Long-shelf-life orange-flavor soft beverage fermented by soybean whey and fermentation method of long-shelf-life orange-flavor soft beverage

Abstract

The invention belongs to the technical field of non-alcoholic beverage fermentation methods, and particularly relates to a long-shelf-life orange-flavor soft beverage fermented by soybean whey and a fermentation method.The method comprises the following steps that the soybean whey and whole-wheat bread are pretreated, a fermented soybean whey base material only containing enterococcus groups is obtained, and the whole-wheat bread is smashed; mixing 92-97% by weight of the fermented soybean whey base material and 3-8% by weight of crushed whole-wheat bread to obtain a mixed fermentation material, and adding hops accounting for 0.03-0.08% by weight of the mixed fermentation material or a hops product containing 0.03-0.08% by weight of the hops for mixing to obtain the mixed fermentation material; naturally fermenting the mixed fermentation material until the pH value is lower than 4.5 to obtain fermentation liquor; and filtering insoluble substances in the fermentation liquor, canning the fermentation liquor, and performing pasteurization to terminate fermentation so as to obtain the soft soybean milk beverage with citrus fragrance. The citrus flavor soft drink is free of fruit and vegetable juice or essence, generates citrus fragrance through fermentation, and can be preserved for 6 months at normal temperature after low-temperature sterilization.

Description

Technical Field

[0001] This invention belongs to the technical field of non-alcoholic beverage fermentation methods, specifically relating to a long-shelf-life citrus-flavored soft drink made from fermented soybeans and a fermentation method thereof. Background Technology

[0002] In recent years, with the increasing demand from consumers for nutrition and health, the market share of functional flavored soft drinks has been growing. Soybean whey is a liquid product obtained during the processing of soy products by using enzymes, salts, and acids to coagulate, precipitate, and separate proteins. Soybean whey is rich in active ingredients such as soy protein, soy oligosaccharides, soy isoflavones, soy saponins, and various minerals. Using soy whey to prepare soft drinks can largely retain the nutrients in the soy whey, solve the problem of soy whey resource waste, and reduce the pressure on enterprises to treat wastewater, resulting in significant economic benefits.

[0003] However, due to the perishable nature and short shelf life of soymilk products, as well as their severe beany odor, the industry primarily extends their shelf life through high-temperature, high-pressure heat treatment or the addition of preservatives. Unpleasant flavors are often masked by adding large amounts of flavorings, fragrances, or fruit and vegetable juices. These processing methods are costly, resulting in significant investment for manufacturers. Furthermore, excessive processing leads to substantial nutrient loss, and consumers often have a negative perception of the use of large amounts of additives, resulting in relatively low product acceptance. Currently, there are no additive-free, low-temperature sterilized soymilk beverages on the market with a long shelf life. Summary of the Invention

[0004] Based on this, the present invention utilizes natural fermentation technology to prepare a long-shelf-life citrus-flavored soy milk soft drink. The product contains no added fruit or vegetable juices or flavorings, and produces a citrus aroma through fermentation. After low-temperature sterilization, it can be stored at room temperature for 6 months.

[0005] The technical solution adopted by this invention to solve its technical problem is:

[0006] According to a first aspect of the present invention, a fermentation method for a long-shelf-life citrus-flavored soft drink made from fermented soybean whites is provided, comprising the following steps:

[0007] Pre-treatment of soy protein and whole wheat bread yields fermented soy protein base and whole wheat bread crumbs containing only enterococcal flora.

[0008] By weight percentage, 92%-97% of fermented soybean base is mixed with 3%-8% of whole wheat bread crumbs to obtain a mixed fermentation material, and 0.03%-0.08% of hops or hop products containing 0.03%-0.08% hops are added to the mixed fermentation material and mixed to obtain a mixed fermentation material.

[0009] The mixed fermentation material is naturally fermented to a pH value lower than 4.5 to obtain a fermentation liquor;

[0010] After filtering insoluble substances in the fermentation liquor, the fermentation is terminated by canning and pasteurization of the fermentation liquor to obtain a bean clear soft drink with citrus aroma.

