Fresh spirulina fermentation liquor with rose fragrance and preparation method thereof

Abstract

The invention discloses fresh spirulina fermentation liquor with rose fragrance and a preparation method of the fresh spirulina fermentation liquor. The method is characterized in that 5% glucose, lactic acid bacteria, saccharomycetes and spore lactobacillus (10: 2: 1: 1) are adopted for composite fermentation of a bacterial liquid, dynamic pH regulation (4.5-6.0) and gradient fermentation (2-6 days) are combined, and synthesis of phenethyl alcohol flavor substances is promoted; the optimal fermentation process conditions are established, the phenethyl alcohol content reaches up to 547.52 mu g / kg, the oxidation resistance is remarkably improved, and the aroma and nutritional ingredients can be stably kept for 12 months under the refrigeration condition. According to the method, the problem that the fishy smell of algae is shielded is solved, and a novel aroma conversion path is constructed through strain metabolism regulation, so that the spirulina fermentation liquor has the natural flower fragrance and the nutrient enrichment characteristic. The method is low in cost, green, environmentally friendly and free of chemical additives, conforms to the development trend of healthy food, provides a new technical normal form for deep processing of algae, and has remarkable market competitiveness and industrialization value.

Description

Technical Field

[0001] The present invention relates to the field of food processing and fermentation technology, and in particular to a method for preparing fresh spirulina fermentation liquid with rose aroma by fermenting fresh spirulina with fermentation liquid containing different effective microorganisms. The preparation method has simple process, low cost and environmental protection, and realizes the dual improvement of functionality and sensory quality through the synergistic effect of multiple fermentation strains, and has good industrial application prospects. Background Art

[0002] Spirulina (Spirulina platensis) is a microalgae rich in high protein, polysaccharides, vitamins and minerals. In recent years, it has gradually become a source of attractive, natural and sustainable functional foods. However, spirulina is often limited by its special fishy smell and poor bioavailability, which often affects its eating experience. In order to reduce the unpleasant odor associated with algae such as spirulina, researchers have tried a variety of physical, chemical and biological methods, and found that liquid fermentation may be a good solution to improve the fishy smell and increase the content of bioactive substances. In recent years, the use of fermentation technology to improve the sensory quality and functional properties of spirulina has received increasing attention. Many liquid fermented products are well-liked by the public for their unique flavor and aroma characteristics. The characteristics and nutritional value of fermented products vary depending on the type of microorganisms used for fermentation. Therefore, in order to maximize the innovative fermented ingredients available in the spiral, choosing the right fermentation strain is a key step.

[0003] Lactic acid bacteria, yeast and spore lactobacillus play an important role in the formation of flavor substances, improving nutritional value and inhibiting the growth of spoilage bacteria in food fermentation. The three strains form a consortium of beneficial microorganisms, showing stronger potential and advantages in food fermentation, with the advantages of high fermentation efficiency, high safety and low cost. In most of the current studies on algae fermentation, it is found that fermentation is carried out by protease hydrolysis and single fermentation, and the effects of mixed strain fermentation and gradient differentiated concentration fermentation on nutritional quality, antioxidant quality and sensory quality are largely ignored, and there is still a lack of research on aroma. Chinese patent application CN202210738203.5 discloses "a strain of plant lactobacillus HSF-LAB-1303 and its use", mentioning that the DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, superoxide anion scavenging rate and total reducing power of spirulina fermentation products prepared by using plant lactobacillus HSF-LAB-1303 strain are significantly improved. The core is the discovery and isolation of a new strain of Lactobacillus plantarum HSF-LAB-1303, but the aroma-producing ability of this strain has not been reported. Chinese patent application CN201710653194.9 discloses "a health food rich in spirulina kinase and its preparation method", proposing to inoculate Lactobacillus subtilis and conduct deep liquid aeration fermentation. The obtained spirulina health food not only has high spirulina kinase activity, but also has a small algae smell, good taste, and is easy to be accepted by people. Compared with traditional spirulina tablets, fermented spirulina can improve its thrombolytic and anti-thrombotic properties, regulate blood lipids, and change blood rheology. The overall process cost of this method is relatively high, especially when the spirulina fermentation matrix is ​​prepared, sterilization treatment is carried out, and physical unit operations such as drying and crushing are destructive to the nutritional value and functional properties. There is no public report on the flavor characteristics and sensory characteristics of this fermented product.

[0004] It can be seen that there are few studies on the fermentation of fresh spirulina by mixed fermentation strains, especially the lack of a systematic method to optimize the fermentation conditions to maximize the application potential of spirulina. Therefore, based on the above situation, the present invention successfully prepared a fresh spirulina fermentation liquid with rose aroma by studying the optimization of fermentation liquids of different concentrations (0%, 1% and 3%) and fermentation time (2 days, 4 days and 6 days), providing technical support for its in-depth application in the fields of food and health. Summary of the invention

[0005] In view of this, the present invention aims to solve the problems in the prior art regarding insufficient improvement of functional characteristics of fresh spirulina during fermentation, difficulty in removing fishy smell, and low utilization rate of active ingredients, and provides a method for preparing fresh spirulina fermentation liquid with rose aroma by fermenting fresh spirulina with fermentation liquid containing different kinds of effective microorganisms. The spirulina fermentation liquid prepared by the method has a strong rose aroma and is rich in nutrition, meets the market demand for natural aroma and healthy food, can be used as a nutritional supplement and antioxidant for food and beverage, and has good industrial application prospects.

