Complex microbial inoculant for increasing content of aroma-producing substances of barley bran and preparation method and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant for increasing the content of aroma-producing substances of barley bran as well as a preparation method and application of the complex microbial inoculant. The complex microbial inoculant is formed by compounding plant lactobacillus dy-1 and Enderbernella ferruginea Pro208 according to the viable count ratio of 5: 2. The preservation number of the plant lactobacillus dy-1 is CGMCC (China General Microbiological Culture Collection Center) No.6016; according to the present invention, the Feibian Escherichia coli (Pro208) has a preservation number of GDMCC 64830; the complex microbial inoculant can grow in a barley bran system, and the aroma and volatile substance composition of barley bran and the content of a key aroma-producing substance butyl acetate are remarkably improved; the complex microbial inoculant can utilize pyruvate dehydrogenase and esterifying enzyme to convert glucose into butyl acetate; in addition, the obtained complex microbial inoculant can be used for preparing the fermented barley bran beverage and remarkably improving the aroma of the fermented barley bran beverage, and has wide application prospects in the food industry.

Description

Technical Field

[0001] The invention belongs to the field of food biotechnology, and specifically relates to a composite bacterial agent for increasing the content of aromatic substances in barley bran, and a preparation method and application thereof. Background Art

[0002] Barley (Hordeum vulgare L.) is a whole grain, rich in starch, protein, minerals, vitamins and other nutrients. Barley contains a variety of bioactive substances, which have anti-inflammatory, antioxidant, cholesterol-lowering, blood sugar-lowering and lipid-lowering effects. Due to problems such as poor flavor and poor edibility, barley products have not yet been widely accepted by the public. Improving the flavor of barley products based on new food processing technologies is of great significance to promoting the further application of barley in the functional food industry.

[0003] Lactic acid bacteria and yeast are common in fermented foods. They use self-generated functional enzymes to convert sugars, fats, proteins and other substances into pleasant aromatic substances such as aromatic esters, alcohols, and aldehydes, thereby enhancing the aroma and taste of food. Direct fermentation is green, efficient, and highly operable, and can be used as an effective way to improve the flavor of grains. Compared with single-strain fermentation, the use of composite bacterial agents can give foods better flavors. At the same time, the defects of specific strains in the fermentation process can be complemented. Existing barley fermentation mostly relies on a single strain, and the flavor improvement effect is often not significant. In addition, there are also studies on the use of multiple strains for barley fermentation, but they are still limited by problems such as unclear flavor substance production pathways and weak strain complementarity. Barley bran is a by-product of barley processing. It is mainly the outermost epidermal component of barley and has a high content of dietary fiber and polyphenols. Existing barley bran has obvious sourness and insignificant aroma. Using composite bacterial agents with clear strain composition and complementary metabolic pathways to ferment barley bran is conducive to the targeted improvement of characteristic aroma components.

[0004] In addition, the invention patent application CN201310261606.6 filed by the applicant earlier disclosed a Lactobacillus plantarum dy-1 (deposit number CGMCC No. 6016), whose barley fermentation product has the function of significantly inhibiting tumor growth and improving immunity. However, there has been little research on its ability to enhance the flavor of barley products. Summary of the invention

[0005] In view of the problem that the existing barley fermentation strains are unclear and the aroma component composition is unclear, resulting in the unsatisfactory flavor of the fermentation product, the present invention provides a composite bacterial agent for increasing the aroma content of barley bran, and a preparation method and application thereof, by using the lactic acid bacteria dy-1 cultivated in the early stage and the newly cultivated Fabian Seberlindner yeast Pro208 in a reasonable ratio to prepare a composite bacterial agent for fermenting barley bran, so that the final product has a better taste and a better aroma effect. The specific technical scheme is as follows:

[0006] Firstly, the invention provides a composite bacterial agent for improving the content of aromatic substances in barley bran. The composite bacterial agent is compounded by Lactobacillus plantarum dy-1 and Fabianseberlindnerella pro208 in a ratio of 5:2 in terms of live bacteria count; the Lactobacillus plantarum dy-1 has a preservation number of CGMCC No.6016; the Fabianseberlindnerella pro208 has a preservation number of GDMCC 64830.

[0007] The aforementioned composite bacterial agent for increasing the content of aromatic substances in barley bran, wherein the number of viable bacteria of Lactobacillus plantarum dy-1 is 2×10 7 CFU / mL, the viable count of the Fabianseber Lindnerella Pro 208 was 2×10 6 CFU / mL.

