Fermented foxtail millet and preparation method thereof

Abstract

The invention belongs to the technical field of food processing, and particularly relates to a foxtail millet leavening and a preparation method thereof, and the preparation method comprises the following steps: carrying out enzymolysis on foxtail millet, and sterilizing to obtain foxtail millet enzymatic hydrolysate; inoculating a mixed seed solution of non-saccharomyces cerevisiae and lactic acid bacteria into the millet enzymatic hydrolysate, and fermenting to obtain a millet fermented product; according to the preparation method provided by the invention, the content of nutritional ingredients such as total phenols, flavones and the like of the foxtail millet is increased, the foxtail millet has various flavor substances, can be used as a raw material of prebiotics or synbiotics, is further processed into synbiotic products, and has a wide application prospect in the fields of food industry and health care products.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and particularly relates to a millet ferment and a preparation method thereof. Background Art

[0002] With the continuous pursuit of healthy diet by people, cereal raw materials with rich nutritional value have received more and more attention. Unprocessed cereal raw materials contain rich dietary fiber, protein, fat, vitamin E, calcium, iron, zinc and other minerals, and also contain secondary metabolites such as flavonoids and polyphenols, which have various health care functions such as lowering blood sugar, lowering blood lipids, promoting intestinal peristalsis, and enhancing antioxidant capacity. Generally, the nutritional substances will be lost and the health benefits will decline during the hulling and fine processing of cereals.

[0003] As a main cereal food, millet contains bioactive substances such as flavonoids and phenolic acids, which have health benefits such as antioxidant. Since the taste of directly eating millet is not good enough and the flavor is poor, consumers usually eat millet obtained after hulling millet. At present, the main processing methods of millet are physical processing or heat treatment processing. Physical processing methods such as milling and dehulling, ultrafine grinding may lead to the loss of millet nutrients due to overprocessing, accelerated oxidation, and easy rancidity of lipids; heat treatment processing methods such as steaming, baking, extrusion and puffing may cause protein denaturation in millet, destruction of heat-sensitive nutrients, and easy generation of Maillard reaction by-products (such as acrylamide, which is neurotoxic to both humans and animals). Therefore, it is necessary to develop more processing methods for millet to improve the nutritional value of millet products and improve the flavor of millet. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a millet ferment and a preparation method thereof. Using millet as a raw material, first enzymatically hydrolyze the millet, and obtain a millet enzymatic hydrolysate after sterilization, then inoculate a mixed seed solution of non-Saccharomyces yeast and lactic acid bacteria into the millet enzymatic hydrolysate, and obtain a millet ferment after fermentation. The preparation method of the present invention improves the flavor, antioxidant capacity, and the content of nutritional components such as total phenols and flavonoids of millet. The obtained millet ferment can be used as a raw material for prebiotics or synbiotics, and further processed into synbiotic products, which has broad application prospects in the food industry and the health product field.

[0005] The first aspect of the present invention provides a preparation method of a millet ferment, comprising the following steps: Enzymatically hydrolyze the millet, and obtain a millet enzymatic hydrolysate after sterilization; Mix the non-Saccharomyces yeast seed solution and the lactic acid bacteria seed solution, and inoculate them into a domestication medium for culture to obtain a mixed seed solution of non-Saccharomyces yeast and lactic acid bacteria; Inoculate the mixed seed solution of non-Saccharomyces yeast and lactic acid bacteria into the millet enzymatic hydrolysate for fermentation to obtain the millet ferment; The viable count of non - Saccharomyces yeast in the non - Saccharomyces yeast seed liquid is at least 10 6 CFU / mL; the viable count of lactic acid bacteria in the lactic acid bacteria seed liquid is at least 10 6 CFU / mL.

[0006] Preferably, the non - Saccharomyces yeast is Wickerhamomyces anomalus strain Wa 27, Pichia guilliermondii strain Pg6 - 2, Pichia kudriavzevii strain Pk 4 - 2, Pichia kudriavzevii strain Pk 2 - 3, Pichia kluyveri strain Pkl 8 - 3 or Metschnikowia pulcherrima strain Mp 6; the lactic acid bacteria is Lactobacillus plantarum Lp 9 - 1 strain.

[0007] Preferably, the non - Saccharomyces yeast is Wickerhamomyces anomalus strain Wa 27.

[0008] Preferably, the inoculation amount of the mixed seed liquid is 2% v / v - 10% v / v; the fermentation temperature is 28°C - 36°C; the fermentation time is 0.5 d - 30 d.

[0009] Preferably, the inoculation amount of the mixed seed liquid is 6% v / v; the fermentation temperature is 32°C; the fermentation time is 4 d.

[0010] Preferably, the volume ratio of the non - Saccharomyces yeast seed liquid to the lactic acid bacteria seed liquid is 0.1 - 10:1.

[0011] Preferably, the enzymatic hydrolysis step is to sequentially add α - amylase, glucoamylase, cellulase, and protease to the millet slurry; based on the millet slurry, the addition amount of α - amylase is 10 U / mL - 30 U / mL, the addition amount of glucoamylase is 100 U / mL - 300 U / mL, the addition amount of cellulase is 65 U / g - 85 U / g, and the addition amount of protease is 400 U / g - 600 U / g.

[0012] Preferably, the addition amount of α - amylase is 20 U / mL, the addition amount of glucoamylase is 250 U / mL, the addition amount of cellulase is 80 U / g, and the addition amount of protease is 450 U / g.

[0013] Preferably, the enzymatic hydrolysis temperature of α - amylase is 70°C - 90°C, and the enzymatic hydrolysis time of α - amylase is 0.5 h - 8 h.

[0014] Preferably, the enzymatic hydrolysis temperature of α - amylase is 80°C; the enzymatic hydrolysis time of α - amylase is 1 h.

[0015] Preferably, the enzymatic hydrolysis temperature of the glucoamylase, cellulase and protease is 40°C to 65°C; the enzymatic hydrolysis time of the glucoamylase, cellulase and protease is 1 h to 24 h.

[0016] Preferably, the enzymatic hydrolysis temperature of the glucoamylase, cellulase and protease is 55°C; the enzymatic hydrolysis time of the glucoamylase, cellulase and protease is 4 h.

