the Invention

[0007] To address the problems mentioned in the prior art mentioned in the background section, this invention provides a preparation method that couples the functions of *Pediococcus lactis*, other types of lactic acid bacteria (*Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Bifidobacterium infantis*) with beet juice, thus preparing a safe, food-grade raw material with potential application value in functional products such as those for lowering blood sugar and uric acid. This invention is achieved through the following technical solution: This application provides a method for preparing fermented beetroot pigment with dual functions of lowering blood sugar and lowering uric acid, comprising the following steps: Beetroot pretreatment: Wash, slice, and crush the beetroot to obtain beetroot pulp; the moisture content of the beetroot pulp should be controlled to be 100-150% of its solid content. Beetroot fermentation: Bacteria are inoculated into the beetroot slurry, and cellulase, pectinase and white sugar are added. After stirring evenly, fermentation is carried out under intermittent multi-frequency ultrasound to obtain fermentation broth. The frequency combination of the intermittent multi-frequency ultrasound is at least two of 20KHz, 40KHz, 60KHz, and 80KHz; the bacteria is Pediococcus lactis HJ516 or a combination of bacteria, wherein the combination of bacteria includes Pediococcus lactis HJ516 and other types of lactic acid bacteria, wherein the other types of lactic acid bacteria are Lactobacillus plantarum, Lactobacillus acidophilus, or Bifidobacterium infantis; Pigment powder preparation: Maltodextrin and acidic substances are added to the fermentation broth, dissolved and mixed, and then dried to obtain the fermented beetroot pigment powder; wherein the acidic substances are ascorbic acid and / or citric acid.

[0008] In some embodiments, the intermittent multi-frequency ultrasound is performed under the following conditions: a dual-frequency combination of 40 kHz and 60 kHz is used, and the ultrasound time is 10 to 30 seconds (this ultrasound time is the time of a single ultrasound and does not include the interval time).

[0009] In some embodiments, the amount of *Pediococcus lactis* HJ516 added is 0.5 to 2% of the weight of the beetroot slurry.

[0010] In some embodiments, the composite bacterial strain combination consists of Pediococcus lactis HJ516 and other types of lactic acid bacteria, wherein the other types of lactic acid bacteria are Lactobacillus plantarum; wherein the mass ratio of the other types of lactic acid bacteria to Pediococcus lactis HJ516 is 1:(1-2).

[0011] In some embodiments, the amount of *Pediococcus lactis* HJ516 added is 1% of the mass of beetroot slurry, and the mass ratio of *Lactobacillus plantarum* to *Pediococcus lactis* HJ516 is 1:1.

[0012] In some embodiments, the amount of cellulase added is 0.5-1.5% of the mass of beet root slurry; wherein the mass ratio of pectinase to cellulase is 1.5:(0.5-1.5). Preferably, the amount of cellulase added is 1% of the mass of beet root slurry; the mass ratio of pectinase to cellulase is 1.5:1.

[0013] In some embodiments, the acidic substances are ascorbic acid and citric acid; wherein the amount of ascorbic acid added is 1-3‰ of the mass of the fermentation broth, and the mass ratio of ascorbic acid to citric acid is (1-3):(0.5-1). Preferably, the amount of ascorbic acid added is 2‰ of the mass of the fermentation broth, and the amount of citric acid added is 0.5‰ of the mass of the fermentation broth.

[0014] In some embodiments, the amount of white sugar added is 0.5-1% of the mass of beetroot slurry; the amount of maltodextrin added is 5‰ of the mass of fermentation broth; and the amount of ascorbic acid added is 1-3‰ of the mass of fermentation broth.

[0015] In some embodiments, during the preparation of the pigment powder: maltodextrin and ascorbic acid are added to the fermentation broth, dissolved and mixed, and then spray-dried to obtain the final product.

[0016] This application also provides a fermented beetroot pigment powder prepared by the method described above.

[0017] This application also provides the application of the fermented beetroot pigment powder as described above in the preparation of baked goods.

[0018] In some embodiments, the baked food is bread; the fermented beetroot pigment powder is added to the bread at an amount of 1 to 4% of the flour mass.

[0019] Based on the above, compared with the prior art, this application has the following technical effects: 1. This invention is the first to combine a specific combination of lactic acid bacteria (Pediococcus lactis HJ516 and other commercially available conventional lactic acid bacteria) with intermittent multi-frequency ultrasonic fermentation technology for the preparation of beetroot pigments. This combination not only significantly improves the yield of betaine and shortens the fermentation time, but more importantly, it endows the final product with excellent inhibitory activities against α-amylase, α-glucosidase, and xanthine oxidase, achieving a dual coupling of hypoglycemic and uric acid-lowering functions.

[0020] 2. By optimizing the ultrasonic frequency and fermentation strain, the best effect of enhancing biological activity was achieved, and its inhibition rate was significantly higher than that of single strain or single frequency ultrasonic treatment.

[0021] 3. The prepared pigment powder has good storage stability and can be used as a natural pigment and functional ingredient in foods such as bread. It can effectively reduce the content of rapidly digestible starch (RDS) in bread, increase the proportion of resistant starch (RS), and slow down the rise in postprandial blood sugar.