[0011] The soft drink prepared from bean clear according to the application can retain the nutritional elements in the bean clear to a great extent, solves the problem of waste of bean clear resources, reduces the pressure of enterprises on sewage treatment, and has remarkable economic benefits. Moreover, the long shelf life bean clear soft drink on the market at home and abroad is mainly prepared by fermentation of exogenous strains or by blending other fruit and vegetable juices, essences and spices, and then by high-temperature sterilization. The application mainly uses bean clear, bread and hops as base materials, and obtains the product by natural fermentation and low-temperature sterilization, thereby solving the problems of high processing cost, excessive processing intensity and serious loss of nutrients; and the product provided by the application is completely free of bean smell and has a pleasant citrus flavor, so the consumer acceptance is high.

[0012] According to the embodiment of the application, the fermented bean clear base material is obtained by pasteurizing the bean clear at 60-75 DEG C for 20-30 min; preferably, the bean clear is treated under the sterilization condition of 65 DEG C for 20 min to inhibit miscellaneous bacteria while retaining suitable self-bacteria of the bean clear; the embodiment of the application is obtained after the bean clear is pretreated by the method, but the bean clear can also be fully sterilized and then added with enterococcus group.

[0013] According to the embodiment of the application, the whole wheat bread crumbs are obtained by baking whole wheat bread into crisp at 100 DEG C and then crushing.

[0014] According to the embodiment of the application, the bean clear is fresh bean clear produced in the pressing process of brine bean curd, and is obtained after removing suspended solids by rough filtration; the protein content in the bean clear is 0.93 mg / mL, the total sugar content is 12.15 mg / mL, and the total flavonoid content is 173.15 mg / L.

[0015] Bean clear is a by-product produced in the processing of bean curd, soybean protein isolate and other soybean products, and is rich in soluble protein, sugar and various active ingredients, but is currently regarded as waste water treatment due to heavy bean smell and easy spoilage.

[0016] According to the embodiment of the application, the whole wheat bread is preferably whole wheat brioche. The baked brioche can compensate for the carbon source and nitrogen source in the bean clear, and the Maillard reaction occurs during the baking of the bread, so that the addition of the bean clear for fermentation can increase the sweet and fragrant flavor of the product.

[0017] According to embodiments of the present invention, the enterococci include at least one of Enterococcus faecalis, Enterococcus faecium, and Enterococcus lactis.

[0018] In this invention, the enterococci generated during the self-fermentation of soybean whey can oxidize beany odor components such as hexanal, hexanol, 1-octen-3-ol, and (E)-2-hexenal into corresponding acids, thereby reducing the beany odor of the product. During soybean whey fermentation, enterococci utilize the carbon and nitrogen sources provided by whole wheat bread, as well as branched-chain amino acids such as leucine and isoleucine in the soybean whey, to generate large amounts of ethyl 2-methylbutyrate, ethyl butyrate, methyl 2-methylbutyrate, and isoamyl acetate under the catalysis of esterases. These substances can impart a unique and rich citrus fruit aroma to the product, stimulating the olfactory nerves and bringing a pleasant feeling, thus increasing the product's appeal. The glycosides in hops promote esterification reactions, effectively increasing the content of these substances. At the same time, hops have a significant antibacterial effect against miscellaneous bacteria, which is conducive to enterococci dominating fermentation and reducing off-flavor sources.

[0019] According to an embodiment of the present invention, the hops are dried hop granules or powder with low alpha acid content;

[0020] The hop products contain hops with low alpha acid content.

[0021] According to an embodiment of the present invention, the hops are preferably French Zinda hops, which have a low alpha acid content of only about 6.0%, and large amounts of dry hopping will not bring bitterness, and the hops blend more harmoniously with the bean aroma and other flavors. Other hops with low alpha acid content can also be used.

[0022] According to an embodiment of the present invention, the fermentation temperature of the natural fermentation is 40-44℃, and the fermentation time is 20-24h.

[0023] According to an embodiment of the present invention, the natural fermentation conditions are a semi-closed environment (allowing only air circulation, isolating bacteria and dust) and stirred fermentation (stirring rate of 60 rpm) to promote the conversion and generation of volatile compounds related to citrus aroma.

[0024] According to an embodiment of the present invention, the sterilization conditions for terminating fermentation by pasteurization after the fermentation broth is bottled are: sterilization temperature of 90 ℃ and sterilization time of 20-30 min.

[0025] According to an embodiment of the present invention, a 200-mesh filter can be used to filter insoluble matter in the fermentation broth.

[0026] According to a second aspect of the present invention, a citrus-flavored soft drink prepared by a fermentation method for a long-shelf-life citrus-flavored soft drink through soybean fermentation is provided.

