A pre-harvest physiological management method for improving the color and odor quality of Spirulina

Abstract

The present invention relates to a pre-harvest physiological management method for improving the color and odor quality of Spirulina algae, belonging to the field of microbial culture technology. The present invention establishes a unique method for improving the sensory quality of Spirulina algae by combining in-situ illumination co-cultivation with muddy dark light co-cultivation. Specifically, when Spirulina is cultured to the late logarithmic growth period or the stable period, glucose and acetate are added to the culture medium, and yeast is inoculated. After the algae are collected and dried, a Spirulina powder with a bright green color and a rich floral and fruity aroma can be obtained. The method disclosed in the present invention solves the problem of poor color and odor acceptance in Spirulina applications through biological means, and has good prospects for industrial application.

Description

Technical Field

[0001] The invention relates to a pre-harvest physiological management method for improving the color and smell quality of spirulina, and belongs to the technical field of microbial cultivation. Background Art

[0002] Compared to traditional food crops, microalgae boast high photosynthetic efficiency, a short growth cycle, and the ability to avoid arable land occupation. They can also efficiently capture and convert carbon using natural seawater and saline water, as well as high-salt wastewater, flue gases, and waste steam generated by production activities. Therefore, they are internationally recognized as a new low-carbon food ingredient and a powerful force in agricultural carbon sequestration. The significant market potential of microalgae-based foods is closely related to their rich and diverse nutritional and functional components, such as phycocyanin, astaxanthin, and polyunsaturated fatty acids.

[0003] Spirulina is currently the world's most produced edible microalgae, but its application in food products presents numerous challenges. Its deep blue-green color, with a dark, cool hue, can create an unappealing color palette for foods like pasta, meat, and dairy products containing spirulina. Spirulina also has a distinct fishy odor, which can make it unpleasant for consumers. These issues have significantly hindered the widespread use of spirulina in food systems and hampered the further development of the microalgae industry.

[0004] The currently reported methods for removing the fishy smell of spirulina are all based on post-harvest physical, chemical or biological treatments, such as protease hydrolysis, activated carbon adsorption, β-cyclodextrin encapsulation, heat treatment, vacuum deodorization, flavor masking, organic solvent extraction and yeast fermentation. However, the deodorization effect is often less than ideal, and is accompanied by problems such as deterioration of color and taste, generation of other odors, and introduction of exogenous additives. Therefore, it has not been widely used in actual production. Summary of the Invention

[0005] In response to the above-mentioned industry status quo, the present invention has developed a pre-harvest physiological management method for Spirulina based on pre-harvest carbon flow distribution, light and dark control, and biological interaction with yeast in an alkaline environment. This method significantly improves the color and odor quality of Spirulina. The process conditions are mild, do not destroy the nutritional value of the algae, and no glucose and acetic acid remain. The yeast can be recycled after use, and the process time can be controlled within 24 hours. This method meets the actual requirements of Spirulina production and has good application prospects.

[0006] The present invention aims to provide a pre-harvest physiological management method for improving the color and odor quality of Spirulina. Specifically, the method combines in-situ light co-cultivation with sludge dark light co-cultivation. The in-situ light co-cultivation method involves adding glucose and acetate to the culture medium and inoculating yeast when Spirulina reaches the late logarithmic growth phase or the stationary phase.

[0007] The technical solution adopted by the present invention is as follows:

[0008] (1) When Spirulina is grown in Zarrouk's minimal or modified medium to the late logarithmic growth phase or the stationary phase, 3-7 g / L glucose and / or 0.5-1.5 g / L acetate are added to the medium, and yeast is inoculated at 0.1%-10% of the Spirulina biomass. The culture is then cultured under light and aeration conditions for 12-36 h.

[0009] (2) The co-culture solution is filtered through 400 mesh to remove 80%-90% of the culture medium to obtain a mixed slurry of Spirulina and yeast, which is then co-cultured for 12-36 hours in the dark.

[0010] (3) After the spirulina and yeast mixture is centrifuged, the yeast and spirulina are separated and precipitated. The algae are collected and dried to obtain spirulina powder with a bright green color and a rich floral and fruity aroma.

[0011] Preferably, in step (1) of the present invention, the amount of glucose added is 5 g / L, the amount of acetate added is 1 g / L, the yeast inoculation amount is 1% of the Spirulina biomass, and the co-cultivation time is 24 h.

[0012] Preferably, the co-culture duration in step (2) of the present invention is 24 hours.