[0017] Preferably, the domestication medium is a medium obtained by mixing rice slurry supernatant and water in a volume ratio of 0.25 to 4:1 and sterilizing.

[0018] Preferably, the volume ratio of the rice slurry supernatant to water in the domestication medium is 1:2.

[0019] Preferably, the rice slurry supernatant is the supernatant after centrifuging the millet enzymatic hydrolysate at 3500 - 4500 r / min for 8 - 12 min.

[0020] Preferably, the rice slurry supernatant is the supernatant after centrifuging the millet enzymatic hydrolysate at 4000 r / min for 10 min.

[0021] Preferably, the material - liquid ratio of millet to water in the millet slurry is 1 g:6 - 10 mL; the millet slurry is obtained by pre - cooking millet and then pulping or directly pulping millet; pre - cooking can soften the husk and grains of millet, facilitating subsequent pulping, enzymatic hydrolysis and microbial fermentation.

[0022] Preferably, the material - liquid ratio of millet to water in the millet slurry is 1 g:8 mL.

[0023] In the second aspect of the present invention, there is provided a fermented millet product prepared by the preparation method of any one of the above.

[0024] In the third aspect of the present invention, there is provided the application of the above - mentioned fermented millet product in the preparation of synbiotics.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: Based on the problems of nutritional value loss and poor flavor in the products developed from current millet, the present invention provides a processing method for millet. The present invention uses unhulled millet as raw material, which can retain more nutrients; first, the millet is ground into slurry, and the millet slurry is successively enzymolyzed by α-amylase, glucoamylase, cellulase and protease to decompose macromolecular substances such as starch and protein in the millet into small molecules that are more easily utilized by microorganisms, thereby improving the fermentation efficiency and releasing nutrients such as polyphenols in the millet through biotransformation by microorganisms to improve the nutritional value. Subsequently, sterilization is carried out to eliminate the interference of miscellaneous bacteria and ensure a pure fermentation environment; after inoculating a mixed seed liquid of non-Saccharomyces cerevisiae and lactic acid bacteria into the millet enzymolyzed liquid and fermenting, a millet fermented product is obtained. The present invention discovers through experiments that, compared with the fermentation by a single Lactobacillus plantarum Lp 9-1 strain, the mixed fermentation of non-Saccharomyces cerevisiae (Wickerhamomyces anomalus strain Wa 27, Pichia guilliermondii strain Pg 6-2, Pichia kudriavzevii strain Pk 4-2, Pichia kudriavzevii strain Pk 2-3, Pichia kluyveri strain Pkl 8-3 or Metschnikowia pulcherrima strain Mp6) and lactic acid bacteria (Lactobacillus plantarum Lp 9-1) can improve the contents of nutrients such as polyphenols in millet and the antioxidant capacity, and increase the contents of aroma substances such as higher alcohols and esters, making the fermented product have richer fruit-like aromas and more complex tastes.

[0026] The present invention discovers through experiments that after inoculating a mixed seed fermentation liquid of Wickerhamomyces anomalus strain Wa 27 and Lactobacillus plantarum Lp 9-1 strain into the millet enzymolyzed liquid and fermenting, the total phenol content and ABTS + radical scavenging ability of the millet fermented product are significantly stronger than those of other fermentation combinations, and can increase the contents of aldehydes, ketones, volatile phenols and terpenes, with lower higher alcohol content and appropriate ester content, enhancing the aroma complexity and taste richness of the millet fermented product. On the one hand, the present invention discovers for the first time that Wickerhamomyces anomalus strain can ferment to produce 5-nonanol, 1-nonanal and acetal, which can provide grassy aroma, citrus fruit flavor and freshness for the fermented product, improving the flavor level of the millet fermented product, and the Wa 27 + Lp 9-1 combined fermentation can produce hexanol, 5-nonanol, ethyl palmitate, acetal and 1-nonanal that other fermentation combinations do not have. On the other hand, the Wa 27 + Lp 9-1 combined fermentation increases the content of 1-nonanol and is accompanied by the production of 1-hexanol, providing banana, floral aroma and sweetness for the fermentation system and enriching the aroma of the fermented liquid; moreover, the content of the terpenoid compound geraniol after the combined fermentation of this combination reaches 16.20 ± 2.73 μg / L. Geraniol not only has a pleasant rose aroma, but also has various physiological activities such as antioxidant, anti-inflammatory and anti-cancer potential.

[0027] Compared with the millet ferment obtained by the same preparation steps using millet as the raw material, the total phenol content, flavonoid content, ABTS ＋ and DPPH free radical scavenging ability of the foxtail millet ferment are higher than those of the millet ferment, indicating that the foxtail millet ferment has higher nutritional value than the millet ferment. The foxtail millet ferment provided by the present invention can be used as a raw material for prebiotics or synbiotics and further processed into synbiotic products, having broad application prospects in the food industry and the health product field. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Effect of the solid-to-liquid ratio on the enzymatic hydrolysis effect of the foxtail millet slurry.

[0029] Figure 2 Effect of the addition amount of α-amylase on the enzymatic hydrolysis effect of the foxtail millet slurry.

[0030] Figure 3 Effect of the addition amount of glucoamylase on the enzymatic hydrolysis effect of the foxtail millet slurry.

[0031] Figure 4 Effect of the addition amount of cellulase on the enzymatic hydrolysis effect of the foxtail millet slurry.

[0032] Figure 5 Effect of the addition amount of protease on the enzymatic hydrolysis effect of the foxtail millet slurry.

[0033] Figure 6 Effect of the inoculum size on the titratable acid content, total phenol content and ABTS + scavenging ability.

[0034] Figure 7 Effect of the fermentation temperature on the titratable acid content, total phenol content and ABTS + scavenging ability.

[0035] Figure 8 Effect of the fermentation time on the titratable acid content, total phenol content and ABTS + scavenging ability.

[0036] Figure 9 Effect of different strain combination fermentation systems on the titratable acid content of the foxtail millet ferment.

[0037] Figure 10 Effect of different strain combination fermentation systems on the total phenol content of the foxtail millet ferment.

[0038] Figure 11 Effect of different strain combination fermentation systems on the ABTS + free radical scavenging ability of the foxtail millet ferment.