[0006] In order to achieve the above-mentioned invention object, the present invention provides the following technical scheme: a fresh spirulina fermentation liquid with rose fragrance and a preparation method thereof, comprising the following steps:

[0007] 1) Raw material preparation: harvest fresh, pollution-free spirulina, filter it with a 600-mesh nylon filter, wash it with distilled water, and drain the water;

[0008] 2) Activation of fermentation bacteria: The fermentation solution was prepared with 5% glucose, lactic acid bacteria, yeast and spore lactobacillus in a volume ratio of 10:2:1:1, activated at 30±2° C. for 24 hours, and fermentation solutions of different concentrations (0%, 1%, 3%) were prepared;

[0009] 3) Preparation of fermentation liquid and fermentation: the cleaned Spirulina and fermentation liquid were mixed evenly in a mass ratio of 2:1, and fermented at 35±2°C for 2 days, 4 days or 6 days;

[0010] 4) Adjusting the pH value of fermentation: Dynamically adjusting the pH value of fermentation (4.5-6.0 range) during the fermentation process;

[0011] 5) Terminating the fermentation: After the fermentation is finished, the fresh spirulina is filtered out to obtain a fresh spirulina fermentation liquid with a rose fragrance;

[0012] 6) Packaging and storage: The fermented liquid is vacuum packed and stored in a refrigerator.

[0013] The 600-mesh nylon filter in step 1) of the present invention is not limited to the preparation method and is a commercially available nylon filter.

[0014] The present invention is not limited to the type of distilled water in step 1), and distilled water has a better effect.

[0015] Preferably, the glucose in step 2) is obtained from commercial sources unless otherwise specified. The glucose preferably used in the present invention is purchased from Shandong Xiwang Sugar Co., Ltd.

[0016] Preferably, the lactic acid bacteria in the fermentation strain in step 2) are Lactobacillus bacteria; the lactic acid bacteria strains used in the present invention are not specifically limited to the strains; the lactic acid bacteria strains preferentially used in the present invention are from the China Industrial Microbiological Culture Collection Administration Center, with the collection number: CICC 22172.

[0017] Preferably, the yeast in the fermentation strain in step 2) is a fungus of the genus Saccharomyces cerevisiae; the yeast strain used in the present invention is not specifically limited to the strain; the yeast strain preferentially used in the present invention is from the China Industrial Microorganism Culture Collection Administration Center, and the collection number is: CICC 32364.

[0018] Preferably, the Sporolactobacillus in the fermentation strain in step 2) is a bacterium of the genus Sporolactobacillus; the Sporolactobacillus strain used in the present invention is not specifically limited to the strain; the Sporolactobacillus strain preferentially used in the present invention is from the China Industrial Microbiological Culture Collection Administration Center, with a collection number of CICC 25281.

[0019] Preferably, the activation condition in step 2) is 30±2° C. for 24 hours in a constant temperature and humidity incubator.

[0020] Preferably, the fermentation liquid at the inoculation amount of 1% fermentation liquid in step 3) after 4 days of fermentation is light green and has a strong and stable rose aroma, and its sensory quality reaches the best, and the antioxidant capacity is significantly improved.

[0021] Preferably, the fermentation pH value is dynamically adjusted in step 4) using 1% citric acid solution and 1% sodium bicarbonate solution.

[0022] Preferably, the pH value in step 4) is in the range of 4.5-6.0, and the more preferred pH value is 5.5.

[0023] Preferably, in step 5), fresh spirulina is filtered out using a 600-mesh nylon filter.

[0024] Preferably, in step 5), the rose aroma components of the fermentation broth are detected using GC-MS technology, wherein the highest phenylethanol content is 547.52 μg / kg.

[0025] Preferably, the optimal fermentation condition of the fresh spirulina fermentation broth with rose fragrance in step 5) is fermentation for 4 days at a 1% fermentation broth inoculation amount.

[0026] Preferably, the fresh spirulina fermentation broth with rose aroma in step 5) is not detected in the fermentation broth with 0% inoculation amount of fermentation bacteria, and the aroma components can be stably maintained for 12 months under refrigerated conditions.