[0027] Beneficial effects

[0028] The present invention provides a long-shelf-life citrus-flavored soft drink made from fermented soybeans and a fermentation method thereof, which have the following advantages compared with the prior art:

[0029] The present invention provides a method for obtaining a long-shelf-life citrus-flavored soft drink through soybean fermentation. This method makes full use of the dominant microbial resources in soybeans and significantly reduces production costs. After soybeans are pasteurized at 65°C for 20 minutes, no external microorganisms need to be added. By controlling the nutritional components and fermentation conditions in soybeans, the mixture is naturally fermented under semi-closed conditions with intermittent stirring.

[0030] This invention utilizes natural fermentation technology to prepare a long-shelf-life citrus-flavored soft drink made from soybeans. The product contains no added fruit or vegetable juices or flavorings. The citrus aroma is produced through fermentation. After being sterilized by low-temperature pasteurization, the product can be stored at room temperature without the nutrients being destroyed by excessive heat treatment. It can be stored at room temperature for 6 months after low-temperature sterilization.

[0031] The long-shelf-life citrus-flavored soft drink made from fermented soybeans provided by this invention has a unique and rich aroma of citrus fruits, such as the freshness of lemon, the sweetness of orange, and the elegance of grapefruit. This aroma can stimulate the olfactory nerves, bringing a pleasant feeling and is very attractive. Attached Figure Description

[0032] Figure 1 The results show the pH changes during the fermentation process of Examples 1-3 and Comparative Examples 1-9 of this invention.

[0033] Figure 2 This is a stacked diagram showing the percentage of species composition at the level of unfermented pasteurized soybean germ in Example 1 of the present invention.

[0034] Figure 3 This is the hemolytic phenotype of four Enterococcus strains and Staphylococcus aureus in Example 1 of the present invention. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention should not be limited to the scope of the embodiments described.

[0036] Example 1

[0037] (1) Raw material processing: pasteurize soybeans at 60℃ for 20 minutes to kill pathogens; divide whole wheat rye bread into small pieces and bake at 100℃ until crisp and crush into bread crumbs; crush French sciutto dry hops into hop powder.

[0038] (2) Material mixing: by weight percentage: 97% fermented soybean base material and 3% whole wheat bread crumbs processed in step (1) are mixed to obtain mixed fermented material, and 0.03% of French hops by weight of mixed fermented material are added.

[0039] (3) Natural fermentation: The mixed materials are naturally fermented at 42℃ for 24 hours, with intermittent stirring during the fermentation process.

[0040] (4) Bottled and sterilized: The obtained fermentation broth was filtered through a 200-mesh filter to remove insoluble matter, and then bottled. The sample was sterilized by pasteurization at 90°C for 30 min.

[0041] Example 2

[0042] The other steps are the same as in Example 1, except that...

[0043] In step (1), the raw material is processed by pasteurizing soybeans at 75°C for 20 minutes to kill pathogens.

[0044] In step (2), the proportions of fermented soybean base, whole wheat bread crumbs and French hops are 94%, 6% and 0.06%, respectively.

[0045] Step (3) Natural fermentation: The fermentation temperature for natural fermentation is 44℃ and the fermentation time is 20h.

[0046] Example 3

[0047] The other steps are the same as in Example 1, except that...

[0048] In step (1), the raw material is processed by pasteurizing soybeans at 60°C for 30 minutes to kill pathogens.

[0049] In step (2), the proportions of fermented soybean base, whole wheat bread crumbs and French hops are 92%, 8% and 0.08%, respectively.

[0050] Step (3) Natural fermentation: The fermentation temperature for natural fermentation is 40℃ and the fermentation time is 24h.

[0051] Step (4) Canning and sterilization: Sterilization time is 20 minutes.

[0052] Comparative Example 1

[0053] The other steps are the same as in Example 1, except that in step (2), the proportion of soybean white in the material mixing is 100%.

[0054] Comparative Example 2

[0055] The other steps are the same as in Example 1, except that the hops are removed and the materials are mixed in step (2): 94% soybeans and 6% whole wheat bread.

[0056] Comparative Example 3

[0057] The other steps are the same as in Example 2, except that whole wheat bread is removed and the materials are mixed in step (2): 0.06% hops are added to the soybean whites.