[0039] Figure 12 Effect of different strain combinations on the number of aroma substances in fermented foxtail millet

[0040] Figure 13 Sensory analysis results of fermented foxtail millet obtained from different strain combinations

[0041] Figure 14 Changes in viable cell counts in fermented foxtail millet and fermented millet. Different capital letters represent significant differences between different fermentation time points in fermented foxtail millet, and different lowercase letters represent significant differences between different fermentation time points in fermented millet, p <0.05

[0042] Figure 15 Changes in titratable acid content in fermented foxtail millet and fermented millet. Different capital letters represent significant differences between different fermentation time points in fermented foxtail millet, and different lowercase letters represent significant differences between different fermentation time points in fermented millet, p <0.05

[0043] Figure 16 Changes in reducing sugar content in fermented foxtail millet and fermented millet. Different capital letters represent significant differences between different fermentation time points in fermented foxtail millet, and different lowercase letters represent significant differences between different fermentation time points in fermented millet, p <0.05

[0044] Figure 17 Changes in total phenol content in fermented foxtail millet and fermented millet. Different capital letters represent significant differences between different fermentation time points in fermented foxtail millet, and different lowercase letters represent significant differences between different fermentation time points in fermented millet, p <0.05

[0045] Figure 18 Changes in flavonoid content in fermented foxtail millet and fermented millet. Different capital letters represent significant differences between different fermentation time points in fermented foxtail millet, and different lowercase letters represent significant differences between different fermentation time points in fermented millet, p <0.05

[0046] Figure 19 Changes in free radical scavenging ability in fermented foxtail millet and fermented millet. Different capital letters represent significant differences between different fermentation time points in fermented foxtail millet, and different lowercase letters represent significant differences between different fermentation time points in fermented millet, p <0.05 Detailed implementation methods

[0047] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the protection scope of the present invention is not limited by the specific embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0048] In the present invention, foxtail millet refers to the grain with the hard inedible glume on the outer layer not removed; millet refers to the grain with the outer glume removed.

[0049] As a major cereal food, foxtail millet contains bioactive substances such as flavonoids and phenolic acids, which have health benefits such as antioxidant effects. Since the taste of directly eating foxtail millet is not good and the flavor is poor, consumers usually eat millet obtained after dehulling foxtail millet at present. This conventional operation will reduce the overall nutritional value of foxtail millet. Therefore, it is necessary to develop more processing methods for foxtail millet to improve the nutritional value of foxtail millet products and improve the flavor of foxtail millet.

[0050] The present invention provides a foxtail millet ferment and a preparation method thereof. First, foxtail millet is enzymatically hydrolyzed, and after sterilization, a foxtail millet enzymatic hydrolysate is obtained. Then, a mixed seed solution of non - Saccharomyces yeasts and lactic acid bacteria is inoculated into the foxtail millet enzymatic hydrolysate, and after fermentation, a foxtail millet ferment is obtained. The preparation method of the present invention improves the flavor, antioxidant capacity, and the contents of nutrients such as total phenols and flavonoids of foxtail millet. The prepared foxtail millet ferment can be used as a raw material for prebiotics or synbiotics and can be further processed into synbiotic products, having broad application prospects in the food industry and the health product field.

[0051] Example 1: A preparation method of a foxtail millet ferment I. Experimental materials and methods 1. Test materials Foxtail millet was purchased from Jinzhong, Shanxi, China; α - amylase (155823 U / mL), glucoamylase (153394 U / mL), cellulase (11649 U / g), and protease (150768 U / g) were all purchased from Xiasheng Industrial Group Co., Ltd.

[0052] The present invention selects Lactobacillus plantarum Lp 9 - 1 as the lactic acid bacteria strain; selects Wickerhamomyces anomalus Wa 27, Pichia guilliermondii Pg 6 - 2, Pichia kudriavzevii Pk 4 - 2, Pichia kudriavzevii Pk 2 - 3, Pichia kluyveri Pkl 8 - 3, or Metschnikowia pulcherrima Mp 6 as the non - Saccharomyces yeast strain.

[0053] The strain information of Lactobacillus plantarum Lp 9-1 can be found in "Li, Y., Fu, W., Du, H., et al. Metabolic profile and antioxidant properties of grape juice subjected to long-fermentation with Lactiplantibacillus plantarum strain isolated from traditional pickled vegetables[J]. Food Bioscience, 2024: 105455.”; The strain of Wickerhamomyces anomalus ( Wickerhamomyces anomalus ), referred to as Wa 27 in the present invention, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 12, 2022, with the deposit number CGMCC No. 25528. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The taxonomic name: Wickerhamomyces anomalus .

[0054] The strain information of Metschnikowia pulcherrima Mp 6 can be found in "Bai X, Gao H, Lan M, et al. Metabolomics and flavor diversity of Viognier wines co-fermented with Saccharomyces cerevisiae from different sources of Metschnikowia pulcherrima strains[J]. Food Bioscience, 2025, 64: 105904.”; The strain information of Pichia guilliermondii Pg 6-2, Pichia kudriavzevii Pk 4-2, Pichia kudriavzevii Pk 2-3, and Pichia kluyveri Pkl 8-3 can be found in "Bai, X., Gao, H. F., Li, X., et al. The effect of different non-Saccharomyces strains on the flavour characteristics of mead. Acta Alimentaria, 2024, 53(1): 46-60.”.

[0055] 2. Preparation method of foxtail millet fermentate S1. Remove impurities from foxtail millet, wash it, pre-cook for 30 min, and beat it into a pulp to obtain foxtail millet slurry; S2. Enzymatically hydrolyze the millet slurry, and sterilize it to obtain a millet enzymolysis solution. S3. Inoculate a mixed seed solution of non - Saccharomyces cerevisiae and lactic acid bacteria into the millet enzymolysis solution, and obtain a millet fermentation product after fermentation.

[0056] 3. Exploration of enzymatic hydrolysis test conditions 3.1 Effect of solid - liquid ratio on the enzymatic hydrolysis effect of millet slurry Under the conditions of an α - amylase addition amount of 20 U / mL and an enzymatic hydrolysis temperature of 80 °C, millet slurries with solid - liquid ratios of 1 g:6 mL, 1 g:7 mL, 1 g:8 mL, 1 g:9 mL, and 1 g:10 mL were enzymatically hydrolyzed for 1 h, and then the DE value (glucose value) of the millet enzymolysis solution was measured.