[0027] The present invention has the following beneficial effects:

[0028] The present application provides a method for preparing a fresh spirulina fermentation liquid with a rose aroma by fermenting fresh spirulina with a fermentation liquid of different effective microorganisms, the method improves the sensory quality of spirulina fermented food, significantly reduces the fishy smell of algae, presents a light green color and a strong and stable rose aroma. At the same time, the fermentation liquid is rich in a variety of active substances (proteins, polysaccharides and antioxidant components), showing excellent antioxidant capacity and nutritional enhancement characteristics. The strong rose aroma meets the market demand for natural aroma and healthy food, greatly improves the sensory quality of the fermentation liquid, and enhances its market acceptance. The present invention is efficient and low-consumption, simple in process, easy to operate, and cost-controlled, and has a good prospect for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 This is the appearance picture of the fresh spirulina fermentation broth of Example 2.

[0031] Figure 2 It is a bar graph of the DPPH radical scavenging rate results of Comparative Examples 1-3 and Examples 1-6.

[0032] Figure 3 It is a bar graph of the ABTS free radical scavenging rate results of Comparative Examples 1-3 and Examples 1-6.

[0033] Figure 4 It is a bar graph of the hydroxyl radical scavenging rate results of Comparative Examples 1-3 and Examples 1-6.

[0034] Figure 5 It is a bar graph showing the results of the changes in soluble protein content of Comparative Examples 1-3 and Examples 1-6.

[0035] Figure 6 It is a bar chart showing the change results of the total free amino acid content of Comparative Examples 1-3 and Examples 1-6.

[0036] Figure 7 It is a bar chart showing the changes in the essential amino acid content of Comparative Examples 1-3 and Examples 1-6.

[0037] Figure 8 It is a graph showing the relative content percentage of volatile organic compounds in Comparative Examples 1-3 and Examples 1-6. DETAILED DESCRIPTION

[0038] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0039] Embodiment 1

[0040] This embodiment provides a fresh spirulina fermentation liquid with rose fragrance, comprising the following steps:

[0041] 1) Raw material preparation: harvest fresh, pollution-free spirulina, filter it with a 600-mesh nylon filter, wash it with distilled water, and drain the water;

[0042] 2) Activation of fermentation bacteria: the fermentation solution is prepared by mixing 5% glucose, lactic acid bacteria, yeast and spore lactobacillus in a volume ratio of 10:2:1:1, and activated at 30° C. for 24 hours to prepare a fermentation solution with a concentration of 1%;

[0043] 3) Preparation of fermentation liquid and fermentation: the cleaned Spirulina and fermentation liquid were mixed evenly in a mass ratio of 2:1, and fermented at 37°C for 2 days;

[0044] 4) Adjusting the pH value of fermentation: During the fermentation process, the pH value of fermentation is dynamically adjusted to 5.5;

[0045] 5) Terminating the fermentation: After the fermentation is finished, the fresh spirulina is filtered out to obtain a fresh spirulina fermentation liquid with a rose fragrance;

[0046] 6) Packaging and storage: The fermented liquid is vacuum packed and stored in a refrigerator.

[0047] The present invention has no limitation on the source of the spirulina, and conventional spirulina in the art may be used.

[0048] Example 2

[0049] This embodiment provides a fresh spirulina fermentation liquid with rose fragrance, comprising the following steps:

[0050] Steps 1) to 2) are the same as in Example 1;

[0051] 3) Preparation of fermentation liquid and fermentation: the cleaned Spirulina and fermentation liquid were mixed evenly in a mass ratio of 2:1, and fermented at 37°C for 4 days;

[0052] Steps 4) to 6) are the same as in Example 1.

[0053] Example 3

[0054] This embodiment provides a fresh spirulina fermentation liquid with rose fragrance, comprising the following steps:

[0055] Steps 1) to 2) are the same as in Example 1;

[0056] 3) Preparation of fermentation liquid and fermentation: the cleaned Spirulina and fermentation liquid were mixed evenly in a mass ratio of 2:1 and fermented at 37°C for 6 days;

[0057] Steps 4) to 6) are the same as in Example 1.

[0058] Example 4

[0059] This embodiment provides a fresh spirulina fermentation liquid with rose fragrance, comprising the following steps:

[0060] Step 1) is the same as in Example 1;

[0061] 2) Activation of fermentation bacteria: the fermentation solution is prepared by mixing 5% glucose, lactic acid bacteria, yeast and spore lactobacillus in a volume ratio of 10:2:1:1, and activated at 31° C. for 24 hours to prepare a fermentation solution with a concentration of 3%;

[0062] 3) Preparation of fermentation liquid and fermentation: the cleaned Spirulina and fermentation liquid were mixed evenly in a mass ratio of 2:1, and fermented at 37°C for 2 days;

[0063] Steps 4) to 6) are the same as in Example 1.

[0064] Example 5

[0065] This embodiment provides a fresh spirulina fermentation liquid with rose fragrance, comprising the following steps:

[0066] Step 1) is the same as in Example 1;

[0067] 2) Activation of fermentation bacteria: the fermentation solution is prepared by mixing 5% glucose, lactic acid bacteria, yeast and spore lactobacillus in a volume ratio of 10:2:1:1, and activated at 31° C. for 24 hours to prepare a fermentation solution with a concentration of 3%;

[0068] 3) Preparation of fermentation liquid and fermentation: the cleaned Spirulina and fermentation liquid were mixed evenly in a mass ratio of 2:1, and fermented at 37°C for 4 days;

[0069] Steps 4) to 6) are the same as in Example 1.