[0008] The aforementioned composite bacterial agent for increasing the content of aromatic substances in barley bran can significantly change the overall composition of aromatic substances in barley bran.

[0009] The aforementioned composite bacterial agent for increasing the content of aromatic substances in barley bran can increase the content of butyl acetate, a key aromatic substance in barley bran, from 0.183 μg / L to 0.941 μg / L.

[0010] The aforementioned composite bacterial agent for increasing the content of aromatic substances in barley bran can utilize pyruvate dehydrogenase and esterase to convert glucose in barley bran into butyl acetate.

[0011] Secondly, the present invention provides a method for preparing the aforementioned composite bacterial agent for increasing the content of aromatic substances in barley bran, comprising the following steps:

[0012] 1) Prepare Lactobacillus plantarum dy-1 to have a viable cell count of 2×10 7 CFU / mL fermentation agent 1, for later use;

[0013] 2) Prepare the Fabianseber Lindner yeast Pro208 to have a viable cell count of 2×10 6 CFU / mL fermentation agent 2, for later use;

[0014] 3) Mix the fermentation agent 1 and the fermentation agent 2 in a volume ratio of 1:4 to obtain a composite bacterial agent suspension.

[0015] As a preferred technical solution, the preparation method of the composite bacterial agent for improving the content of aromatic substances in barley bran is as follows: the frozen Lactobacillus plantarum dy-1 is inoculated into MRS culture medium, anaerobically cultured at 37°C for 24 hours, and then subcultured in MRS culture medium for 18 hours, the culture medium of Lactobacillus plantarum dy-1 is taken, centrifuged at 8000rpm for 10 minutes, the supernatant is removed, and the obtained bacterial mud is washed with physiological saline for 2 to 3 times, and then added with distilled water to prepare the Lactobacillus plantarum fermentation bacterial agent, and the number of viable bacteria is 2×10 7 CFU / mL.

[0016] As a preferred technical solution, the preparation method of the composite bacterial agent for improving the content of aromatic substances in barley bran is as follows: the frozen Fabianseberlindner yeast Pro208 is inoculated into the YPD culture medium, anaerobically cultured at a temperature of 30°C for 24 hours, and then subcultured in the YPD culture medium for 18 hours, the culture medium of Fabianseberlindner yeast Pro208 is taken, centrifuged at 8000rpm for 10 minutes, the supernatant is removed, the obtained bacterial mud is washed with physiological saline for 2 to 3 times, and then distilled water is added to obtain the yeast fermentation bacterial agent, and the number of viable bacteria is 2×10 6 CFU / mL.

[0017] Again, the present invention provides an application of the aforementioned composite bacterial agent for increasing the content of aroma substances in barley bran, which is used for fermentation of barley bran to increase the content of aroma substances in the fermented product; specifically comprising the following steps:

[0018] A) washing and soaking the barley, removing the husk, grinding the barley repeatedly, and passing the barley bran through an 80-mesh sieve;

[0019] B) weighing barley bran after UV sterilization, adding the composite bacterial suspension, and stirring evenly; the addition amount of the composite bacterial suspension is 7 mL of the composite bacterial suspension per 1 gram of barley bran;

[0020] C) The barley bran added with the composite bacterial agent was placed at 31°C, pH 5.5, and fermented at 220 rpm on a shaking table for 8 hours. After the fermentation, it was centrifuged at 8000 rpm for 10 minutes. The supernatant was sterilized in an 80°C water bath for 20 minutes and cooled at room temperature.

[0021] The beneficial effects of the present invention are:

[0022] 1) The composite bacterial agent of the present invention has a simple composition and is only composed of Lactobacillus plantarum dy-1 and Fabianseberlindnerella yeast Pro208. During the fermentation process, Lactobacillus plantarum dy-1 converts glucose into acetic acid through pyruvate dehydrogenase, and Fabianseberlindnerella yeast Pro208 converts acetic acid into butyl acetate through esterification.

[0023] 2) The composite bacterial agent of the present invention can increase the content of butyl acetate, a key aromatic substance in the fermented barley bran product, from 0.183 μg / L to 0.941 μg / L. Among them, butyl acetate has a fruity aroma, which makes the fermented barley bran product have a better aroma, which is conducive to the production of food with the required aroma and improves market acceptance.