[0058] Comparative Example 4

[0059] The other steps are the same as in Example 2, except that the soybean white is replaced with pure water. In step (2), the proportions of pure water, whole wheat bread and French hops are 94%, 6% and 0.06%, respectively.

[0060] Comparative Example 5

[0061] The other steps are the same as in Example 2, except that the natural fermentation temperature in step (3) is changed to 37°C.

[0062] Comparative Example 6

[0063] The other steps are the same as in Example 2, except that in step (1), the raw material processing involves completely sterilizing the soybean whites, and in step (1), the material mixing involves adding 3% of a 1×10⁻⁶ solution. 8 CFU / mL of Lactobacillus plantarum LP-45.

[0064] Comparative Example 7

[0065] The other steps are the same as in Example 2, except that in step (1), the raw material processing involves completely sterilizing the soybean whites, and in step (1), the material mixing involves adding 3% of a 1×10⁻⁶ solution. 8 CFU / mL yeast (Angel Yeast).

[0066] Comparative Example 8

[0067] The other steps are the same as in Example 2, except that in steps (1) raw material processing and (2) material mixing, the bread is replaced with white bread.

[0068] Comparative Example 9

[0069] The other steps are the same as in Example 2, except that in steps (1) raw material processing and (2) material mixing, bread is replaced with glucose.

[0070] Experiment Example 1: Sensory Evaluation

[0071] The sensory evaluation panel consisted of 10 non-smokers (6 men and 6 women), aged 20-30. Candidates were all from the College of Food Science and Engineering, Bohai University. The selection, training, and supervision of panel members followed the international standard method for sensory evaluation (ISO 8586: 2012). 100g samples were placed in clean, odorless food-grade containers. The sensory evaluation members assessed the aroma and taste characteristics at room temperature. They were required to repeatedly smell and describe the aroma and taste characteristics of the sample within a certain time period. Through discussion, citrus, bean, beany, grain, and sweet aromas were ultimately determined to represent the overall aroma of the bean-based soft drink. A 5-point scale was used for sensory evaluation: 0 points: no taste; 1-4 points: flavor and taste gradually increase; 5 points: strongest flavor and taste. The evaluation was repeated three times, with a 20-minute rest period between evaluations. The average value of each sensory attribute from the three repeated experiments was used to create a sensory score table. The aroma sensory scoring indicators and definitions are shown in Table 1.

[0072] Table 1. Sensory Evaluation Definitions and Reference Standards for Bean Soft Drinks

[0073]

[0074] As shown in Table 2, no citrus aromas were detected in Comparative Example 1 (fermentation of pure soy whey) and Comparative Example 2 (fermentation of a mixture of soy whey and bread). Comparative Example 3 (fermentation of a mixture of soy whey and hops) and Comparative Example 4 (fermentation of a mixture of water, bread, and hops) exhibited some citrus-like aromas, but the scores were only 0.75–1.01, indicating a low intensity of citrus aromas. In Comparative Example 5, where soy whey, bread, and hops were fermented together at a temperature controlled at 37 ℃ for 24 hours, citrus flavors were also perceptible, but the aroma intensity was low, at only 1.02.

[0075] In the most preferred embodiment 2 of the present invention, soybean whey, bread and hops were mixed for fermentation, the fermentation temperature was controlled at 42 ℃ and the time was 24 h, the final pH reached 4.5, and the citrus flavor score was significantly improved to 3.06 (P<0.05), which was the highest among all samples.

[0076] In Example 2, the soybean whey, bread, and hops fermentation system constructed under natural fermentation conditions at 42 ℃ showed a significant reduction in the sensory score of beany odor to 0.74 compared to single soybean whey fermentation (Comparative Example 1), soybean whey mixed with bread fermentation (Comparative Example 2), or soybean whey mixed with hops fermentation (Comparative Example 3). Simultaneously, the intensity of grain, sweet, and citrus aromas was significantly enhanced, achieving the control of beany odor removal and aroma enhancement in the soybean whey product flavor. Compared to Comparative Example 5, which had the same formula but was fermented at 37 ℃, the 42 ℃ natural fermentation process recommended in this invention further reduced beany odor and significantly enhanced citrus aromas, demonstrating that the defined fermentation temperature range is crucial and essential for obtaining the target citrus flavor.