[0070] Example 5

[0071] This embodiment provides a fresh spirulina fermentation liquid with rose fragrance, comprising the following steps:

[0072] Step 1) is the same as in Example 1;

[0073] 2) Activation of fermentation bacteria: the fermentation solution is prepared by mixing 5% glucose, lactic acid bacteria, yeast and spore lactobacillus in a volume ratio of 10:2:1:1, and activated at 31° C. for 24 hours to prepare a fermentation solution with a concentration of 3%;

[0074] 3) Preparation of fermentation liquid and fermentation: the cleaned Spirulina and fermentation liquid were mixed evenly in a mass ratio of 2:1 and fermented at 37°C for 6 days;

[0075] Steps 4) to 6) are the same as in Example 1.

[0076] Comparative Example 1

[0077] This embodiment provides a fresh spirulina fermentation liquid with rose fragrance, comprising the following steps:

[0078] Step 1) is the same as in Example 1;

[0079] 2) Activation of fermentation bacteria: The fermentation solution was prepared with 5% glucose, without adding fermentation bacteria, and activated at 30° C. for 24 hours to prepare a fermentation solution with a concentration of 0%;

[0080] 3) Preparation of fermentation liquid and fermentation: the cleaned Spirulina and fermentation liquid were mixed evenly in a mass ratio of 2:1, and fermented at 37°C for 2 days;

[0081] Steps 4) to 6) are the same as in Example 1.

[0082] Comparative Example 2

[0083] This embodiment provides a fresh spirulina fermentation liquid with rose fragrance, comprising the following steps:

[0084] Step 1) is the same as in Example 1;

[0085] 2) Activation of fermentation bacteria: The fermentation solution was prepared with 5% glucose, without adding fermentation bacteria, and activated at 30° C. for 24 hours to prepare a fermentation solution with a concentration of 0%;

[0086] 3) Preparation of fermentation liquid and fermentation: the cleaned Spirulina and fermentation liquid were mixed evenly in a mass ratio of 2:1, and fermented at 37°C for 4 days;

[0087] Steps 4) to 6) are the same as in Example 1.

[0088] Comparative Example 3

[0089] This embodiment provides a fresh spirulina fermentation liquid with rose fragrance, comprising the following steps:

[0090] Step 1) is the same as in Example 1;

[0091] 2) Activation of fermentation bacteria: The fermentation solution was prepared with 5% glucose, without adding fermentation bacteria, and activated at 30° C. for 24 hours to prepare a fermentation solution with a concentration of 0%;

[0092] 3) Preparation of fermentation liquid and fermentation: the cleaned Spirulina and fermentation liquid were mixed evenly in a mass ratio of 2:1 and fermented at 37°C for 6 days;

[0093] Steps 4) to 6) are the same as in Example 1.

[0094] All experiments in this example were repeated three times, and the data are expressed as the mean ± standard deviation of the three repetitions. The data were analyzed for significance (p<0.05) using one-way analysis of variance (ANOVA) and Duncan test in SPSS.

[0095] (I) Performance test method:

[0096] Experiments were conducted on the fresh spirulina fermentation broth with rose fragrance of the embodiments and comparative examples, and the fermentation broth was taken to test the changes in color (L*, a*, b* and ΔE), antioxidant activity (DPPH free radical scavenging ability, ABTS free radical scavenging rate and hydroxyl free radical scavenging rate), soluble protein, free amino acid content, essential amino acid content and volatile organic compound components and relative content.

[0097] 1. Color (L*, a*, b* and ΔE)

[0098] The fresh spirulina fermentation liquid with rose fragrance of Example 2 and Comparative Example 2 was tested, and the test method was as follows:

[0099] The color change of the fermentation liquid is directly detected by the colorimeter. The colorimeter is calibrated with a black board and a white board before measurement. Each sample is tested in parallel 3 times and repeated 3 times. In short, the average values ​​of L*, a*, and b* of the fermentation liquid are measured, where L*, a*, and b* are color indicators representing brightness, red-green, and yellow-blue, respectively. The calculation formula of color difference ΔE is as follows:

[0100]

[0101] Among them, L0*, a0*, and b0* are the color indexes of the fermentation liquid without inoculation of fermentation bacteria on day 0 of fermentation.