[0024] 3) The preparation method of the composite bacterial agent of the present invention is simple. The prepared composite bacterial agent can be mixed with barley bran for fermentation. The fermentation time only takes 8 hours, which is lower than conventional fermentation, is conducive to market promotion, and has good practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the colony morphology of Lactobacillus plantarum dy-1;

[0026] Figure 2 This is the colony morphology of Fabianseber Lindnerella Pro208;

[0027] Figure 3 The effect of compound bacterial agent on the aroma of barley bran;

[0028] Figure 4 The effect of different proportions of compound bacterial agents on the aroma of barley bran;

[0029] Figure 5 The changes of pH and viable bacteria count of the compound bacterial agent during the fermentation of barley bran;

[0030] Figure 6 The effect of compound bacterial agent on the overall composition of volatile substances in barley bran;

[0031] Figure 7 The effect of compound bacterial agent on the content of butyl acetate, a key aroma substance in barley bran;

[0032] Figure 8 The effect of Lactobacillus plantarum dy-1 fermentation on the activity of pyruvate dehydrogenase;

[0033] Fig. 9 The effect of yeast Pro208 fermentation on esterase activity;

[0034] Fig.10 The effect of composite bacterial agent on the aroma of barley bran beverage. DETAILED DESCRIPTION

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0036] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0037] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0038] The plant lactobacillus dy-1 (Lactiplantibacillus plantarum dy-1) involved in the embodiments of the present invention, the colony is shown in Figure 1 , which is the plant lactobacillus dy-1 involved in the applicant's previous application CN201310261606.6, and is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with a deposit number of CGMCC No.6016 and a deposit date of April 18, 2012.

[0039] Cyberlindnerafabianii Pro208 involved in the embodiments of the present invention is a newly discovered fermentation agent, which was deposited on July 4, 2024 at the Guangdong Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with a deposit number of GDMCC 64830. The colony is shown in Figure 2 .

[0040] Example 1 Preparation of composite bacterial agent

[0041] The frozen Lactobacillus plantarum dy-1 was inoculated into MRS culture medium and cultured anaerobically at 37°C for 24 h. After subcultured in MRS culture medium for 18 h, the culture medium of Lactobacillus plantarum dy-1 was taken and centrifuged at 8000 rpm for 10 min. The supernatant was removed and the obtained bacterial sludge was washed with physiological saline for 2 to 3 times. Then, distilled water was added to prepare the Lactobacillus plantarum fermentation agent. The number of viable bacteria was 2×10 7 CFU / mL, for future use.

[0042] The frozen Fabianseberlindnerella yeast Pro208 was inoculated into YPD culture medium and cultured anaerobically at 30°C for 24 h. After subculture in YPD culture medium for 18 h, the culture medium of Fabianseberlindnerella yeast Pro208 was taken and centrifuged at 8000 rpm for 10 min. The supernatant was removed and the obtained bacterial sludge was washed with physiological saline for 2 to 3 times. Then, distilled water was added to obtain a yeast fermentation agent. The number of viable bacteria was 2×10 6 CFU / mL, for future use.

[0043] The prepared Lactobacillus plantarum dy-1 and Fabianseber Lindnerella Pro208 are mixed in a required volume ratio, for example, a ratio of 1:4, to obtain a composite bacterial agent for barley bran fermentation.

[0044] Example 2: Composite bacterial agent is used to ferment barley bran

[0045] The barley was washed and soaked, the husk was removed, and the barley was repeatedly ground and passed through an 80-mesh sieve to obtain barley bran. 30 g of UV-sterilized barley bran was weighed, 210 mL of the composite bacterial agent (mixed in a ratio of 1:4) prepared in Example 1 was added, and the mixture was stirred evenly. Fermentation was performed for 8 hours at 31°C, pH 5.5, and a shaking table at 220 rpm; after fermentation, centrifugation was performed at 8000 r / min for 10 minutes, and the supernatant was sterilized in a water bath at 80°C for 20 minutes and cooled at room temperature to obtain fermented barley bran.

[0046] Example 3: Composite bacterial agent is used to enhance the aroma of barley bran

[0047] This example investigates the effect of the composite bacterial agent on improving the aroma of fermented barley bran.