[0022] 4. The preparation method provided by this invention is simple, mild, and easy to scale up, providing safe and effective raw materials for developing functional products that alleviate hyperglycemia and hyperuricemia.

[0023] Other features and beneficial effects of this application will be set forth in the following description and will be apparent in part from the description or may be learned by practicing the application. The purpose and other beneficial effects of this application can be realized and obtained from the description and claims. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. The technical features designed in the different implementations of this application described below can be combined with each other as long as they do not conflict with each other. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0025] In the description of this application, it should be noted that all terms used in this application (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and should not be construed as limiting this application; it should be further understood that the terms used in this application should be understood to have the same meaning as those in the context of this specification and the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this application.

[0026] This application provides the following experiments to verify the effectiveness of the proposed solution: I. Experimental Procedure: 1. The beetroot pigment preparation method of this application employs specific ultrasonic treatment methods and optimized ultrasonic treatment process parameters to achieve the desired effect. The verification experiment is as follows: Verification Experiment 1: The Effect of Ultrasound on Fermentation (1) Pretreatment of beetroot: After washing the beetroot with 5wt% salt water, slice and crush it to obtain beetroot pulp. The moisture content of the beetroot pulp is controlled to be 100-150% of its solid content. (2) Fermentation of beetroot: Add 1 wt% (based on the weight of the pre-treated beetroot) of Pediococcus lactis HJ516, 0.5 wt% of commercially available Lactobacillus plantarum, 1 wt% of cellulase, 1 wt% of pectinase, and 1 wt% of white sugar to the pre-treated beetroot, stir well, and place it in an ultrasonic device for intermittent multi-frequency ultrasonic (single frequency, dual frequency) assisted fermentation.

[0027] The ultrasonic conditions were as follows: a combination of multiple frequencies of 20KHz, 60KHz, and 80KHz (the differences between different experimental groups lie in the ultrasonic conditions, see Table 1 for details), a power of 50%, ultrasonication for 10 seconds every 5 minutes, a fermentation temperature of 28℃, an initial fermentation pH of 5-7, and a fermentation time of 30-48 hours to obtain the fermentation broth. (3) Preparation of beetroot pigment powder: Add 5 wt‰ maltodextrin and 2 wt‰ ascorbic acid to beetroot fermentation liquid, and spray dry while stirring to obtain beetroot pigment powder.

[0028] It should be noted that the fresh beetroots used in this experiment were pre-treated and therefore contained juice, which formed a slurry. If the raw material is drier, it needs to be soaked in water so that the water content of the beetroot slurry is 100-150% of its solid content.

[0029] The specific effects obtained under different ultrasound conditions are shown in Table 1 below: Table 1. Effects of ultrasound on fermentation

[0030] Red beets belong to the genus *Betula* of the family Chenopodiaceae and are rich in beet pigments, phenols, flavonoids, saponins, and other active substances. Red beets possess certain potential in maintaining health and preventing disease, exhibiting pharmacological effects such as improving athletic performance, promoting cardiovascular health, anti-inflammation, antioxidant properties, and lowering blood pressure. They are also believed to have neuroprotective effects and be used to treat central nervous system-related diseases. However, current research on the prevention and treatment of hyperuricemia with red beets is limited, and few scholars have studied the inhibitory effects of lactic acid bacteria fermentation of red beet juice on the activities of α-amylase / α-glucosidase and xanthine oxidase.

[0031] Ultrasonic-assisted extraction (UART) is a popular extraction method in recent years. It utilizes the cavitation and mechanical effects of ultrasound to damage cell walls, increasing the contact area between the target product and the extract, thereby accelerating the dissolution of the target product and significantly improving the yield. UART is characterized by its time-saving, high efficiency, energy saving, and environmental friendliness. UART is based on the application of ultrasound in the 20-100 kHz range. Ultrasound waves induce cavitation bubbles through the solvent. When these cavitation bubbles break on the sample surface, they damage the cell walls, leading to the dissolution of intracellular substances. This enhances solvent penetration into the cells and improves compound release. Compared to traditional extraction techniques, UART exhibits several advantages, such as lower organic solvent consumption, shorter extraction time, and simpler operation. Therefore, UART is widely used to extract bioactive substances from natural products and is applied in industries such as food and pharmaceuticals.

[0032] However, there are no reports on ultrasound promoting microbial growth or the synthesis of certain microbial metabolites (especially the application of ultrasound in the fermentation of beetroot pigments with dual functions of lowering blood sugar and uric acid). Ultrasound-assisted extraction is a commonly used active ingredient extraction technique in recent years, utilizing cavitation to disrupt cell walls and improve extraction efficiency. This application innovatively applies intermittent multi-frequency ultrasound to the microbial fermentation stage, stimulating lactic acid bacteria metabolism through cavitation and mechanical effects, rather than the traditional extraction process. This application innovatively uses intermittent multi-frequency ultrasound to assist microbial fermentation, preparing beetroot fermented pigments with certain functional properties.