[0102] 2. Antioxidant activity (DPPH, ABTS and hydroxyl radical scavenging rate)

[0103] The fresh spirulina fermentation liquid with rose fragrance of Examples 1-6 and Comparative Examples 1-3 was tested, and the test method was as follows:

[0104] 1) DPPH free radical scavenging rate:

[0105] Accurately pipette 2mL of the fermentation liquid to be tested and add it to 2mL of the freshly prepared DPPH solution (0.1mmol / L, the solvent is 70% ethanol), so that the two are fully mixed, keep the reaction at room temperature and avoid light for 30 minutes, and measure its absorbance at a wavelength of 517nm. The higher the absorbance value, the stronger the DPPH free radical scavenging effect, and vice versa, the lower the free radical scavenging rate. The calculation formula is as follows:

[0106]

[0107] Wherein: A0 is the absorbance of 2 mL DPPH solution and 2 mL 70% ethanol; A1 is the absorbance of 2 mL DPPH solution and 2 mL sample solution; A2 is the absorbance of 2 mL 70% ethanol + 2 mL sample solution.

[0108] 2) ABTS free radical scavenging rate:

[0109] 7mmol / L ABTS and 2.45mmol / L potassium persulfate were mixed in equal volumes, and then placed at room temperature and protected from light for 16 hours. After the reaction, the mixed solution was diluted with ethanol. When the absorbance at a wavelength of 734nm was diluted to 0.700±0.005, this was ABTS. + Working fluid.

[0110] Accurately pipette 50 μL of fermentation broth and add 0.15 mL of ABTS + Working solution, and let it stand for 6 minutes at room temperature and away from light, then measure the absorbance value at 734nm wavelength. The calculation formula is as follows:

[0111]

[0112] Where: A blank is the absorbance value when blank; A sample is the absorbance value when the sample is added.

[0113] 3) Hydroxyl free radical scavenging rate:

[0114] Accurately pipette 1.0 mL of sample solution, add 2.0 mL of salicylic acid ethanol solution (6 mmol / L) and 2.0 mL of FeSO4 (6 mmol / L) to mix them thoroughly, then add 0.1 mL of H2O2 solution (6 mmol / L) to mix them thoroughly, then react in a 37°C water bath for 30 minutes, and measure the absorbance at a wavelength of 510 nm. The calculation formula is as follows:

[0115]

[0116] In the formula: A1 is the sample group; A2 is without adding color developing agent H2O2; A0 is the blank control absorbance value.

[0117] 3. Soluble protein

[0118] The fresh spirulina fermentation liquid with rose fragrance of Examples 1-6 and Comparative Examples 1-3 was tested, and the test method was as follows:

[0119] The soluble protein content was determined by Coomassie Brilliant Blue G-250 staining. Accurately measure 1 ml of fermentation broth, centrifuge (10,000 × g, 4 ° C, 10 min) to remove the precipitate, and take the supernatant for use. Mix 1.0 mL of sample supernatant with 5 ml of Coomassie Brilliant Blue G-250 working solution, and measure the absorbance at 595 nm for 15 minutes at dark room temperature using a multifunctional microplate microplate reader. Prepare a 0-100 μg / mL standard solution using bovine serum albumin (BSA) as the standard.

[0120] When quantifying, a calibration curve (y = 2.0417x + 0.2578, R 2 =0.9994), the soluble protein content was calculated according to the sample absorbance value and the standard curve, and the result was expressed in mg / mL. Each group of samples was measured in parallel 3 times, and the average soluble protein content was finally taken.

[0121] 4. Free amino acid content and essential amino acid content

[0122] The fresh spirulina fermentation liquid with rose fragrance of Examples 1-6 and Comparative Examples 1-3 was tested, and the test method was as follows:

[0123] The amino acids in the fermentation broth were separated by chromatography using an Agilent HPLC. Accurately measure 5 mL of 12 mol / L hydrochloric acid and mix with 1 mL of the fermentation sample, place in a magnetic stirrer, and stir at 110°C for 14 h to completely acidify the protein. Then place in an oven at 80°C for 24 h to completely dry it. After derivatization, take 400 μL of the above solution and add 200 μL of 1 mol·L -1 Triethylamine acetonitrile solution and 200 μL 0.1 mol·L -1 The isothiocyanate benzyl cyanide solution was mixed evenly. After being placed at room temperature and away from light for 1 hour, 1 mL of n-hexane was added, vortexed, centrifuged at 5000 r / min for 5 minutes, and the solution was removed to prepare a derivative solution.

[0124] The chromatographic column is C 18 (250 mm × 4.6 mm, 5 μm), column temperature 43 °C, mobile phase 0.1 mol·L -1Sodium acetate buffer (pH 6.5)-acetonitrile (93:7), mobile phase is acetonitrile-water (4:1), flow rate is 1.0mL / min. Detection wavelength is 254nm, injection 10μL. Elution flow rate is 0-17min, solvent A is 100%. 17-18min, solvent B is 0-3%; from 18-40min, solvent B increases to 21%; from 41-45min, solvent B remains at 100%; from 46 to 50 minutes, solvent B decreases from 100% to 0%. The amino acid quantitative method is used for the mixture of 18 amino acids, 18 amino acid reference substances are selected, and the mixed solution is accurately prepared. The amino acid linear curve is R 2 >0.99, amino acid content is expressed in μM.