[0048] In this embodiment, the fermented products involved in the investigation are as follows:

[0049] Fermented product 1: barley bran fermented with the composite bacterial agent of the present invention in Example 2;

[0050] Fermentation product 2: barley bran not inoculated with a starter; the preparation method thereof is as follows: 30 g of the barley bran obtained in Example 2 is taken, 210 mL of distilled water is added, and the mixture is shaken on a shaker at 31° C. and 220 rpm for 8 h, followed by sterilization and cooling;

[0051] Fermentation product 3: barley bran fermented by Lactobacillus plantarum dy-1; the preparation method thereof is as follows: 30 g of barley bran treated in Example 2 was added with 210 mL of Lactobacillus plantarum dy-1 prepared in Example 1, and the mixture was fermented at 31° C. and 220 rpm on a shaking table for 8 h, followed by sterilization and cooling.

[0052] Fermentation product 4: barley bran fermented by Fabianseberlindnerella Saccharomyces Pro208; the preparation method thereof is as follows: 30 g of the barley bran treated in Example 2 was added with 210 mL of Fabianseberlindnerella Pro208 prepared in Example 1, and the mixture was fermented in a shaking table at 31° C. and 220 rpm for 8 h, followed by sterilization and cooling.

[0053] In this example, the odor characteristics of the above four fermentations were compared through aroma descriptive sensory analysis.

[0054] Fifteen sensory assessors (5 males, 10 females; average age 25 years old) who performed well in aroma description and differentiation were selected and regularly trained in aroma description of barley bran fermented with compound bacterial agents. The aroma was freely described, and the descriptors were unified through literature research and group discussion. The descriptive sensory analysis was carried out in a professional sensory laboratory with room temperature controlled at 20°C. The experiment finally determined six aroma descriptors, including fruity, mellow, plant, earthy, fat and sour, and the intensity of the aroma was rated on a 10-point scale ("0" means imperceptible and "10" means strongly perceived).

[0055] The experimental results are as follows Figure 3 As shown in the data, the earthy and fatty smells were more prominent in the unfermented samples. After fermentation with Lactobacillus plantarum and yeast, the earthy and fatty smells were improved, but the aroma of the samples was not significantly improved. Compared with fermentation with Lactobacillus plantarum and yeast, the composite bacterial agent significantly enhanced the fruity and mellow aroma of barley bran. At the same time, the composite bacterial agent can effectively reduce the sour taste caused by Lactobacillus plantarum fermentation. Therefore, compared with fermentation with a single bacterial agent, fermentation with a composite bacterial agent has obvious advantages in enhancing the aroma of barley bran and reducing the sour taste of barley bran.

[0056] Example 4: Study on the Optimal Ratio of Composite Bacterial Agents

[0057] In this embodiment, barley bran fermented with the composite bacterial agent prepared in Example 2 was used as the basis for investigation, and different addition ratios of Lactobacillus plantarum dy-1 and Fabianseberlindnerella Pro208 were set to investigate the aroma of the fermentation product.

[0058] The ratios of Lactobacillus plantarum dy-1 and Fabianseberlindnerella Pro208 used in the barley bran fermented with the composite bacterial agent used in this embodiment are 9:1, 4:1, 1:1, 1:4 (Example 2), and 1:9. The fermentation process of barley bran is consistent with that of Example 2. This embodiment also compares the odor characteristics of barley bran liquid fermented with composite bacterial agents at different ratios through aroma descriptive sensory analysis, and the specific process is consistent with that of Example 3.

[0059] In this embodiment, six aroma descriptors, namely sour, pungent, sweet, plant, mellow and fruity, are finally determined to describe the aroma of the fermented product of barley bran, and the intensity of the aroma is also rated on a 10-point scale (with a scale of 2, "0" representing imperceptible, and "10" representing strong perception).

[0060] The experimental results are as follows Figure 4 As shown in the figure, when the addition ratio of plant lactobacillus fermentation agent to yeast fermentation agent is 9:1 and 4:1, the pungent smell and sour taste of the fermented samples are stronger, and other aromas are weaker; when the addition ratio of plant lactobacillus fermentation agent to yeast fermentation agent is 1:1, 1:4 and 1:9, the sour taste and pungent smell of the fermented samples are weaker; when the addition ratio of plant lactobacillus fermentation agent to yeast fermentation agent is 1:4, compared with the other four fermentation samples, the fruity, sweet and mellow aromas in the samples are significantly increased, the sour taste is milder, the pungent smell is the weakest, and the aroma sensory intensity is ranked as fruity aroma > sweet aroma > mellow aroma > sour taste > plant aroma > pungent smell. It shows that when the addition ratio of plant lactobacillus fermentation agent to yeast fermentation agent is 1:4, the effect of improving the aroma of barley bran is the best.