[0033] In this verification experiment, the fixed bacterial strains were 1 wt% *Pediococcus lactis* HJ516 and 1 wt% *Lactobacillus plantarum*. The ultrasonic frequency was varied, and the betaine content of the fermentation broth and the enzyme inhibitory activity of the resulting pigment powder were compared under different ultrasonic conditions. The experimental results showed that: (1) The pigment powder obtained from probiotic fermentation of beet juice has good α-amylase / α-glucosidase inhibition rate and XOD (xanthine oxidase) inhibition rate. In humans, purine nucleotides are degraded in the liver, intestines, and kidneys to form purine bases, which are further converted into uric acid (UA) by XOD catalysis. Increased purine levels in the body lead to the accumulation of UA in serum and stimulate HUA and gout. Therefore, inhibiting XOD activity is crucial for the prevention of UA-mediated diseases (including HUA).

[0034] (2) Ultrasonic-assisted fermentation exhibits the best ability to promote microbial fermentation, increase the content of betalains in beet pigments, and significantly shorten the fermentation time of probiotics, which has a positive effect on improving production efficiency. The effect can be effectively improved under single frequency. The effect is more significant under dual frequency conditions of 20 / 40KHz, 20 / 60KHz, 20 / 80KHz, 40 / 60KHz, 40 / 80KHz, and 60 / 80KHz. The treatment condition of 40 / 60KHz is the best choice. Using 40 / 60KHz dual-frequency ultrasonic-assisted fermentation, the highest betalain content and the best enzyme inhibition activity can be obtained in a shorter fermentation time (30-36h).

[0035] The reasons for this improvement are likely as follows: Ultrasonic treatment enhances the extraction efficiency of beetroot pigment powder not only because the ultrasound-induced cavitation effect damages the beetroot sample tissue and cell walls, increasing the permeability of the extraction solvent, but also because the ultrasound-induced turbulence significantly increases the solid-liquid mass transfer coefficient. Furthermore, the mechanical effect of ultrasound increases the contact surface area between the solid and liquid phases. This is because ultrasound causes the solid sample to break down, leading to an increase in the total surface area and further improving the solvent extraction efficiency. Therefore, the beetroot pigment powder obtained by the method provided in this application has a good ability to inhibit the activity of α-amylase / α-glucosidase and xanthine oxidase, and has the potential to be used as a raw material for developing functional foods that alleviate hyperglycemia and hyperuricemia.

[0036] 2. The beetroot pigment preparation method in this application uses specific fermentation bacteria to obtain the desired effect. The verification experiment is as follows: Verification Experiment 2: The Effect of Lactic Acid Bacteria on Fermentation (1) Pretreatment of beetroot: Wash the beetroot with 5wt% salt water, slice and crush it, and set aside; (2) Fermentation of beetroot: Pretreated beetroot was mixed with 0.5-2 wt% of bacteria (the differences between experimental groups lie in the strains and dosages, see Table 2 for details), 1 wt% cellulase, 1 wt% pectinase, and 0.5 wt% white sugar. After thorough mixing, the mixture was placed in an ultrasonic device for intermittent multi-frequency ultrasonic-assisted fermentation. The ultrasonic conditions were: 40 / 60 kHz (dual-frequency ultrasound), power 75%, ultrasound every 5 minutes for 30 seconds, fermentation temperature 25-30℃, initial fermentation pH 5-7, and fermentation time 36 hours to obtain the fermentation broth. (3) Preparation of beetroot pigment powder: Add 5 wt‰ maltodextrin and 1 wt‰ ascorbic acid to beetroot fermentation liquid, and spray dry while stirring to obtain beetroot pigment powder. The specific effects obtained under different fermentation conditions are shown in Table 2 below: Table 2. Effects of lactic acid bacteria on fermentation

[0037] Beetroot is rich in flavonoids and other pigments, and it has been shown to have some uric acid-lowering effects, but the activity level is not yet excellent. Therefore, the current issue is how to enhance this functional property or endow it with other active functions. Probiotics have unique functions, and their mechanism of action may be that their metabolites have specific effects, and secondly, they can affect the gut microbiota after being ingested by humans.

[0038] The inventors hypothesized that probiotics, through metabolic activity and with ultrasound promoting this metabolism, might lead to the faster degradation, transformation, or binding of certain active ingredients in fruits and vegetables, thus affecting their functional effects. The experimental results, shown in Table 2, indicate that the addition of lactic acid bacteria increases the exudation of betalains from beetroots and enhances their inhibition rate against three enzymes. This is because during fermentation, beneficial metabolites produced during microbial growth and metabolism, or the metabolism of existing substances by the produced enzymes, alter the chemical composition and properties of beet juice. Furthermore, certain components in beets may alter the metabolic pathways of probiotics, thereby changing the chemical characteristics and molecular weight of active ingredients during fermentation, modifying their structure, and ultimately enhancing their bioactivity.

[0039] This application innovatively employs specific bacteria (other types of lactic acid bacteria + Pediococcus lactis HJ516) combined with intermittent multi-frequency ultrasound-assisted microbial fermentation to obtain beetroot fermented pigments possessing the aforementioned specific functional characteristics. The beetroot pigment powder fermented by these two specific mixed strains exhibits a significant blood sugar-lowering effect. Its mechanism of action involves metabolites secreted by the strains during cultivation that inhibit the activity of α-amylase and α-glucosidase, thereby exerting a blood sugar-lowering effect. Secondly, the enzymes produced by the microorganisms during fermentation can disrupt plant cell walls, thereby promoting the dissolution of active substances or the degradation of macromolecules, even transforming them into soluble small molecules.