[0125] 5. Volatile organic compound components and relative content

[0126] The fresh spirulina fermentation liquid with rose fragrance of Examples 1-6 and Comparative Examples 1-3 was tested, and the test method was as follows:

[0127] HS-SPME-GC / MS was used to determine the volatile aroma compounds in the fermentation broth. First, 6.0 mL of fermentation broth was measured in a 20 mL vial, sodium chloride (3.0 g) was added, and 10 μL of 2,4,6-trimethylpyridine as an internal standard was added for quantification. The vial was capped tightly with a silicon septum and placed in a 40 °C water bath for 20 minutes to equilibrate the volatiles. Then, the SPME needle was inserted into the vial and exposed to the headspace in a 40 °C water bath for 20 minutes. The fiber was immediately removed from the vial and injected into a GC / MS detector consisting of an HP 6890GC and a 7895C mass selective detector. A non-polar column (DB-5MS column) was used. The GC oven temperature will be programmed to 10 °C min -1 The rate of heating was gradually increased from 40°C to 230°C, with the initial and final holding times of 3 and 15 min, respectively. Helium was used as the carrier gas (1.5 mL min -1 ).

[0128] The mass spectrometry selective detector (MSD) conditions are as follows: capillary direct interface temperature 280 ° C; ionization energy 70 eV; mass range 35-350 amu; scan rate 4.45 scan / s. The column effluent will be separated in a 1:1 ratio between the FID and the sniffing port using a deactivated fused silica capillary (90 cm long, 0.25 mm diameter). The FID and the sniffing port are maintained at 250 ° C and 200 ° C, respectively. Identification will be based on the comparison of the mass spectrum of the unknown compound with the National Institute of Standards and Technology (NIST) mass spectral database. The total peak area of ​​the volatile aroma substances will be calculated, and the relative content will be calculated by the area normalization method.

[0129] (II) Performance test results and analysis

[0130] 1. Color (L*, a*, b* and ΔE*)

[0131] The color test results of Comparative Example 2 and Example 2 are shown in Table 1. The color of the fermented product is closely related to the acceptance of consumers. The L*, b* and ΔE values ​​of Comparative Example 2 always maintain a high level, indicating that the system color is brighter. The L*, b* and ΔE values ​​of Example 2 are significantly lower than those of Comparative Example 2, reflecting the significant change in the color of the fermented liquid from light to dark, and the fermented liquid gradually changes from a green tone to a yellow-red tone. This may be because Spirulina is rich in carotenoids (such as β-carotene and lutein), which have a yellow tone and become the main source of the reduction in the b* value. At the same time, the fermentation bacteria may degrade certain proteins and polysaccharides into yellow or brown metabolites (such as oxidation products of organic acids) during the fermentation process, further increasing the yellow tone of the system. It is worth noting that the chromaticity changes of Example 2 at each fermentation stage are relatively mild, which indicates that an appropriate amount of Spirulina can be evenly distributed in the system, and the pigment release and metabolic process are stable. This makes the L* value, a* value and b* value of Example 2 show better balance throughout the fermentation process.

[0132] Table 1. Fermentation broth color test results

[0133]

[0134] 2. Antioxidant activity (DPPH, ABTS and hydroxyl radical scavenging rate)

[0135] 1) DPPH free radical scavenging rate:

[0136] Fermentation technology is considered to be a technology that can promote the accumulation of food nutrients and improve the antioxidant activity of fermentation broth. DPPH free radical scavenging experiment is a common standard technique in antioxidant research. The DPPH free radical scavenging experiment results of Comparative Examples 1-3 and Examples 1-6 are shown in Figure 2. Figure 2 As shown. The DPPH free radical scavenging rate was significantly affected by the proportion of fermentation broth added and the fermentation time (P<0.05). Under different fermentation broth inoculation amounts, the DPPH free radical scavenging ability showed an overall increasing trend with the fermentation time. In Example 2, the maximum value was 78% when the inoculation amount was 1% and fermented for 4 days. Compared with the same fermentation time in Comparative Example 2, the year-on-year increase was 3.0%. This may be because Spirulina is rich in polyphenol compounds, and the addition of an appropriate amount of fermentation broth converts it into small molecule metabolites with antioxidant effects during the fermentation process.

[0137] 2) ABTS free radical scavenging rate:

[0138] The changes in the ABTS free radical scavenging rates of Comparative Examples 1-3 and Examples 1-6 are as follows: Figure 3 As shown. Under the same fermentation broth inoculation amount, with the increase of fermentation time, the ABTS free radical scavenging rate showed a trend of first increasing and then decreasing. Example 2 reached a maximum value of 44.3% after 4 days of fermentation at a bacterial inoculation amount of 1%, an increase of 36.1% compared with Comparative Example 2. The results show that antioxidant substances (such as polysaccharides, polyphenols, carotenoids and phycocyanin, etc.) are generated during the fermentation process, which increases the ABTS clearance rate in the fermentation broth. At 6 days, the metabolites (such as aldehydes and ketone compounds) in the fermentation broth may have a negative effect on antioxidant substances, resulting in a decrease in antioxidant activity, and the addition of excessive fermentation broth can also induce oxidative stress, resulting in the inhibition of the synthesis of antioxidant substances.