[0057] 3.2 Effect of α - amylase addition amount on the enzymatic hydrolysis effect of millet slurry When the solid - liquid ratio is constant and the enzymatic hydrolysis temperature is 80 °C, millet slurries with α - amylase addition amounts of 10 U / mL, 15 U / mL, 20 U / mL, 25 U / mL, and 30 U / mL were enzymatically hydrolyzed for 1 h, and then the DE value of the millet enzymolysis solution was measured.

[0058] 3.3 Effect of glucoamylase addition amount on the enzymatic hydrolysis effect of millet slurry After the α - amylase enzymatic hydrolysis is completed, under the condition of an enzymatic hydrolysis temperature of 55 °C, millet slurries with glucoamylase addition amounts of 100 U / mL, 150 U / mL, 200 U / mL, 250 U / mL, and 300 U / mL were enzymatically hydrolyzed for 4 h, and then the total sugar content of the millet enzymolysis solution was measured.

[0059] 3.4 Effect of cellulase addition amount on the enzymatic hydrolysis effect of millet slurry After the α - amylase enzymatic hydrolysis is completed, under the conditions of a constant glucoamylase addition amount and an enzymatic hydrolysis temperature of 55 °C, millet slurries with cellulase addition amounts of 65 U / g, 70 U / g, 75 U / g, 80 U / g, and 85 U / g were enzymatically hydrolyzed for 4 h, and then the total sugar content of the millet enzymolysis solution was measured.

[0060] 3.5 Effect of protease addition amount on the enzymatic hydrolysis effect of millet slurry After the α - amylase enzymatic hydrolysis is completed, under the conditions of constant glucoamylase and cellulase addition amounts and an enzymatic hydrolysis temperature of 55 °C, millet slurries with protease addition amounts of 400 U / g, 450 U / g, 500 U / g, 550 U / g, and 600 U / g were enzymatically hydrolyzed for 4 h, and then the DH value (degree of hydrolysis) of the millet enzymolysis solution was measured.

[0061] 4. Selection of fermentation conditions 4.1. Preparation of mixed seed liquid: Select Wickerhamomyces anomalus Wa 27 as non-Saccharomyces cerevisiae, activate it and Lactobacillus plantarum Lp9-1 in their respective media, and then culture them in the seed medium for 24 h to ensure that the colony concentration is above 10 6 CFU / mL. Take the activated seed liquid of Wickerhamomyces anomalus Wa 27 (cultured at 28 °C for 24 h) and the activated seed liquid of Lactobacillus plantarum Lp9-1 (cultured at 37 °C for 24 h), mix them in a 1:1 ratio, and inoculate them into the acclimation medium to prepare the mixed seed liquid. Among them, the acclimation medium is prepared by mixing rice slurry supernatant and water in a volume ratio of 1:2. The rice slurry supernatant is the supernatant after centrifuging the millet enzymatic hydrolysate at 4000 r / min for 10 min.

[0062] 4.2. Inoculum size Add 2% (v / v), 4% (v / v), 6% (v / v), 8% (v / v), 10% (v / v) of the mixed seed liquid to the millet enzymatic hydrolysate respectively, and measure the titratable acid content, total phenol content and ABTS ＋ radical scavenging ability of the obtained millet fermented product after constant temperature fermentation at 32 °C for 30 h.

[0063] 4.3. Fermentation temperature Add 6% (v / v) of the mixed seed liquid to the millet enzymatic hydrolysate respectively, and measure the titratable acid content, total phenol content and ABTS ＋ radical scavenging ability of the millet fermented product after constant temperature fermentation at 28 °C, 30 °C, 32 °C, 34 °C, 36 °C for 30 h.

[0064] 4.4. Fermentation time Add 6% (v / v) of the mixed seed liquid to the millet enzymatic hydrolysate respectively, and carry out constant temperature fermentation at 32 °C. Starting from 12 h of fermentation, sample every 6 h and then measure the titratable acid content, total phenol content and ABTS ＋ radical scavenging ability.

[0065] 5. Index determination 5.1. Determination of DE value, total sugar content and DH value Determine the DE value of the fermented product according to the method for the determination of reducing power and glucose equivalent of starch hydrolysis products in GB / T 22428.1-2008; determine the total sugar content of the fermented product according to GB / T 15038-2006; use the formaldehyde titration method for the determination of DH value.

[0066] 5.2. Determination of titratable acid content Mix 5 mL of the sample with 25 mL of distilled water, and titrate with 0.05 mol / L NaOH until the pH value of the mixed solution reaches 8.2. At the same time, use distilled water for a blank test, and the result is calculated as the content of tartaric acid (g / L).

[0067] 5.3 Total phenol content Based on the principle of the Folin-phenol colorimetric method, add 0.2 mL of the sample to 0.5 mL of 0.2 mol / L Folin-phenol solution, then add 1 mL of 17 w / w% sodium carbonate solution, and then add 2.3 mL of distilled water. At the same time, use distilled water for a blank control. After placing it in the dark for 1 h, measure the absorbance value at 765 nm, establish a standard curve between the gallic acid concentration and the absorbance value, and the formula is y = 0.0008x + 0.0617, R 2 = 0.998, where x represents the gallic acid concentration and y represents the absorbance value. Use the standard curve prepared with gallic acid to convert the measured absorbance value into the total phenol content.

[0068] 5.4 ABTS + and DPPH radical scavenging ability The antioxidant activity was determined using an ABTS radical scavenging ability detection kit and a DPPH radical scavenging ability detection kit.

[0069] 5.5 Detection of volatile substances Place 8 mL of the millet fermentation sample in a headspace vial, then add 2.4 g of sodium chloride and 20 μL of 2-octanol (12 μL / 100 mL) into the headspace vial, place it in a water bath at 40 °C for extraction for 30 minutes, and use gas chromatography-mass spectrometry (GC-MS) to detect volatile compounds.