[0077] In a soybean whey, bread, and hops system inoculated with lactic acid bacteria fermentation (Comparative Example 6) and yeast fermentation (Comparative Example 7), the citrus flavor scores were 0.5 and 0.42, respectively. Furthermore, when whole wheat bread was replaced with white bread toast (Comparative Example 8) and glucose (Comparative Example 9), the citrus flavor scores were only 0.42 and 0.22, respectively. The sensory intensity of the citrus flavor was very weak, indicating that the metabolic activity of Enterococcus during the natural fermentation of the soybean whey and the nutrients provided by the whole wheat bread are key factors in the production and enhancement of citrus as an aroma species.

[0078] Table 2 Sensory comparison results of soybean soft drinks in Examples 1-3 and Comparative Examples 1-9

[0079]

[0080] Therefore, this invention not only effectively solves the problem of soybean residue having a strong beany taste and being difficult to use directly, but also transforms it into a fermented beverage or flavor base with a distinct citrus flavor, realizing the high-value utilization of soybean residue by-products.

[0081] Experimental Example 2:

[0082] (1) Qualitative and quantitative analysis of volatile components in the sample.

[0083] Gas chromatography-ion mobility spectrometry (GC-IMS) was used for qualitative and quantitative analysis of volatile components in the samples. 5.00 g of sample was weighed and added to 20 mL headspace vials, which were incubated at 40 °C for 15 min. The injection volume was 500 μL, the IMS temperature was 45 °C, the injection needle temperature was 85 °C, and the column temperature was 60 °C. High-purity nitrogen was used as the carrier gas, with flow rates of 2 mL / min (0–2 min), 2–10 mL / min (2–10 min), and 10–100 mL / min (10–20 min). The analysis time was 21 min, and the drift gas flow rate was 75 mL / min. Qualitative analysis was performed using GC-IMS library search software with an IMS database. The linear retention index was determined using C4–C9 ketones.

[0084] The internal standard method was used to quantitatively analyze the volatile components in the sample. 5.00 g of sample was weighed and added to a 20 mL headspace vial, along with 5 μL of 2-methyl-3-heptanone solution (0.8 mg / mL). A standard curve was plotted with the ratio of the peak area of ​​each flavor compound standard to the peak area of ​​the internal standard as the ordinate, and the ratio of the mass concentration of the flavor compound standard to the mass concentration of the internal standard as the abscissa.

[0085] (2) The aroma contribution of volatile flavor components was determined by using the aroma activity value (OAV) method.

[0086] The OAV value is calculated using the following formula:

[0087] OAV=C / OT

[0088] Where C is the concentration of the compound in the sample, and OT is its respective threshold in water or the test matrix. It is generally believed that flavor compounds with OAV ≥ 1 have aroma activity, that is, they can be perceived by smell and make an important contribution to the overall flavor. The larger the OAV, the greater its flavor contribution.

[0089] The results are shown in Tables 3 and 4.

[0090] Table 3. GC-IMS identification of citrus aroma components in soybean soft drinks from Examples 1-3 and Comparative Examples 1-9.

[0091]

[0092] Table 4. OAV values ​​of citrus aroma components in soybean soft drinks from Examples 1-3 and Comparative Examples 1-9 identified by GC-IMS

[0093]

[0094] As shown in Tables 3 and 4, Comparative Example 1 is a system of pure soybean whey fermented naturally at 42 ℃. The contents of all four esters are 0, and the corresponding OAV is also 0, indicating that it is difficult to generate ester aroma components with citrus aroma by relying solely on the self-fermentation of soybean whey.

[0095] In Comparative Example 2, after fermentation of soybeans and bread, the contents of ethyl 2-methylbutyrate and isoamyl acetate were 0.65 μg / kg and 0.32 μg / kg, respectively, with OAV values ​​of 8.13 and 2.13, respectively. The system could detect a certain citrus flavor, but the aroma intensity was weak.

[0096] In Comparative Example 3, ethyl 2-methylbutyrate, ethyl butyrate, methyl 2-methylbutyrate, and isoamyl acetate were all detected in the mixed fermentation system of soybean whey and bread. The OAV values ​​of ethyl 2-methylbutyrate, methyl 2-methylbutyrate, and isoamyl acetate were 15.62, 8.48, and 3.6, respectively, with a total OAV of 27.7, significantly higher than that of Comparative Example 2. This indicates that hops are an important source of citrus aroma precursors, but the intensity of the citrus aroma remains limited in the absence of bread.