[0040] In this verification experiment, the ultrasound conditions were fixed at 40 / 60 kHz dual frequency and 75% power, while the types and amounts of lactic acid bacteria were varied. The results are shown in Table 2. The results show that there are significant differences in the effects of different bacterial species. Using other types of lactic acid bacteria alone (Lactobacillus plantarum, Bifidobacterium infantis, and Lactobacillus acidophilus) did not significantly improve the inhibition rate of α-amylase / α-glucosidase, while using Pediococcus lactis HJ516 alone or in combination with other types of lactic acid bacteria significantly improved the inhibition rate of all three enzymes. Furthermore, when the mixed bacterial ratio was 1 wt% Lactobacillus plantarum + (1-2) wt% Pediococcus lactis HJ516, the extraction effect was improved. The combination of 1 wt% Lactobacillus plantarum + 1 wt% Pediococcus lactis HJ516 was the optimal one, with an XOD activity inhibition rate as high as 65.3%, showing the best synergistic effect.

[0041] The possible reasons are as follows: In the fermentation system, molecular collisions easily occur between multiple components, and the secondary metabolites produced by microorganisms during fermentation react with the active ingredients of traditional Chinese medicine, generating new and diverse active substances. However, other types of lactic acid bacteria do not significantly improve the α-amylase inhibition rate and α-glucosidase inhibition rate of beetroot pigment powder. This indicates that there are functional differences among the strains themselves, and only the strains specifically designed in this application can achieve the desired results.

[0042] 3. The beetroot pigment preparation method in this application uses a specific enzyme preparation to achieve the desired effect. The verification experiment is as follows: Verification Experiment 3: The Effect of Enzyme Preparations on Fermentation (1) Pretreatment of beetroot: Wash the beetroot with 5wt% salt water, slice and crush it, and set aside; (2) Fermentation of beetroot: 1 wt% of *Pediococcus lactis* HJ516, 1 wt% of commercially available *Lactobacillus plantarum*, cellulase and pectinase (the difference between different experimental groups lies in the addition of different enzyme preparations, see Table 3 for details), and 0.5 wt% of white sugar were added to the pretreated beetroot. After stirring evenly, the mixture was placed in an ultrasonic device for intermittent multi-frequency ultrasonic-assisted fermentation. The ultrasonic conditions were: 40 / 60 kHz (dual-frequency ultrasound), power of 75%, ultrasonication for 30 seconds every 5 minutes, fermentation temperature of 30℃, initial fermentation pH of 5-7, and fermentation time of 36 hours to obtain the fermentation broth. (3) Preparation of beetroot pigment powder: Add 5 wt‰ maltodextrin and 1 wt‰ ascorbic acid to beetroot fermentation liquid, and spray dry while stirring to obtain beetroot pigment powder. The specific effects obtained under different conditions are shown in Table 3 below: Table 3 Effects of enzyme preparations on fermentation

[0043] Beetroot contains some fiber, and its pericarp contains pectin, which binds some pigments. In order to increase the content of beetroot red in the fermentation broth, different enzyme preparations are used in order to increase the content of active ingredients.

[0044] In the verification experiment, the bacterial strains (1 wt% Pediococcus lactis HJ516 and 1 wt% Lactobacillus plantarum) and ultrasonic conditions (40 / 60 kHz dual frequency) were fixed, while the amounts of cellulase and pectinase added were varied. The experimental results are shown in Table 3. It can be seen that: A combination of 1.5 wt% pectinase and (0.5–1.5) wt% cellulase yielded high betalain content and improved XOD inhibition rate, but had no significant effect on the inhibition rates of α-amylase and α-glucosidase. The optimal combination of 1.5 wt% pectinase and 1 wt% cellulase was found to achieve the highest betalain content and improved XOD inhibition rate. This indicates that the addition of enzymes can slightly affect the composition of the mixture, thereby influencing the activity of the final fermented beetroot pigment powder.

[0045] 4. Verify the effect of the pigment powder prepared in this application on the digestibility of bread: Verification Experiment 4: The Effect of Pigment Powder on Bread Digestibility (1) Pretreatment of beetroot: Wash the beetroot with 5wt% salt water, slice and crush it, and set aside; (2) Fermentation of beetroot: Pretreated beetroot was mixed with 1 wt% of *Pediococcus lactis* HJ516, 1 wt% of commercially available *Lactobacillus plantarum*, 1.5 wt% of cellulase, 1 wt% of pectinase, and 0.5 wt% of white sugar. After thorough mixing, the mixture was placed in an ultrasonic device for intermittent multi-frequency ultrasonic-assisted fermentation. The ultrasonic conditions were: 40 / 60 kHz (dual-frequency ultrasound), power 75%, ultrasonic interval of 30 seconds every 5 minutes, fermentation temperature of 30℃, fermentation pH of 5-7, and fermentation time of 36 hours to obtain the fermentation broth. (3) Preparation of beetroot pigment powder: Add 5wt‰ maltodextrin and 1‰ ascorbic acid to beetroot fermentation liquid, dissolve them completely, and then spray dry while stirring to obtain beetroot pigment powder; (4) Application of beetroot pigment powder in baked goods: Weigh out 250g of high-gluten flour, 10g of sugar, 8g of fresh yeast, 125g of water, and add or omit 2.5-10g of beetroot coloring powder. Slowly stir for 10 minutes until the dough is smooth. Cover with plastic wrap and ferment at 26-28℃ for 45-60 minutes to obtain fermented dough. Divide the fermented dough, roll it into balls, shape it, and place it in a proofing box at 38℃ and 80% humidity for 30 minutes. Finally, bake it in an oven at 210℃ for 35 minutes to obtain bread.