[0061] Example 5: Selection of the Optimal Conditions for Fermentation of Barley Bran with Composite Bacterial Agent

[0062] In this example, the fermentation conditions of barley bran fermented with a composite bacterial agent were investigated, including the effects of fermentation time and pH value of the fermentation liquid on the growth of the composite bacterial agent.

[0063] In this embodiment, the barley bran fermented with the composite bacterial agent prepared in Example 2 is used as the basis for investigation. The investigation method is: from the beginning of fermentation to the end of fermentation, a sample is taken every 4 hours. 3 mL of fermentation liquid is transferred with a pipette, and the pH value of the fermentation liquid in each time period is measured with a pH meter; 0.5 mL of fermentation liquid is transferred with a pipette, and 4.5 mL of 0.9% saline is added to prepare a bacterial suspension. After a series of gradient dilution operations, 100 μL of dilutions with appropriate dilutions are taken for coating on MRS agar medium (natamycin is added to inhibit yeast growth) and YPD agar medium (chloramphenicol is added to inhibit plant lactobacillus growth) for observing and analyzing the changes in the number of microbial colonies during the fermentation process.

[0064] The experimental results are as follows Figure 5 As shown in the figure, the pH of the fermentation liquid gradually decreased during the fermentation of barley bran with the composite bacterial agent, and the pH at the end of the fermentation was 3.5; at 8h of fermentation, the colony number of Lactobacillus plantarum dy-1 increased by an order of magnitude to 10 8 CFU / mL; the yeast colony count reached a plateau at 8h of fermentation, and the colony count increased by an order of magnitude to 10 7 CFU / mL, after 8 hours, the number of colonies increased from 10 7CFU / mL decreased to 10 5 CFU / mL. This shows that during the fermentation of the composite microbial agent, the colony count of Lactobacillus plantarum dy-1 increased, resulting in increased acid production and decreased pH; when the pH was lower than 4, the number of live yeast cells decreased significantly. Therefore, 8h is the appropriate time for the composite microbial agent to ferment barley bran, and the pH value should be controlled above 4 during the fermentation process.

[0065] Example 6: Analysis of volatile aroma substances in barley bran fermented with composite bacterial agent

[0066] This example is based on the fermentation products 1-4 in Example 3. This example uses headspace solid phase microextraction-gas chromatography-mass spectrometry to detect volatile substances in barley bran liquid.

[0067] Extraction of volatile components: Weigh 1.5 g NaCl, 5.0 mL sample, and 6 μL internal standard 2-octanol with a mass concentration of 5 mg / L, and place them in a 20 mL headspace bottle, tighten the lid, mix, place in an automatic injection plate, keep equilibrium at 40°C for 1 min, then perform headspace solid phase microextraction at 40°C for 20 min, and analyze on GC-MS. Each sample was repeated 3 times independently.

[0068] Chromatographic conditions were set as follows: Rtx-WAX column (30.0 m × 0.25 mm, 0.25 μm); carrier gas was helium (99.999%), flow rate was 1.0 mL / min; temperature program: 40°C for 4 min, then increased to 150°C at a rate of 6°C / min for 5 min, then increased to 245°C at a rate of 7°C / min for 10 min; injection port temperature was 250°C. MS conditions: ion source temperature was 230°C, energy was 70 eV, full scan mode, mass number range was 35-450u.

[0069] Qualitative analysis of volatile components: extract the mass spectrum of volatile components, compare and search with the NIST mass spectrum database, and refer to the retention time of C8-C20 alkane mixed standard to perform qualitative analysis of volatile components using the retention index method.

[0070] Quantitative analysis of volatile components: The internal standard semi-quantitative method is used for quantitative analysis, that is, the relative concentration of the component to be tested is calculated based on the peak area ratio of the internal standard and each volatile component in the sample:

[0071]

[0072] Wherein, CX is the content of unknown volatile substance, μg / mL; f is the relative correction factor, f=1; C0 is the mass concentration of the internal standard substance, μg / mL; V0 is the volume of the added internal standard 2-octanol, mL; SX is the peak area of ​​the unknown volatile substance; S0 is the peak area of ​​the added internal standard substance; V is the sample volume, mL.