[0097] Comparative Example 4 used water instead of soy whey. Although trace amounts of ethyl 2-methylbutyrate and methyl 2-methylbutyrate were still detectable, with OAV values ​​of 6.75 and 2.96 respectively, the OAV values ​​of the other two esters were 0, resulting in a total OAV of only 9.71, far lower than the system containing soy whey (Example 2). This indicates that soy whey is not a simple solvent, but rather a key nitrogen source and precursor substrate for the formation of citrus esters. It is difficult to obtain a strong and full-bodied citrus aroma solely from the bread and hops system.

[0098] Compared to Example 1, Example 2 increased the amount of bread added to 6% and hops to 0.06%. The fermentation system constructed in the soybean whey matrix significantly promoted the synergistic formation of various citrus esters, resulting in a total OAV several times higher than other control samples. This is a key formulation condition for forming a strong citrus aroma. However, when the amount of hops added increased to 0.08%, the high concentration of hops affected the growth of microorganisms to some extent, inhibiting fermentation and reducing the production of citrus flavor components. Under the same raw material ratio, natural fermentation at 42°C was significantly better than constant temperature fermentation at 37°C. This was more conducive to the metabolism of the soybean whey's own microbial community and the bread hops, promoting the synthesis and accumulation of citrus esters. This is one of the necessary process conditions for achieving a high-intensity citrus aroma in this invention.

[0099] Similarly, Comparative Examples 6 and 7 show that they have low OAV values ​​for citrus aroma compounds, and lactic acid bacteria and yeast cannot produce large amounts of citrus aroma compounds in this system. Comparative Examples 8 and 9 show that white bread and glucose, as simple carbon sources, also cannot produce large amounts of citrus aroma compounds.

[0100] Experimental Example 3: pH Measurement

[0101] The pH values ​​of the soybean soft drinks in Examples 1-3 and Comparative Examples 1-9 were measured using a pH meter during fermentation. The results are as follows: Figure 1 As shown.

[0102] like Figure 1It can be seen that, except for Comparative Example 3, which is a mixture of soybean whey and hops fermentation, Comparative Example 4, which is a mixture of water, bread and hops fermentation, Comparative Example 7, which is yeast fermentation, and Example 3, which has an addition amount of 92% soybean whey, 8% bread and 0.08% hops, other soybean whey soft drinks can achieve rapid acid production, and the pH reaches 4.5 or below when fermented for 16 hours.

[0103] Experiment Example 4: Detection of Total Bacterial Count

[0104] The total bacterial count of the soybean soft drinks in Examples 1-3 and Comparative Examples 1-9 was determined during fermentation. The method was in accordance with the national standard GB4789.2-2022 Food Safety National Standard for Microbiological Examination of Food - Determination of Total Bacterial Count. The results are shown in Table 5.

[0105] Table 5. Colony count results of soybean soft drinks in Examples 1-3 and Comparative Examples 1-9 during storage.

[0106]

[0107] The colony count results are shown in Table 5. Colonies were detected in Comparative Example 3, which was a mixture of soybean whey and hops fermentation, and Comparative Example 4, which was a mixture of water, bread, and hops, on day 30. All other soybean whey soft drinks remained sterile. This indicates that the product of this invention can be stored at room temperature after being sterilized by low-temperature pasteurization, and the nutritional components will not be destroyed by excessive heat treatment.

[0108] Experiment Example 5: Determination of Microbial Community Composition

[0109] Whole-genome sequencing of the microorganisms in the unfermented pasteurized soybean extract from Example 1 was performed using the Illumina platform (Shanghai Meiji Biotechnology Co., Ltd.). The results are as follows: Figure 2 As shown, the main bacterial flora in naturally fermented soybean soft drinks is enterococci, including Enterococcus faecalis, Enterococcus faecium, and Enterococcus lactis. Four enterococci strains were screened from the samples: Enterococcus faecium B-1, Enterococcus faecalis B-2, Enterococcus faecalis D-1, and Enterococcus lactis D-2.

[0110] Experiment Example 6: Antibiotic Susceptibility Test

[0111] The following six antibiotics (vancomycin, ampicillin, penicillin, erythromycin, chloramphenicol, and tetracycline) were selected for antibiotic susceptibility testing using the disk spectrophotometry method to determine the antibiotic susceptibility of the enterococci obtained in Example 1 of Experiment 5. The enterococcal bacterial suspension concentration was corrected to 0.5 McFarland turbidity tubes using physiological saline. The bacterial suspension was spread onto MHA agar plates using a cotton swab, and antibiotic susceptibility test discs were placed on the surface. After standing for 5 minutes, the plates were inverted and incubated at 37 °C. After 24 hours, the diameter of the inhibition zone was measured using a ruler, as shown in Table 6.