[0046] The performance of breads with different amounts of beetroot coloring powder was tested, and the results are shown in Table 4 below: Table 4. Effects of beetroot pigment powder on bread digestibility

[0047] Starchy foods provide the energy necessary for human survival. However, the potential risk of high blood sugar is associated with high glycemic index (GI) foods. Starch in food is gradually hydrolyzed into glucose by α-amylase and pancreatic enzymes, leading to elevated blood sugar levels. Long-term consumption of high-GI starchy staples may also lead to health problems such as obesity and high blood sugar.

[0048] Because slow-digesting starch (SDS) and resistant starch (RS) are not easily digested by amylase, they cannot be rapidly broken down into glucose after entering the human digestive system, and therefore do not cause a sudden rise in blood sugar levels. This is considered beneficial for people who are sensitive to blood sugar levels or for diabetic patients.

[0049] Table 4 shows that the addition of beetroot pigment powder can inhibit the digestibility of starch in bread to a certain extent. This may be because the starch binds with the pigment, inhibiting the binding of starch with amylase. Furthermore, the pigment has a certain inhibitory effect on amylase, thus reducing the digestibility of starch. Therefore, the beetroot pigment powder obtained in this application can be used as a raw material in functional food processing.

[0050] 5. To verify the effect of ascorbic acid on the pigment powder prepared in this application when applied to bread: Verification Experiment 5: Effect of Ascorbic Acid on the Storage Stability of Beetroot Pigments (1) Pretreatment of beetroot: Wash the beetroot with 5wt% salt water, slice and crush it, and set aside; (2) Fermentation of beetroot: Pretreated beetroot was mixed with 1 wt% of *Pediococcus lactis* HJ516, 1 wt% of commercially available *Lactobacillus plantarum*, 1.5 wt% of cellulase, 1 wt% of pectinase, and 0.5 wt% of white sugar. After thorough mixing, the mixture was placed in an ultrasonic device for intermittent multi-frequency ultrasonic-assisted fermentation. The ultrasonic conditions were: 40 / 60 kHz (dual-frequency ultrasound), ultrasonic time of 10 s, power of 75%, ultrasonic interval of 30 s every 5 min, fermentation temperature of 30℃, fermentation pH of 5-7, and fermentation time of 36 h to obtain the fermentation broth. (3) Preparation of beetroot pigment powder: Add maltodextrin at a mass ratio of 5 wt‰, ascorbic acid and / or citric acid (0.5‰–1‰) at a mass ratio of 1-3‰ to beetroot fermentation broth, and spray dry while stirring to obtain beetroot pigment powder; The experimental groups with different amounts of ascorbic acid were tested, and the results are shown in Table 5 below: Table 5. Effects of ascorbic acid on the storage stability of beetroot pigments

[0051] A common problem with pigments is their sensitivity to the environment, and light generally promotes their decomposition, affecting their color. Ascorbic acid is a common color-protecting agent that can maintain the stability of food color and prevent browning of fruits and vegetables: when added to fruit and vegetable juices, jams, and freshly cut fruits and vegetables, it can inhibit the activity of polyphenol oxidase. It is also a common antioxidant, effectively delaying the oxidative deterioration of food. Table 5 shows the effect of adding different doses of ascorbic acid before spray drying on beetroot red pigments during storage. Ascorbic acid can improve the stability of pigments, especially when used in combination with citric acid. The color-protecting effect is most stable when the addition amounts of ascorbic acid and citric acid are 2‰ and 0.5‰, respectively.

[0052] It should be noted that: In the verification experiment of this paper, commercially available Lactobacillus plantarum, Lactobacillus acidophilus, and Bifidobacterium infantis can be used for other types of lactic acid bacteria. The key point is that a specific type of Pseudococcus lactis HJ516 is used in combination with one of Lactobacillus plantarum, Lactobacillus acidophilus, or Bifidobacterium infantis. For other types of lactic acid bacteria, it is not limited to this. When implementing the scheme of this application, other commercially available strains of other types of lactic acid bacteria (Lactobacillus plantarum, Lactobacillus acidophilus, and Bifidobacterium infantis) can also be used.

[0053] In summary, the solution provided in this application has at least the following design concept and beneficial effects: I. Core Design Concept The core of this application lies in the ingenious construction of a synergistic system of "physical-biological-chemical" interaction, which can be broken down into the following three levels: 1. Physical level (method): Intermittent multi-frequency ultrasound Objective: Not traditional auxiliary extraction, but auxiliary microbial fermentation.

[0054] Concept: Utilizing the cavitation and mechanical effects of specific frequency combinations (at least two of 20kHz, 40kHz, 60kHz, and 80kHz, especially 40 / 60kHz dual-frequency) ultrasound, the fermentation system of beetroot fermentation by lactic acid bacteria is continuously and gently disturbed at the microscopic level. This not only disrupts the beetroot cell wall and promotes substrate release, but more importantly, it stimulates the metabolic activity of lactic acid bacteria, acting as a "physical catalyst."