[0070] 5.6 Sensory evaluation Ten judges (5 females and 5 males) conducted a sensory evaluation of the fermented product: Take 50 mL of the fermented product sample and place it in a colorless, transparent, clean, and dry beaker, and evaluate the fermented product from five aspects: acidity, odor, acceptance, chromaticity, and taste using a 9-point system.

[0071] 6 Statistical analysis Use Excel 2021 version to organize the experimental data, and use IBM SPSS Statistics 26.0 to perform statistical analysis on the data, p <0.05 is considered to have a significant difference; the graphs are drawn using Origin 2021.

[0072] II. Results and analysis 1. Enzymatic pretreatment of millet slurry 1.1 Effect of solid-liquid ratio on the enzymatic hydrolysis effect of millet slurry The influence of the solid-liquid ratio on the enzymatic hydrolysis effect of millet slurry is shown in Figure 1 . When the enzymatic hydrolysis temperature (80 °C), the addition amount of α-amylase (20 U / mL), and the enzymatic hydrolysis time (1 h) are constant, when the solid-liquid ratio increases from 1 g:6 mL to 1 g:7 mL, the DE value has no obvious change. It is speculated that when the moisture content is too low, the slurry during the gelatinization process will be too viscous, which is not conducive to stirring. When the solid-liquid ratio increases from 1 g:7 mL to 1 g:8 mL, the DE value shows a significant increase and reaches the peak value ( p <0.05). As the solid-liquid ratio increases, the DE value decreases. Therefore, the most suitable solid-liquid ratio is 1 g:8 mL.

[0073] 1.2 Influence of the addition amount of α-amylase on the enzymatic hydrolysis effect of millet slurry The influence of the addition amount of α-amylase on the enzymatic hydrolysis effect of millet slurry is shown in Figure 2 . When the enzymatic hydrolysis temperature (80 °C), the solid-liquid ratio (1 g:8 mL), and the enzymatic hydrolysis time (1 h) are constant, as the addition amount of α-amylase increases from 10 U / mL to 20 U / mL, the interaction between α-amylase and starch in the millet substrate is continuously enhanced, thereby converting more starch into reducing sugars, resulting in a significant increase in the DE value at this stage ( p <0.05). After reaching the maximum value when the addition amount is 20 U / mL, it tends to be stable. Therefore, the addition amount of amylase selected is 20 U / mL.

[0074] 1.3 Influence of the addition amount of glucoamylase on the enzymatic hydrolysis effect of millet slurry: The influence of the addition amount of glucoamylase on the enzymatic hydrolysis effect of millet slurry is shown in Figure 3 . After the enzymatic hydrolysis of α-amylase is completed, when the solid-liquid ratio (1 g:8 mL), the enzymatic hydrolysis temperature (55 °C), and the enzymatic hydrolysis time (4 h) are constant, as the addition amount of glucoamylase increases, the starch and starch-like substances in the system are further converted into monosaccharides that can be utilized by microorganisms for fermentation, thereby increasing the total sugar content in the system. When the addition amount of glucoamylase is 250 U / mL, the total sugar content reaches the maximum value ( p <0.05). Therefore, the optimal addition amount of glucoamylase is 250 U / mL.

[0075] 1.4 Influence of the addition amount of cellulase on the enzymatic hydrolysis effect of millet slurry The influence of the addition amount of cellulase on the enzymatic hydrolysis effect of millet slurry is shown in Figure 4。When the enzymatic hydrolysis of α - amylase ends, when the solid - liquid ratio (1 g: 8 mL), enzymatic hydrolysis temperature (55 °C), addition amount of glucoamylase (250 U / mL) and enzymatic hydrolysis time (4 h) are fixed, as the addition amount of cellulase increases from 65 U / g to 80 U / g, lower - molecular - weight sugars and other degradation products will be formed in the system, and the total sugar content at this stage increases significantly ( p <0.05), and the total sugar content reaches the maximum value when the addition amount is 80 U / g. Therefore, the optimal addition amount of cellulase is 80 U / mL.

[0076] 1.5. Influence of the addition amount of protease on the enzymatic hydrolysis effect of millet slurry Protease can cleave proteins into smaller fragments, making them easier to be absorbed by cells. The influence of the addition amount of protease on the enzymatic hydrolysis effect of millet slurry is shown in Figure 5 。

[0077] When the enzymatic hydrolysis of α - amylase ends, when the solid - liquid ratio (1 g: 8 mL), enzymatic hydrolysis temperature (55 °C), addition amount of glucoamylase (250 U / mL), addition amount of cellulase (80 U / g) and enzymatic hydrolysis time (4 h) are fixed, adding protease can hydrolyze the proteins in the millet slurry into free peptides and amino acids. When the addition amount of protease increases from 400 U / g to 450 U / g, the DH value shows a significant increase ( p <0.05), and it tends to be stable as the addition amount increases. On the basis of comprehensively considering the raw material cost and enzymatic hydrolysis effect, the appropriate dosage of protease was determined to be 450 U / g for the next experiment.

[0078] 2. Influence of combined fermentation conditions on millet fermented products 2.1. Inoculum size When the fermentation temperature (32 °C) and fermentation time (30 h) are fixed, as the inoculum size of the mixed seed liquid of the strains increases, the total phenol content and ABTS + radical scavenging ability in the fermentation system both show a trend of increasing first and then decreasing ( Figure 6 ). Within a certain range, increasing the inoculum size allows the strains to quickly utilize the fermentation substrates for metabolism and promote the synthesis of phenols, thereby improving the antioxidant capacity of the millet fermented products. However, too high an inoculum size will lead to nutritional competition among the strains on limited substrates, thus affecting the synthesis of phenolic substances. Therefore, the inoculum size of the mixed seed liquid is preferably 6%.

[0079] 2.2. Fermentation temperature The optimal fermentation temperatures of Lactobacillus plantarum and non - Saccharomyces cerevisiae are generally different. Low temperature has a certain impact on the activity of Lactobacillus plantarum, and too high a temperature will inhibit the growth and metabolic activity of non - Saccharomyces cerevisiae. From Figure 7It can be seen that when the inoculation amount (6%) and the fermentation time (30 h) are fixed, at 32 °C, the titratable acid content, total phenol content and ABTS + radical scavenging ability of the millet fermented product all reach the maximum value.