[0139] 3) Hydroxyl free radical scavenging rate:

[0140] The changes in the hydroxyl radical scavenging rates of Comparative Examples 1-3 and Examples 1-6 are as follows: Figure 4 As shown. Among all fermentation broth samples, the hydroxyl radical scavenging rate of Examples 1-3 was significantly higher than that of Comparative Examples 1-3 and Examples 4-6. Although the scavenging rate of Examples 4-6 was improved in the early stage of fermentation, the scavenging ability began to decline in the later stage of fermentation. On the 6th day of fermentation, the scavenging rate of Example 6 was only 51.73%, significantly lower than 57.02% of Example 3. The results show that the hydroxyl scavenging rate in Examples 1-3 with the addition of an appropriate amount of 1% inoculum fermentation broth was significantly higher than that in Comparative Examples 1-3. The hydroxyl radical scavenging ability is mainly attributed to the release of bioactive peptides and amino acids (arginine, glycine and proline). Spirulina protein is decomposed into amino acids such as histidine, tyrosine, and cysteine, which can react with hydroxyl radicals as electron donors and convert them into more stable products, thereby terminating the free radical chain reaction. When fermented for 6 days, the products of Examples 3 and 6 may have reduced hydroxyl radical scavenging rates due to lipid accumulation, which inhibited the expression of the antioxidant activity of microorganisms.

[0141] 3. Soluble protein

[0142] The changes of soluble proteins in Comparative Examples 1-3 and Examples 1-6 are as follows: Figure 5As shown. The soluble protein content in food, feed and biological products is an important indicator for evaluating the quality of fermented products. The soluble protein content of Example 6 is 0.48 mg / mL, reaching the lowest value. This may be because the fermentation liquid decomposes the cell wall of Spirulina in the early stage of fermentation, promotes the degradation of protein macromolecules, and generates small soluble proteins, which leads to a higher soluble protein content in the early and middle stages of fermentation. In particular, the protein substrate and bacterial metabolism of Example 2 reach an optimal equilibrium state, so that the protein hydrolysis rate reaches a peak value. Compared with Example 3, the reason why soluble protein decreases in the later stage of fermentation and the degree of protein hydrolysis is significantly reduced is that as the fermentation time increases, the accumulation of metabolic waste in the system inhibits the metabolic activity and enzyme secretion of microorganisms.

[0143] 4. Free amino acids and essential amino acids content

[0144] The free amino acids released by the large-scale degradation of proteins during the fermentation process help improve the taste of the fermented product. The total free amino acid content and essential amino acid content of Comparative Examples 1-3 and Examples 1-6 are as follows: Figure 6 and Figure 7 As shown. The results show that as the fermentation time increases, the contents of Gly, Ser, Pro, Thr, Cys, Asp, Met, His, Phe, Lys, Tyr, Ala and Leu in the fermentation broth of Examples 1-6 show a significant trend of first increasing and then decreasing, and the total amino acid content and essential amino acid content of the fermentation broth obtained by Implementation 2 reach the highest peak values, which are 1568.54 mg / mL and 288.20 mg / mL, respectively. Thereafter, as the fermentation time is extended to 6 days, the total amino acid content and essential amino acid content of the fermentation broth obtained by Implementation 3 decrease slightly. This phenomenon can be attributed to the fact that the low inoculation amount (1%) in the initial stage of fermentation can maintain the metabolic activity of the microorganisms for a long time, the protease activity is high, and the effective release of essential amino acids and the accumulation of flavor amino acids are effectively promoted. In the later stage, the total amount decreases due to the metabolic consumption of amino acids by the bacterial flora or their conversion into other metabolites (such as organic acids).

[0145] Compared with the 1% fermentation broth inoculation amount in Examples 1-3, the total amount of amino acids in the 3% fermentation broth inoculation amount in Examples 4-6 increased at a relatively slow rate, with a relatively delayed peak value, and then also showed a downward trend. This shows that the higher fermentation broth inoculation amount (3%) weakened the active metabolism of the fermentation flora due to the large flora density and the consumption of substrates, and the release and accumulation of amino acids were limited, and the peak content was lower than that of Examples 1-3. For essential amino acids, the proportion of essential amino acids in the total amount of amino acids increased significantly during the fermentation process, which shows that lactic acid bacteria, yeast and spore lactobacillus in the fermentation broth can effectively release high nutritional value components in spirulina protein during the fermentation process, especially His, Lys, Leu and Ile, which is conducive to the release of essential amino acids and plays an important role in contributing to the rose-scented flavor of the fermentation broth.