[0055] 2. Biological level (core): Combination of specific functional strains Objective: To achieve "division of labor and collaboration" of functions.

[0056] Concept: Lactic acid bacteria were not used arbitrarily, but rather a carefully selected combination of *Pediococcus lactis* HJ516 and other commercially available lactic acid bacteria (*Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Bifidobacterium infantis*). Data shows that both are indispensable: HJ516 is primarily responsible for producing hypoglycemic-related active substances (efficiently inhibiting α-amylase / α-glucosidase), while the synergistic effect of both greatly enhances the uric acid-lowering effect (inhibiting XOD). Experiments show that when the mixed bacterial ratio is 1 wt% *Lactobacillus plantarum* + (1-2) wt% *Pediococcus lactis* HJ516, the extraction effect is improved. The optimal combination is 1 wt% *Lactobacillus plantarum* + 1 wt% *Pediococcus lactis* HJ516, with an XOD activity inhibition rate as high as 65.3%, demonstrating the best synergistic effect.

[0057] 3. Chemical and process level (optimization and assurance): Enzymatic hydrolysis-assisted stabilization treatment Objective: To maximize yield and ensure product usability.

[0058] Concept: Adding cellulase and pectinase at the initial stage of fermentation pre-degrades the cell wall structure, providing more sufficient substrate for subsequent microbial fermentation and ultrasonic treatment. Adding ascorbic acid before drying solves the industry pain point of unstable beet red pigment, ensuring the quality of the final product during storage. In particular, the optimized use of ascorbic acid and citric acid in a specific ratio provides the most stable color protection effect and excellent quality retention during storage.

[0059] In summary, the concept of this application is: Using multi-frequency ultrasound designed with a specific frequency as the "engine," a specific mixture of lactic acid bacteria is used as the "converter" to efficiently biotransform beetroots that have undergone enzyme pretreatment. Finally, through stabilization treatment, a new functional raw material with high color value, high stability, and dual biological activity is obtained.

[0060] II. Beneficial Effects 1. Significantly improves core indicators (yield and activity) (1) Betalain content: Under specific conditions (specific dual-frequency ultrasound, specific mixed strains, specific complex enzymes), the betalain content in the fermentation broth was significantly increased, proving that the synergistic system can greatly promote the dissolution and transformation of the target components. Especially under the optimal ultrasound conditions (40 / 60kHz dual frequency, fermentation for 36h), the betalain content in the fermentation broth reached 17.3±0.3 mg / 100mL; if the enzyme preparation is further optimized (1.5wt% cellulase + 1wt% pectinase), the betalain content can be increased to 17.6±0.9mg / 100mL, which is much higher than that of the non-ultrasound group (7.6±0.1 mg / 100mL), proving that the synergistic system can greatly promote the dissolution and transformation of the target components.

[0061] (2) Bioactivity: The obtained pigment powder exhibits excellent enzyme inhibitory activity.

[0062] Blood sugar reduction: The obtained pigment powder has excellent α-amylase inhibition rate. Under optimal conditions, the α-amylase inhibition rate is up to 34.5% and the α-glucosidase inhibition rate is up to 36.8%.

[0063] Uric acid reduction: The obtained pigment powder has a good xanthine oxidase inhibition rate, reaching up to 65.9% under optimal conditions. This achieves the dual potential of a single product to significantly lower blood sugar and uric acid, which is rare in existing technologies.

[0064] 2. Significantly improve production efficiency With the assistance of dual-frequency ultrasound, fermented beetroot pigments with both hypoglycemic and uric acid-lowering functions can be obtained in a shorter fermentation time. In particular, with the assistance of 40 / 60 kHz dual-frequency ultrasound, the fermentation time is shortened from 48 hours to 30-36 hours, which can achieve or even exceed the effect of traditional fermentation of 48 hours, thereby increasing production capacity and reducing energy consumption and time costs.

[0065] 3. Imbue terminal products with functionality Adding the fermented beetroot pigment prepared in this application, which has dual functions of lowering blood sugar and uric acid, to bread can dose-dependently reduce the proportion of rapidly digestible starch and increase the proportion of resistant starch. For example, adding 5g of pigment powder can reduce RDS from 86.3% to 83.1% and increase RS from 6.9% to 9.3%. This directly transforms a natural pigment into a functional ingredient that can regulate the glycemic index of food.

[0066] 4. Greatly improves product storage stability After adding 2‰ ascorbic acid, the betalain retention rate of the pigment powder increased from 61.1% to 94.3% after 180 days of storage under light, solving the problem of the commercial application of easily degradable natural betalain pigment.

[0067] III. Novelty of this application: 1. This application is the first to apply "intermittent multi-frequency ultrasound" technology to the specific system of "lactic acid bacteria fermentation of beetroot," rather than for simple extraction. The background art also clearly points out that ultrasound is mostly used for extraction, and research on its use in promoting microbial fermentation and enhancing the function of its metabolites has not been reported. This application groundbreakingly discovers that a specific combination of *Pediococcus lactis* HJ516 with other types of lactic acid bacteria in a specific ratio exhibits unexpected effects in the ultrasonic fermentation system for the synergistic production of active substances that inhibit α-amylase / α-glucosidase and XOD (data shows that the mixed bacteria effect is far superior to the single bacteria).