[0013] Beneficial effects of the present invention:

[0014] (1) The pre-harvest biological treatment method adopted in the present invention is simple to operate, has mild process conditions, does not destroy the nutritional value of the algae, and does not require the use of exogenous additives.

[0015] (2) The spirulina powder prepared by the present invention has a higher β-carotene content and higher nutritional value.

[0016] (3) The spirulina powder prepared by the present invention has a bright green color and a milder tone.

[0017] (4) The spirulina powder prepared by the present invention has a significant fruity and floral aroma, no algae smell, and is more easily accepted by consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The specific implementation manner and beneficial effects of the present invention will be further described in detail below with reference to the accompanying drawings.

[0019] Figure 1 Screening diagram of the optimal conditions for in situ illumination co-cultivation; (a) Biomass concentration and optical density (OD) at different inoculation ratios of Spirulina platensis and Saccharomyces cerevisiae 560(a) Growth curve of Spirulina platensis based on cell counting; (b) Microscopic images after 24 hours of culture under different co-culture conditions. Scale bar = 50 μm; (d) Growth curve of Saccharomyces cerevisiae based on cell counting.

[0020] Figure 2 Characterization of the illumination monoculture and in situ illumination co-culture system of Spirulina platensis and Saccharomyces cerevisiae at a yeast to microalgae biomass ratio of 10:1000; (a) Optical density (OD 560 ) and total biomass; (b) the changing dynamics of lipid accumulation; (c) the changing dynamics of glucose concentration; (d) the changing dynamics of dissolved oxygen; (e) the extracellular organic acid content of Saccharomyces cerevisiae; (f) the changing dynamics of pH and acetic acid in the culture medium during in situ illumination co-cultivation.

[0021] Figure 3 Diagrams evaluating the color appearance, aroma quality, and photosynthetic pigments of microalgae; (a) Appearance of wet and freeze-dried biomass of monocultured and (b) co-cultured Spirulina platensis; (c) Colorimetric measurement; (d) Radar plot of eight odor attributes; (e) Contents of chlorophyll a (Chl.a), chlorophyll b (Chl.b), and carotenoids in wet biomass; (f) Contents of phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE) in wet biomass.

[0022] Figure 4 Figure 3. Types and contents of carotenoids in Spirulina grown in monoculture and in situ light-treated coculture.

[0023] Figure 5 Effects of supplementation of culture medium with acetic acid (AC, 1.0 g / L), lactic acid (LC, 1.0 g / L), and yeast extracellular solution (YES) on the biomass yield of Arthrospira platensis; (a) biomass yield; (b) volatile flavor profile; (c) chlorophyll a (Chl. a), chlorophyll b (Chl. b), and carotenoids; (d) phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE). Asterisks indicate significant differences (***P < 0.001, **P < 0.01).

[0024] Figure 6 A circular chromosome representation of the Spirulina platensis FACHB-902 genome; from the outside in, circles 1 and 2 represent clockwise and counterclockwise transcribed coding sequences, respectively; circles 3 and 4 represent GC content and GC skewness, respectively; and circle 5 represents the genomic sequence information.

[0025] Figure 7Figure 3 Genome sequencing and transcriptome analysis of Spirulina platensis; (a) KEGG pathways and gene numbers enriched in the transcriptome; fragments per kilobase per million mapped fragments (FPKM) values of exon models; (b) carotenoid biosynthesis genes, such as phytoene desaturase (crtI)-1, crtI-2, phytoene dehydrogenase (pds)-1, pds-2, and phytoene synthase (crtB); (c) carotenoid cleavage genes, such as carotenoid cleavage dioxygenase (ccd), β-carotene hydroxylase (bch), cytochrome P450 enzyme (cyp)-1 and cyp-2; (d) phycobiliprotein biosynthesis genes, such as phycocyanobilin cleavage enzyme β subunit (cpcF) and phycocyanobilin-ferredoxin oxidoreductase (pcyA); (e) chlorophyll biosynthesis genes, such as cytochrome P 450 (por) oxidoreductase (por), light-independent protochlorophyllide reductase L subunit (chlL), light-independent protochlorophyllide reductase N subunit (chlN), light-independent protochlorophyllide reductase β subunit (chlB), and Mg-protoporphyrin IX methyltransferase (chlG); (f) fatty acid biosynthesis genes, such as acetyl-CoA carboxylase (ACCase)-1, ACCase-2, stearoyl-CoA9-desaturase (scd), and oleoyl-CoA12-desaturase (fad6); (g) fatty acid degradation genes, such as alcohol dehydrogenase (adh)-1, adh-2, cyp-1, and cyp-2; (h) Schematic diagram of the aroma compound precursor accumulation and release pathway. Values with different superscript letters within the same gene were statistically different (P < 0.05) using one-way analysis of variance and Tukey's test for multiple comparisons.