[0073] The experimental results are as follows Figure 6 As shown in the figure, the variance contribution rates of PC1 and PC2 are 81.4% and 13.8% respectively, and the cumulative variance contribution rate reaches 95.2%, indicating that the PCA model is reliable. The PCA results show that the overall composition of the volatile components of the four samples is significantly different.

[0074] According to the analysis of the test results: A total of 70 volatile components were detected in all samples, including 14 alcohols, 15 aldehydes, 9 acids, 13 esters, 4 ketones and 15 other compounds. In terms of the content of volatile substances, the content of the compound bacterial fermentation sample was 122.685μg / L, the content of the unfermented sample was 51.195μg / L, the content of the plant lactobacillus fermentation sample was 66.507μg / L, and the content of the yeast fermentation sample was 79.609μg / L. This shows that compared with single bacterial fermentation, compound fermentation can significantly increase the total amount of volatile substances in the sample.

[0075] PLS-DA analysis showed that butyl acetate was the key differentiating substance between unfermented samples and fermented samples. The specific results are shown in Table 1.

[0076] Table 1 Key aroma substances in barley bran samples fermented with composite bacterial agents

[0077]

[0078] Example 7: Effect of composite bacterial agent on the content of butyl acetate, a key aromatic substance in barley bran fermentation

[0079] The qualitative and quantitative analysis of volatile components is the same as in Example 6.

[0080] The experimental results are as follows Figure 7 As shown, the composite bacterial fermentation increased the content of butyl acetate, a key aroma substance in barley bran, from 0.183 μg / L to 0.941 μg / L, and increased the fruity aroma compared with the unfermented samples and yeast fermented samples.

[0081] Example 8: The composite bacterial agent can synergistically convert glucose into butyl acetate.

[0082] During the fermentation process, glucose is converted into pyruvate through the glycolysis pathway, pyruvate can be converted into acetic acid through pyruvate dehydrogenase, and acetic acid is converted into butyl acetate through esterase. The pyruvate dehydrogenase (PDH) activity detection kit was used to determine the enzyme activity of pyruvate dehydrogenase in the fermentation system of Lactobacillus plantarum, and the p-nitrophenol method was used to determine the enzyme activity of esterase in the fermentation system of yeast.

[0083] The experimental results are as attached Figure 8 , 9 , 10. Compared with the unfermented sample, the fermentation of Lactobacillus plantarum increased the activity of pyruvate dehydrogenase from 2.166U / mL to 12.026U / mL, and the fermentation of yeast increased the activity of esterase from 1.361U / mL to 32.232U / mL. Combined with the above enzyme activity determination results, it shows that the composite bacterial agent can promote the production of butyl acetate through the pyruvate dehydrogenase and esterase pathways.

[0084] Example 9: Composite bacterial agent can ferment and improve the aroma of barley bran beverage

[0085] Preparation of barley bran beverage fermented with composite bacteria: weigh 30g of barley bran sterilized by ultraviolet light, mix the plant lactobacillus fermentation bacteria and the yeast fermentation bacteria in a ratio of 1:4, add to the barley bran and stir evenly, the ratio of barley bran to suspension is 30g:210mL. Ferment on a shaker at 31°C and 220rpm for 8h, filter with gauze after fermentation, and collect the filtrate. Add sucralose at a ratio of 4% and xanthan gum at a ratio of 1% to the filtrate, sterilize in a water bath at 80°C for 20min, and put the prepared fermented barley bran beverage into a sterile packaging container.

[0086] Preparation of unfermented barley bran beverage: weigh 30 g of UV sterilized barley bran, add 210 mL of water to the barley bran, shake on a shaker at 31°C and 220 rpm for 8 hours, filter with gauze, and collect the filtrate. Add 4% sucralose and 1% xanthan gum to the filtrate, sterilize in a water bath at 80°C for 20 minutes, and put the prepared unfermented barley bran beverage into a sterile packaging container.

[0087] Descriptive sensory analysis was the same as in Example 3, comparing the odor characteristics of the unfermented barley bran beverage and the barley bran beverage fermented with the composite inoculant.

[0088] The experimental results are as follows Fig.10 As shown in the results, compared with the unfermented samples, the fat and earthy smells of the samples fermented with the composite bacteria were significantly reduced; and the fruity, mellow and sour flavors in the samples fermented with the composite bacteria were significantly increased, giving the barley bran beverage a unique fermented aroma. This shows that composite bacteria fermentation can improve the aroma of barley bran beverages.