[0080] 2.3. Fermentation time The fermentation time affects the reproduction and metabolism of the strain. As Figure 8 shown, when the inoculation amount (6%) and the fermentation temperature (32 °C) are fixed, with the continuous extension of the fermentation time, the titratable acid content of the millet fermented product shows a stable increase, and the polyphenol content and ABTS + radical scavenging ability show a fluctuating state. The titratable acid content, polyphenol content and ABTS + radical scavenging ability indexes all reach the maximum value at 96 hours of fermentation. Continuing to extend the fermentation time, the titratable acid content of the millet fermented product has no significant change, but the polyphenol content and ABTS + radical scavenging ability significantly decreases (<0.05). Therefore, the fermentation time of 96 hours is selected for subsequent fermentation experiment exploration. p <0.05). Therefore, the fermentation time of 96 hours is selected for subsequent fermentation experiment exploration.

[0081] 3. Co-fermentation of Lactobacillus plantarum and different non-Saccharomyces cerevisiae strains 3.1. Titratable acid content The influence of different co-fermentation systems on the titratable acid content of the millet fermented product is as Figure 9 shown. After 24 - 48 hours of fermentation, the titratable acid content of the millet fermented product obtained by all fermentation combinations significantly increases (<0.05), because Lactobacillus plantarum fermentation can produce a large amount of lactic acid. When the fermentation is extended to 96 hours, the titratable acid content of the millet fermented product obtained by the Pk 4-2 + Lp 9-1, Pk 2-3 + Lp 9-1, Pkl 8-3 + Lp 9-1 co-fermentation combinations significantly decreases (<0.05), indicating that Pichia pastoris may affect the growth and fermentation of Lactobacillus plantarum. On the contrary, the remaining combinations continue to increase significantly (<0.05). p <0.05), indicating that Pichia pastoris may affect the growth and fermentation of Lactobacillus plantarum. On the contrary, the remaining combinations continue to increase significantly (<0.05). p <0.05), indicating that Pichia pastoris may affect the growth and fermentation of Lactobacillus plantarum. On the contrary, the remaining combinations continue to increase significantly (<0.05). p <0.05).

[0082] 3.2. Total phenol content Figure 10 shows the influence of different co-fermentation systems on the total phenol content of the millet fermented product. From 24 to 96 hours, the total phenol content of the millet fermented product obtained by all fermentation combinations is significantly higher than that of the Lactobacillus plantarum single-strain fermented product (<0.05), indicating that co-fermentation is beneficial to increasing the content of polyphenolic substances in the millet fermented product. Especially, the Wa 27 + Lp 9-1 combination reaches the highest value at 96 hours. p <0.05), indicating that co-fermentation is beneficial to increasing the content of polyphenolic substances in the millet fermented product. Especially, the Wa 27 + Lp 9-1 combination reaches the highest value at 96 hours.

[0083] 3.3, ABTS ＋ Free radical scavenging ability ABTS ＋ Free radical scavenging ability is an important indicator of antioxidant activity. For example Figure 11 , ABTS ＋ The variation pattern of free radical scavenging ability is inconsistent with that of total phenol content, which may be due to the different antioxidant properties of different phenolic substances and the generation of other secondary metabolites with strong antioxidant ability. The ABTS + free radical scavenging ability of most millet fermentates all began to increase significantly from 48 h to 96 h of fermentation ( p <0.05), and only the combined fermentation of Mp 6 + Lp 9-1 reached the maximum at 48 h of fermentation. When fermented for 96 h, the ABTS + free radical scavenging ability of the Wa 27 + Lp 9-1 group was significantly stronger than that of the millet fermentates in other fermentation combinations ( p <0.05).

[0084] 3.4, Volatile Compounds and Sensory Evaluation In this study, the inventors focused on exploring the antioxidant properties exhibited after fermentation with different strain combinations. Since after optimizing the fermentation conditions, different strain combinations generally had high antioxidant activity at 96 hours of fermentation, the millet fermentates at 96 hours of fermentation were used as the research object to explore the differences in volatile substances and further study the interaction between volatile substances and metabolites. By such a research method, the antioxidant characteristics of fermenting millet slurry with different strain combinations were explored, providing a basis for the subsequent development of healthy beverages.

[0085] 3.4.1, Volatile Compounds In this study, a total of 32 aroma substances were detected in 6 different fermentation combinations and the single SC9-1 strain fermentation control group, including alcohols, esters, acids, aldehydes, ketones, phenols, terpenes and others. For details, see Table 1 and Figure 12 . The number of aroma substances in the fermentate of the combination of Pichia guilliermondii and Lactobacillus plantarum (Pg 6-2 + Lp 9-1) was lower than that in the Lactobacillus plantarum fermentation group. The number of aroma substances in the fermentates of the remaining combined fermentation groups was higher than that in the control group of single Lactobacillus plantarum fermentation. The number of aroma substances in the fermentate of the combination of Wickerhamomyces anomalus and Lactobacillus plantarum (Wa 27 + Lp 9-1) was the highest, and the amounts of esters and aldehydes in the Wa 27 + Lp9-1 combination were significantly higher than those in the other combinations.

[0086] Table 1 Concentrations of aroma substances in fermentates obtained from different fermentation combinations (μg / L) Note: "ND" in the table represents not detected, and "NF" represents not found. Different lowercase letters (abcde) in the same row indicate significant differences in the content of the same volatile compound among different strain fermentation combinations ( p <0.05).

[0087] Higher alcohols have a fresh fragrance and play a decisive role in the sensory quality of the fermented product. As shown in Table 1, there are 6 kinds of alcohols in the fermented millet of different mixed fermentation groups. Isoamyl alcohol and phenethyl alcohol are present in all the fermented millet, providing rose and malt aromas for the fermentation system. The contents of isoamyl alcohol and phenethyl alcohol in the fermented product of the combination of non-Saccharomyces cerevisiae and Lactobacillus plantarum are significantly higher than those in the single strain fermentation group of Lactobacillus plantarum ( p <0.05), probably because non-Saccharomyces cerevisiae can promote the Ehrlich pathway of yeast during mixed fermentation. Among them, both the Pk 2-3 + Lp 9-1 group and the Pk 4-2 + Lp 9-1 group are the combined fermentations of Pichia kudriavzevii + Lactobacillus plantarum, and the fermented products of both groups show relatively high contents of isoamyl alcohol and phenethyl alcohol ( p <0.05), and only contain these two higher alcohols. The combined fermentation of Wa 27 + Lp 9-1 can increase the content of 1-nonanol and is accompanied by the production of 1-hexanol, providing banana and floral aromas for the fermentation system and enriching the aroma of the fermented product. Research shows that when the content of higher alcohols exceeds 400 mg / L, it will have a negative impact on the aroma characteristics of the product. In this study, the content range of higher alcohols is 97.06 - 6236.94 μg / L, significantly lower than 400 mg / L.