[0112] Table 6: Antibiotic Resistance in Enterococci

[0113]

[0114] Note: S: Sensitive, I: Moderate, R: Resistant

[0115] Table 6 shows that all four Enterococcus strains were sensitive to the six antibiotics and could be inhibited by them.

[0116] Experiment Example 7: Hemolytic Experiment

[0117] Sterilized Columbia agar medium was thoroughly mixed with 5% (v / v) sterile sheep blood. Activated enterococci and Staphylococcus aureus were streaked onto Columbia agar plates and incubated at 37 °C for 48 h. Staphylococcus aureus served as a positive control. The presence of a clear zone around the colonies indicated β-hemolytic activity; the absence of a clear zone indicated γ-hemolytic activity, meaning the strain was not hemolytic.

[0118] The results are as follows Figure 3 The results showed that, with Staphylococcus aureus as a positive control, none of the four Enterococcus strains obtained in Example 1 of Experiment 5 exhibited hemolytic activity, indicating that they did not produce hemolytic toxins. In conclusion, the Enterococcus in naturally fermented soybean soft drink has a certain degree of safety.

[0119] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0120] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A fermentation method of a long shelf-life citrus flavored soft drink fermented with soy whey, characterized by, The method comprises the following steps: Pre-treating bean dreg and whole wheat bread to obtain fermented bean dreg base containing only Enterococcus group and whole wheat bread crumbs; Mixing the fermented bean dreg base 92%-97% and the whole wheat bread crumbs 3%-8% to obtain mixed fermentation material, and adding 0.03%-0.08% of hops or a hops product containing 0.03%-0.08% of hops to the mixed fermentation material to obtain mixed fermentation material; Carrying out natural fermentation on the mixed fermentation material until the pH value is lower than 4.5 to obtain fermented liquid; After filtering the insoluble substances in the fermented liquid, the fermented liquid is canned, pasteurized and the fermentation is terminated to obtain a bean dreg soft drink with citrus aroma.

2. The fermentation method of a fermented long shelf life citrus flavored soft drink of soybean according to claim 1, characterized in that, The fermented bean dreg base is obtained by pasteurizing the bean dreg at 60-75 ℃ for 20-30 min. The whole wheat bread crumbs are obtained by baking whole wheat bread into small pieces at 100 ℃ until dry and then crushing.

3. The fermentation method of a fermented long shelf life citrus flavored soft drink of soybean according to claim 2, characterized in that, The bean dreg is fresh bean dreg produced in the pressing process of brined bean curd, and is obtained after removing suspended solids by coarse filtration. The whole wheat bread is whole wheat stollen.

4. The fermentation method of a fermented long shelf life citrus flavored soft drink of soybean according to claim 1, characterized by, The Enterococcus includes at least one of Enterococcus faecalis, Enterococcus faecium and Enterococcus lactis.

5. The fermentation method of a fermented long shelf life citrus flavored soft drink of soybean according to claim 1, characterized by, The hops are low-alpha acid content dry hop pellets or powder; The hops product contains low-alpha acid content hops.

6. The fermentation method of a fermented long shelf life citrus flavored soft drink of soybean according to claim 5, characterized in that, The hops are French Saaz hops.

7. The fermentation method of a fermented long shelf life citrus flavored soft drink of soybean according to claim 1, characterized by, The fermentation temperature of the natural fermentation is 40-44 ℃, and the fermentation time is 20-24 h.

8. The fermentation method of a fermented long shelf life citrus flavored soft drink of soybean according to claim 1, characterized by, The natural fermentation is carried out in a semi-closed environment and by stirring.

9. The fermentation method of fermented long shelf life citrus flavored soft drink of soybean according to claim 1, characterized in that, The pasteurization conditions for terminating the fermentation after the fermented liquid is canned are: pasteurization temperature is 90 ℃, and pasteurization time is 20-30 min.

10. A citrus-flavored soft drink with long shelf life prepared by the fermentation method of the fermented bean dreg long shelf life citrus-flavored soft drink according to any one of claims 1-9.

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