[0068] 2. The technical solution of this application organically integrates ultrasound, lactic acid bacteria fermentation, and enzymatic hydrolysis into a novel and mutually reinforcing process chain, producing unexpected technical effects: through the above combination, a synergistic effect of "1+1+1>3" is achieved. This is reflected in: (1) It not only improved the pigment yield, but also “created something out of nothing” or “greatly enhanced” its blood sugar and uric acid lowering biological activity (as can be seen from the comparison of “no bacteria” and “single bacteria” groups).

[0069] (2) While shortening the fermentation time, products with higher levels of active ingredients were obtained, which broke the traditional understanding of fermentation.

[0070] Therefore, this solution solves a long-standing technical problem: efficiently obtaining a safe, natural beetroot pigment food ingredient that has both blood sugar and uric acid lowering functions and good stability.

[0071] 3. This application is applicable to large-scale industrial production and can be widely used in functional products and other fields. The method described in this application uses readily available raw materials, has clear steps, and uses equipment (ultrasonic equipment and spray dryer) that are common in the food industry, making it easy to industrialize.

[0072] The pigment powder prepared in this application can be directly used as a natural pigment in baked goods such as bread, and can simultaneously endow them with the health attributes of "low GI" and "lowering uric acid", thus meeting the huge market demand for natural and healthy functional foods.

[0073] In summary, this application has pioneered a novel method for preparing beetroot pigment powder that combines high color value, high stability, and clear dual biological activities of lowering blood sugar and uric acid. It is suitable for large-scale industrial production and can be widely used in functional products (such as food).

[0074] It should be noted that: The Pediococcus acidilactici HJ516 strain is an existing strain, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 31438 and deposit date of July 24, 2024.

[0075] 2. Testing Method: The specific test methods used in the verification experiments described in this application are as follows: (1) Determination of beet pigment content According to the method of Li Yao et al., the sample was centrifuged at 5000 r / min for 15 min, and the supernatant was used for analysis. 1 mL of sample was diluted in 30 mL of distilled water and shaken for 10 s to mix thoroughly. Values ​​were read at 476 nm and 538 nm using a UV / IS spectrophotometer to analyze betalains and betalains, respectively. Additionally, a wavelength of 600 nm was used to correct for any potential impurities. The following formula was used for calculation:

[0076] Where A is the difference between the absorbance reading at 476 nm or 538 nm and the reading at 600 nm; FD is the dilution factor; I is the optical path length of the cuvette; MM is the molecular weight of the pigments: betalain 550 g / mol, betaflavin 308 g / mol; δ is the molar absorptivity: betalain 60000 L / (mol·cm), betaflavin 48000 L / (mol·cm).

[0077] The method used by Li Yao et al. specifically comes from: Li Yao, Guo Rui, Yan Mingzhe, et al. Changes in quality and antioxidant capacity of red beets during natural fermentation by isolated lactic acid bacteria [J]. Modern Food Science and Technology, 2021, 37(1):207-215.

[0078] (2) Determination of α-glucosidase inhibitory activity First, add 25 μL of 0.1 mol / L PBS buffer (pH 6.8) to the wells of a 96-well plate, then add 25 μL of 20 mmol / L PNPG and 25 μL of sample (CFS); incubate at 37°C for 10 min, then add 50 μL of 0.2 U / mL α-glucosidase; react at 37°C for 15 min, and finally add 100 μL of 0.1 mol / L Na₂CO₃ solution to terminate the reaction. Measure the absorbance (A) of the reaction solution at 405 nm using a microplate reader. Each group was set up in triplicate, with 1 mg / mL acarbose as a positive control. The inhibition rate is shown in the following formula:

[0079]

[0080] In the formula: The blank is PBS + PNPG + PBS + enzyme + Na2CO3; The blank control was PBS + PNPG + PBS + PBS + Na2CO3; The sample consisted of PBS + PNPG + sample + enzyme + Na2CO3; Sample blank is PBS + PNPG + sample + PBS + Na2CO3 (3) α-Amylase inhibitory activity The α-amylase inhibition rate of strains exhibiting good α-glucosidase inhibition rate was determined. 0.25 mL of sample solution was mixed with 1 mg / mL α-amylase solution at a 1:1 volume ratio and incubated at 37 °C for 10 min. The reaction solution was then added to 0.5 mL of 1.5% soluble starch solution at 37 °C and reacted at 37 °C for 5 min. Next, 1 mL of 3,5-dinitrosalicylic acid (DNS) solution was added, and the mixture was reacted in a boiling water bath for 5 min. The mixture was then rapidly cooled to room temperature, and 8 mL of PBS was added. After standing for 30 min, the absorbance was measured at 540 nm. PBS solution (0.1 mol / L, pH = 6.8) was used as a blank control for both the α-amylase solution and the test sample. Three replicates were set up for each group.

[0081]

[0082] Among them, A is the sample group, which contains the sample solution and α-amylase solution; B is the blank sample group, which contains the sample solution but not the α-amylase solution; C is the control group, which does not contain the sample solution but contains the α-amylase solution; and D is the blank group, which does not contain the sample solution or the α-amylase solution.