[0088] Esters have a unique floral fragrance and play a positive role in the formation of the aroma of the fermented product. Compared with the single strain fermentation control group of Lactobacillus plantarum, the types and contents of esters in the mixed fermentation combinations are significantly increased ( p <0.05), indicating that mixed fermentation promotes the enrichment of esters in the fermented millet. Among them, the contents of acetate and fatty acid esters in the combination of Pichia kluyveri (Pkl 8-3 + Lp 9-1) are significantly higher than those of the other combinations ( p <0.05). The number of esters during the fermentation process in the combination of Wickerhamomyces anomalus (Wa 27 + Lp 9-1) is higher than that in the combination of Pichia kluyveri (Pkl 8-3 + Lp 9-1).

[0089] The content of acid is important for the balance of the fermented product, and excessive acid will cause rancidity or sour taste. The Pk 4-2 + Lp 9-1 group, the Pkl 8-3 + Lp 9-1 group, and the Pk 2-3 + Lp 9-1 group all produced isovaleric acid and caprylic acid after fermentation, and the fatty acid content is significantly higher than that of the other combinations ( p(<0.05). Both strains Pk 4-2 and Pk 2-3 are Pichia kudriavzevii, indicating that the fermentation characteristics of Pichia kudriavzevii are to regulate acidity rather than enhance aroma. At the same time, the ester contents in the fermented products of the Pk 4-2 + Lp 9-1 group, the Pkl 8-3 + Lp 9-1 group, and the Pk 2-3 + Lp 9-1 group are also significantly higher than those of the other combinations, indicating that the increase in fatty acid concentration may increase the synthesis of corresponding esters.

[0090] As an important aromatic substance, aldehydes have a very low threshold. In this invention, a total of 5 aldehydes were detected. The content of decanal in the fermented product of the Wa27 + Lp 9-1 group is higher than that of the single strain fermentation of Lactobacillus plantarum, indicating that the fermentation of Wickerhamomyces anomalus has a good promoting effect on the production of decanal. In addition, 1-nonaldehyde produced by the fermentation of the Wa 27 + Lp 9-1 group can provide grassy and sweet flavors for the fermented product of foxtail millet. In addition, ketones contribute to floral and fruity aromas. The content of 2-octanone in the fermented product of foxtail millet after fermentation by the Wa 27 + Lp 9-1 group is significantly higher than that of the other combinations, and its content is 37.40 ± 16.46 μg / L.

[0091] Volatile phenolic substances are aromatic compounds, but exceeding a certain concentration will affect the sensory quality. Two volatile phenols, 4-ethylphenol and 2,4-di-tert-butylphenol, were detected in the fermented product, and their concentration ranges from 48.22 to 228.34 μg / L, which is significantly lower than the sensory threshold of 725 μg / L, so it will not have an adverse effect on the fermentation system.

[0092] Geraniol, a terpene compound detected in the fermented product of foxtail millet, not only has a pleasant rose aroma, but also has various physiological activities such as antioxidant, anti-inflammatory, and anti-cancer potential. Among them, the geraniol contents in the fermented products of foxtail millet after fermentation by the Wa 27 + Lp 9-1 group and the Pk 4-2 + Lp 9-1 group are both significantly higher than those of the single strain fermentation of Lactobacillus plantarum ( p (<0.05), especially in the Wa 27 + Lp 9-1 group, the geraniol content reached 16.20 ± 2.73 μg / L after fermentation.

[0093] In summary, through the aroma determination of the fermented product of foxtail millet, it was found that on the basis of the single strain fermentation of Lactobacillus plantarum, adding different types of non-Saccharomyces yeasts can significantly promote its aroma and fruity aroma. In particular, the co-fermentation of Lactobacillus plantarum and Wickerhamomyces anomalus can increase the contents of aldehydes, ketones, volatile phenols, and terpenes, and has a lower content of higher alcohols and an appropriate amount of esters, enhancing the complexity of the fermented product.

[0094] 3.4.2, Sensory analysis Sensory analysis showed that during the exploration of the co-fermentation of non-Saccharomyces yeasts and Lactobacillus plantarum, compared with the traditional single-strain fermentation method of Lactobacillus plantarum, the co-fermentation technology greatly promoted the production of fruity and floral odors in the fermented products. Especially for the Wa 27+Lp 9-1 combination, the fermented millet products exhibited a more intense and rich odor ( Figure 13 ), and the enhancement of the herbaceous odor was consistent with the significant increase in aldehyde content. Neither the Pg 6-2+Lp 9-1 group nor the Mp 6+Lp 9-1 group had obvious aroma characteristics. Due to the excessive concentration of esters in the Pkl 8-3+Lp 9-1 combination, a special odor similar to "nail polish" was produced, which had a certain impact on the odor of the fermented product. The fermentation of the Wa 27+Lp 9-1 combination increased the acidity of the fermented product compared with the single-strain fermentation of Lactobacillus plantarum. In terms of taste, the Wa 27+Lp 9-1 combination performed excellently, with better taste intensity and chromaticity than other combinations, while the Pk 4-2+Lp 9-1 group and the Pk 2-3+Lp 9-1 group had a bitter taste.

[0095] In summary, the fermented products of the Wa 27+Lp 9-1 combination were superior to the other combinations in terms of aroma complexity and taste richness.

[0096] 4. Fermentation of millet or proso millet by Lactobacillus plantarum and Wickerhamomyces anomalus Since the fermented products of the Wa 27+Lp 9-1 combination were superior to the other combinations in terms of aroma complexity and taste richness, this study further explored the physicochemical characteristics and flavor substance properties of the fermented products of Lactobacillus plantarum and Wickerhamomyces anomalus after fermenting millet and proso millet.