[0146] 5. Volatile organic compound components and relative content

[0147] The volatile organic compound components and relative contents in the fermentation broths of Comparative Examples 1-3 and Examples 1-6 were analyzed and identified by using the method of HS-SPME combined with GC-MS. The relative contents of volatile organic compounds are shown in FIG. Figure 8 shown.

[0148] On the whole, the volatile odor substances in Comparative Examples 1-3 are mainly esters and aldehydes. As the fermentation time increases, the proportion of esters and aldehydes in the fermentation broth gradually decreases, and the proportion of alcohols and acids in Examples 1-6 gradually increases, and alcohols account for most of the overall volatile substances. During the fermentation process, the fermentation broth of Examples 1-6 has a strong rose fragrance, which is attributed to the enhanced degradation of fatty acids and amino acids in the metabolism of microorganisms such as lactic acid bacteria and yeast in the fermentation broth. During the fermentation process, the volatile compounds in Examples 1-6 have the highest content of phenylethanol. As a phenylalanine derivative with a strong rose and honey aroma, phenylethanol plays an important role and significance in improving the fishy smell of spirulina. The content of phenylethanol volatile substances is shown in Table 2. In Comparative Examples 1-3, the content of phenylethanol is almost undetectable or extremely trace, indicating that the spirulina solution without the addition of fermentation broth itself does not produce this compound. Under the treatment of 1% fermentation broth, the content of phenylethanol changes from 194.35 μg / kg in Example 1 to 547.52 μg / kg in Example 2, reaching the highest value. In the treatment of 3% fermentation broth, the content of phenylethanol changes from 122.47 μg / kg in Example 4 to 310.82 μg / kg in Example 6. This trend shows that the concentration of the fermentation broth and the fermentation time are the key factors for the production of phenylethanol, which is related to the concentration of the fermentation broth and the enhanced activity of metabolic pathways related to the microbial strains. In Example 2, the fermentation broth with a concentration of 1% broth showed the most significant accumulation of phenylethanol during the 4-day fermentation period. This may be because the lactic acid bacteria, yeast and spore lactobacillus in the fermentation broth produce amino acid decarboxylase through the action of multiple enzymes, which accelerates the degradation and conversion of precursor substances such as amino acids and fatty acids in Spirulina. At the same time, esterase combines fatty acids and alcohol substances through esterification to produce phenylethanol.

[0149] Table 2. Results of phenylethanol content in fermentation broth

[0150]

[0151] This also confirms that using the fermentation liquid of the present application to ferment fresh spirulina can add rose fragrance and sweet flavor to the fermentation liquid, reduce its original strong grass smell, and improve the sensory properties of spirulina. The optimal fermentation conditions are those of Example 2.

[0152] As mentioned above, the above examples are only used to explain the technical solutions of the present invention, rather than to limit the present invention; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some technical features equivalently; therefore, such changes or substitutions will not cause the essence of the respective technical solutions to deviate from the spirit and scope of each implementation method in the present invention.

Claims

1. A fresh spirulina fermentation liquid with rose fragrance and a preparation method thereof, characterized in that Prepared by the following steps: 1) Raw material preparation: harvest fresh, pollution-free spirulina, filter it with a 600-mesh nylon filter, wash it with distilled water, and drain the water; 2) Activation of fermentation bacteria: The fermentation solution was prepared with 5% glucose, lactic acid bacteria, yeast and spore lactobacillus in a volume ratio of 10:2:1:1, activated at 30±2° C. for 24 hours, and fermentation solutions of different concentrations (0%, 1%, 3%) were prepared; 3) Preparation of fermentation liquid and fermentation: the cleaned Spirulina and fermentation liquid were mixed evenly in a mass ratio of 2:1, and fermented at 35±2°C for 2 days, 4 days or 6 days; 4) Adjusting the pH value of fermentation: Dynamically adjusting the pH value of fermentation (4.5-6.0 range) during the fermentation process; 5) Terminating the fermentation: After the fermentation is finished, the fresh spirulina is filtered out to obtain a fresh spirulina fermentation liquid with a rose fragrance; 6) Packaging and storage: The fermented liquid is vacuum packed and stored in a refrigerator.

2. The method according to claim 1, characterized in that The lactic acid bacteria in the fermentation strains are bacteria of the genus Lactobacillus, the yeasts are fungi of the genus Saccharomyces cerevisiae, and the spore lactobacillus is bacteria of the genus Sporolactobacillus.

3. The method according to claim 1, characterized in that When the fermentation liquid was inoculated with 1% of the fermentation liquid for 4 days, the fermentation liquid showed a light green color and had a strong and stable rose aroma. Its sensory quality reached the best and its antioxidant capacity was significantly improved.

4. The method according to claim 1, characterized in that: The GC-MS technology was used to detect the aroma components of the fermentation broth, among which the phenylethanol content was the highest at 547.52 μg / kg. The aroma and nutritional components could be stably maintained for 12 months under refrigerated conditions.

5. The fresh spirulina fermentation liquid obtained according to any one of the preparation methods of claims 1-4.

Images