[0089] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

[0090] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-restrictive from any point of view. In addition, it should be understood that although this specification is described in accordance with the implementation mode, it does not include only one technical solution. This narrative mode of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in the embodiments can also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A composite bacterial agent for increasing the content of aromatic substances in barley bran, characterized in that: The composite bacterial agent is composed of Lactobacillus plantarum dy-1 and Fabian Seberlindner's yeast Pro208 in a ratio of 5:2 in terms of live bacteria count. The Lactobacillus plantarum dy-1 has a deposit number of CGMCC No.6016; The Fabiensebrinella Saccharomyces cerevisiae Pro208 has a deposit number of GDMCC 64830.

2. The composite bacterial agent for increasing the content of aromatic substances in barley bran according to claim 1, characterized in that: In the composite bacterial agent, the number of viable bacteria of Lactobacillus plantarum dy-1 is 2×10 7 CFU / mL, the viable count of the Fabianseber Lindnerella Pro208 was 2×10 6 CFU / mL.

3. The composite bacterial agent for increasing the content of aromatic substances in barley bran according to claim 2, characterized in that: The composite bacterial agent can significantly change the overall composition of the aroma substances in barley bran.

4. The composite bacterial agent for increasing the content of aromatic substances in barley bran according to claim 3, characterized in that: The composite bacterial agent can increase the content of butyl acetate, a key aroma substance in barley bran, from 0.183 μg / L to 0.941 μg / L.

5. The composite bacterial agent for increasing the content of aromatic substances in barley bran according to claim 4, characterized in that: The composite bacterial agent can utilize pyruvate dehydrogenase and esterase to convert glucose in barley bran into butyl acetate.

6. A method for preparing a composite bacterial agent for increasing the content of aromatic substances in barley bran according to claims 1-5, characterized in that: The steps include: 1) Prepare Lactobacillus plantarum dy-1 to have a viable cell count of 2×10 7 CFU / mL fermentation agent 1, for later use; 2) Prepare the Fabianseber Lindner yeast Pro208 to have a viable cell count of 2×10 6 CFU / mL fermentation agent 2, for later use; 3) Mix fermentation agent 1 and fermentation agent 2 in a volume ratio of 1:

4.

7. The method for preparing the composite bacterial agent for increasing the content of aromatic substances in barley bran according to claim 6, characterized in that: The preparation method of the fermentation agent 1 is as follows: frozen Lactobacillus plantarum dy-1 is inoculated into MRS culture medium, anaerobically cultured at 37°C for 24 hours, and then subcultured in MRS culture medium for 18 hours, the culture medium of Lactobacillus plantarum dy-1 is taken, centrifuged at 8000rpm for 10 minutes, the supernatant is removed, the obtained bacterial sludge is washed with physiological saline for 2 to 3 times, and then added with distilled water to prepare the Lactobacillus plantarum fermentation agent, and the number of viable bacteria is 2×10 7 CFU / mL.

8. The method for preparing the composite bacterial agent for increasing the content of aromatic substances in barley bran according to claim 6, characterized in that: The preparation method of the fermentation agent 2 is as follows: frozen Fabianseberlindnerella Saccharomyces Pro208 is inoculated into YPD culture medium, anaerobically cultured at 30°C for 24 hours, and then subcultured in YPD culture medium for 18 hours, then the culture medium of Fabianseberlindnerella Saccharomyces Pro208 is taken, centrifuged at 8000rpm for 10 minutes, the supernatant is removed, the obtained bacterial sludge is washed with physiological saline for 2 to 3 times, and then distilled water is added to obtain a yeast fermentation agent, and the number of viable bacteria is 2×10 6 CFU / mL.

9. An application of the composite bacterial agent for increasing the content of aromatic substances in barley bran according to claims 1-6, characterized in that: Used for the fermentation of barley bran to increase the content of aromatic substances in the fermented product; specifically includes the following steps: A) washing and soaking barley, removing the husk, grinding the barley repeatedly, and passing the barley bran through an 80-mesh sieve; B) weighing barley bran after UV sterilization, adding the composite bacterial agent, and stirring evenly; the amount of the composite bacterial agent added is 7 mL per 1 gram of barley bran; C) The barley bran added with the composite bacterial agent was placed at 31°C, pH 5.5, and fermented at 220 rpm on a shaking table for 8 hours. After the fermentation was completed, it was centrifuged at 8000 rpm for 10 minutes. The supernatant was sterilized in an 80°C water bath for 20 minutes and cooled at room temperature.

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