[0097] 4.1 Viable cell count As Figure 14 shown, during the initial stage of fermentation from 0h to 72h, the viable cell counts of lactic acid bacteria and Wickerhamomyces anomalus in the fermented millet products and fermented proso millet products increased significantly and reached the maximum at 72h of fermentation ( p <0.05), indicating that using millet as the fermentation substrate was beneficial to the growth of lactic acid bacteria and non-Saccharomyces yeasts. As the fermentation time extended, the growth rate of lactic acid bacteria and Wickerhamomyces anomalus slowed down, and the viable cell count decreased.

[0098] 4.2 Titratable acid content As Figure 15 shown, during the fermentation stage from 72h to 96h, the titratable acid content in the fermented millet products was higher than that in the fermented proso millet products, and this change trend was consistent with the increase in the viable cell count of lactic acid bacteria. In the later stage of fermentation, microorganisms would use some organic acids for metabolic activities to maintain balance, and the gradually decreasing available raw materials and the weakening of microbial activity would lead to a decrease in the titratable acid content.

[0099] 4.3, Reducing sugar content As can be seen from Figure 16 , with the start of fermentation, the reducing sugar content of foxtail millet fermented product and millet fermented product decreased significantly ( p < 0.05). Among them, the reducing sugar content in the foxtail millet fermented product remained at a lower level compared to the millet fermented product, possibly because foxtail millet provided a more abundant nutrient matrix for microorganisms compared to millet, thus accelerating the metabolic rate of reducing sugars.

[0100] 4.4, Total phenol content As Figure 17 shown, during the fermentation process, the total phenol content of the foxtail millet fermented product was higher than that of the millet fermented product, especially during 96 h to 15 d of fermentation. This is because phenolic compounds in foxtail millet mainly exist in the bran, so the content of phenolic substances in foxtail millet is more abundant. Therefore, during the long-term fermentation process, the total phenol content of the foxtail millet fermented product has an advantage compared to the millet fermented product.

[0101] 4.5, Flavonoid content As Figure 18 shown, during the fermentation process, the flavonoid content of the foxtail millet fermented product was significantly higher than that of the millet fermented product ( p < 0.05), indicating that dehulling has a direct impact on the flavonoid content, and the release of flavonoids was significantly promoted within the first 96 h of the fermentation period.

[0102] 4.6, ABTS ＋ and DPPH free radical scavenging ability As can be seen from Figure 19 , after a short fermentation period of 24 h by the combination of Lactobacillus plantarum and Wickerhamomyces anomalus, the ABTS + free radical scavenging ability and DPPH free radical scavenging ability of the fermented product increased significantly ( p < 0.05). Then, the scavenging ability of the foxtail millet fermented product started to decline from 96 h of fermentation, and the scavenging ability of the millet fermented product started to decline at 72 h of fermentation. Similar to the change in flavonoid content, the antioxidant ability of the foxtail millet fermented product was higher than that of the millet fermented product.

[0103] It should be noted that when the present invention involves a numerical range, it should be understood that any value between the two endpoints of each numerical range and either of the two endpoints can be selected. Since the steps and methods adopted are the same as those in the embodiments, in order to avoid redundancy, the present invention describes the preferred embodiments. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0104] Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. A method for preparing millet fermentation product, characterized in that: The following steps are involved: Performing enzymatic hydrolysis on millet and obtaining millet enzymatic hydrolysate after sterilization; The non-brewer's yeast seed liquid and the lactic acid bacteria seed liquid are mixed and inoculated into an acclimation medium for cultivation to obtain a mixed seed liquid of the non-brewer's yeast and the lactic acid bacteria; The millet fermentation product is obtained by inoculating a mixed seed liquid of non-brewer's yeast and lactic acid bacteria into the millet enzymatic hydrolysate for fermentation; The number of viable non-brewery yeast cells in the non-brewery yeast seed liquid is at least 10 6 CFU / mL; the number of viable lactic acid bacteria in the lactic acid bacteria seed liquid is at least 10 6 CFU / mL.

2. The preparation method according to claim 1, characterized in that: The non-brew yeast is aberrant Wickham yeast strain Wa 27, Pichia guilliermondii strain Pg 6-2, Pichia kudrida strain Pk 4-2, Pichia kudrida strain Pk2-3, Pichia kluyveri strain Pkl 8-3 or Maggi yeast strain Mp 6; the lactic acid bacteria is Lactobacillus plantarum strain Lp9-1.

3. The preparation method according to claim 1, characterized in that: The inoculation amount of the mixed seed liquid is 2% v / v~10% v / v; the fermentation temperature is 28°C~36°C; and the fermentation time is 0.5d~30d.

4. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis step comprises sequentially adding α-amylase, glucoamylase, cellulase and protease to the millet slurry; based on the millet slurry, the α-amylase addition amount is 10 U / mL~30 U / mL, the glucoamylase addition amount is 100 U / mL~300 U / mL, the cellulase addition amount is 65 U / g~85 U / g, and the protease addition amount is 400 U / g~600 U / g.

5. The preparation method according to claim 4, characterized in that: The enzymatic hydrolysis temperature of the α-amylase is 70° C. to 90° C.; the enzymatic hydrolysis time of the α-amylase is 0.5 h to 8 h.

6. The preparation method according to claim 4, characterized in that: The enzymolysis temperature of the glucoamylase, cellulase and protease is 40°C to 65°C; the enzymolysis time of the glucoamylase, cellulase and protease is 1h to 24h.

7. The preparation method according to claim 1, characterized in that: The acclimation culture medium is obtained by sterilizing a mixture of rice milk supernatant and water in a volume ratio of 0.25-4:1; the rice milk supernatant is the supernatant of millet enzymatic hydrolysate after centrifugation at 3500-4500 r / min for 8-12 minutes.

8. The preparation method according to claim 1, characterized in that: The solid-liquid ratio of millet to water in the millet slurry is 1g:6~10mL.

9. A millet fermentation product prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the millet fermentation product according to claim 9 in preparing synbiotics.

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