[0083] (4) XOD inhibition rate Following the method of Chen et al., XOD assay kits (Mengxi Biotechnology Co., Ltd., Suzhou) were used to determine xanthine oxidase (XOD) activity to assess the role of beetroot pigments in reducing uric acid. Specific procedures for determining XOD activity in samples were performed according to the manufacturer's instructions.

[0084] For details of the method used by Chen et al., please refer to: Chen, H., He, J., Li, W., Wang, Z., Du, M., & Kan, J. (2025). Structural and functional characteristics of esterified starch and microencapsulation for urate-lowering probiotic: Effect of hydrophobic side-chain length. Carbohydrate Polymers, 358, 123529. (5) Determination of digestibility The in vitro digestibility of different bread samples was measured using a modified version of the classic method described by Englyst et al. 1 g of bread was heated in a boiling water bath for 10 minutes. After cooling and stabilization in a 37°C water bath, an enzyme preparation was added. The enzyme preparation contained trypsin and amylase prepared according to the Englyst assay procedure. During digestion, the reaction mixture was removed at 20, 40, and 120 min and mixed with twice the volume of ethanol. These mixtures were used as stock solutions for glucose determination. The GOPOD procedure was performed using a resistant starch kit.

[0085] For details on the method used by Englyst et al., please refer to: Englyst, HN, Kingman, SM, & Cummings, JH (1992). Classification and measurement of nutritionally important starch fractions. European journal of clinical nutrition, 46, S33-50. It should be noted that: In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0086] Furthermore, those skilled in the art should understand that although many problems exist in the prior art, each embodiment or technical solution of this application can be improved in only one or a few aspects, without necessarily solving all the technical problems listed in the prior art or background art simultaneously. Those skilled in the art should understand that any content not mentioned in a claim should not be construed as a limitation on that claim.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for preparing a fermented beetroot pigment, characterized in that, Includes the following steps: Beetroot pretreatment: Wash, slice, and crush the beetroot to obtain beetroot pulp; the moisture content of the beetroot pulp should be controlled to be 100-150% of its solid content. Beetroot fermentation: Bacteria are inoculated into the beetroot slurry, and cellulase, pectinase and white sugar are added. After stirring evenly, fermentation is carried out under intermittent multi-frequency ultrasound to obtain fermentation broth. The frequency combination of the intermittent multi-frequency ultrasound is at least two of 20KHz, 40KHz, 60KHz, and 80KHz; the bacteria is Pediococcus lactis HJ516 or a combination of bacteria, wherein the combination of bacteria includes Pediococcus lactis HJ516 and other types of lactic acid bacteria, wherein the other types of lactic acid bacteria are Lactobacillus plantarum, Lactobacillus acidophilus, or Bifidobacterium infantis; Pigment powder preparation: Maltodextrin and acidic substances are added to the fermentation broth, dissolved and mixed, and then dried to obtain the fermented beetroot pigment powder; wherein the acidic substances are ascorbic acid and / or citric acid.

2. The preparation method according to claim 1, characterized in that, The intermittent multi-frequency ultrasound is characterized by the use of a dual-frequency combination of 40kHz and 60kHz.

3. The preparation method according to claim 1, characterized in that, The amount of *Pediococcus lactis* HJ516 added is 0.5-2% of the weight of the beet root slurry.

4. The preparation method according to claim 3, characterized in that, The composite bacterial strain combination consists of Pediococcus lactis HJ516 and other types of lactic acid bacteria, wherein the other types of lactic acid bacteria are Lactobacillus plantarum; wherein the mass ratio of the other types of lactic acid bacteria to Pediococcus lactis HJ516 is 1:(1-2).

5. The preparation method according to claim 4, characterized in that, The amount of *Pediococcus lactis* HJ516 added is 1% of the mass of beetroot pulp, and the mass ratio of *Lactobacillus plantarum* to *Pediococcus lactis* HJ516 is 1:

1.

6. The preparation method according to claim 1, characterized in that, The amount of cellulase added is 0.5-1.5% of the weight of the beet root slurry; wherein the mass ratio of the pectinase to the cellulase is 1.5:(0.5-1.5). The acidic substances are ascorbic acid and citric acid; wherein the amount of ascorbic acid added is 1 to 3‰ of the mass of the fermentation broth, and the mass ratio of ascorbic acid to citric acid is (1 to 3): (0.5 to 1).

7. The preparation method according to claim 6, characterized in that, The amount of cellulase added is 1% of the mass of beet root slurry; the mass ratio of pectinase to cellulase is 1.5:

1. The amount of ascorbic acid added is 2‰ of the mass of the fermentation broth, and the amount of citric acid added is 0.5‰ of the mass of the fermentation broth; The amount of white sugar added is 0.5-1% of the mass of beetroot slurry; the amount of maltodextrin added is 0.5% of the mass of fermentation liquid.

8. A fermented beetroot pigment powder prepared by any one of claims 1 to 7.

9. The application of the fermented beetroot pigment powder as described in claim 8 in the preparation of baked goods.

10. The application according to claim 9, characterized in that, The baked goods are bread; the fermented beetroot pigment powder is added to the bread at a rate of 1-4% of the flour mass.

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