[0026] Figure 8 The results of odor sensory evaluation and volatile flavor compound determination were as follows: (a) odor sensory evaluation of mud-like dark-light co-cultivation, (b) odor sensory evaluation of algal powder yeast fermentation process, and (c) changes in algal terpene odor compounds during mud-like dark-light co-cultivation. DETAILED DESCRIPTION

[0027] Example 1: This example provides a method for in situ co-cultivation of Spirulina with Saccharomyces cerevisiae under illumination before harvesting

[0028] The specific technical solution steps are as follows:

[0029] First, spirulina is cultured until it grows to the late logarithmic growth stage or the stable phase. At this point, nutrients at specific concentrations are added to the culture medium, i.e., 3-7 g / L of glucose and 0.5-1.5 g / L of acetate are added to provide a suitable nutritional environment for the subsequent co-cultivation process. Next, cerevisiae is inoculated at a ratio of 0.1%-10% of the spirulina biomass to ensure that the inoculation ratio of the two is within a reasonable range to promote synergy between each other. Finally, the inoculated system is placed under light and ventilation conditions for co-cultivation, and the co-cultivation time is controlled at 12-36 hours. In this way, spirulina can be co-cultivated with cerevisiae in situ before harvesting to achieve a specific culture effect.

[0030] To ensure production quality, commercial microalgae are usually harvested in the late logarithmic growth phase, when biomass yield is sufficient and the culture cycle is relatively short. In the present invention, the pre-harvest culture of Spirulina platensis in the late logarithmic growth phase was inoculated with Saccharomyces cerevisiae at three yeast to microalgae biomass ratios (1:1000, 10:1000, and 100:1000), and 5 g / L of glucose was added to the co-cultivation system. Based on the OD value during the 36-hour co-cultivation period, the culture medium was inoculated with Saccharomyces cerevisiae at OD value of 1:1000, 10:1000, and 100:1000. 560 Values and biomass concentration change dynamics ( Figure 1 a) Inoculation of Saccharomyces cerevisiae at yeast to microalgae biomass ratios of 1:1000 and 10:1000 significantly increased the biomass yield of the co-culture (P<0.01), whereas addition of excessive Saccharomyces cerevisiae at an inoculation ratio of 100:1000 significantly decreased the biomass yield of the co-culture (P<0.05).

[0031] like Figure 1 As shown in b, at inoculation ratios of 1:1000 and 10:1000, Spirulina platensis continued to proliferate throughout the co-culture period, but at an inoculation ratio of 100:1000, it gradually disappeared from the co-culture system. Figure 1 c) shows that at an inoculation ratio of 100:1000, the platensis Spirulina cells were broken, confirming that in the presence of excessive yeast, platensis Spirulina gradually died. Figure 1 d shows that at all three inoculation ratios, Saccharomyces cerevisiae proliferated during the entire co-culture period. These results indicate that excessive yeast can cause survival pressure on microalgae, which may explain why in the co-culture system with an inoculation ratio of 100:1000, OD 560 The values were significantly lower than those at lower inoculation ratios ( Figure 1 a).

[0032] Based on the above results, a pre-harvest co-culture system of Spirulina platensis and Saccharomyces cerevisiae was established using a yeast to microalgae biomass ratio of 10:1000. Figure 2As shown in a and 2b, the OD of the co-culture system was higher than that of the single culture of Spirulina platensis and Saccharomyces cerevisiae. 560 The values, biomass concentration, and lipid accumulation in the biomass were significantly higher, indicating that the co-culture had excellent biomass and lipid yields.

[0033] Example 2: This example provides a method for co-cultivating Spirulina and Saccharomyces cerevisiae in a dark light sludge state;

[0034] The specific technical solution steps are as follows:

[0035] First, the culture system containing spirulina and Saccharomyces cerevisiae is filtered through a 400-mesh filter. This filtration step effectively removes 80%-90% of the culture medium components, resulting in a mixed slurry of spirulina and yeast. This mixed slurry is then placed in the dark for continued co-cultivation, with the duration of the co-cultivation strictly controlled within the range of 12-36 hours. In this dim environment, the spirulina and Saccharomyces cerevisiae interact to achieve specific cultivation goals, laying the foundation for subsequent related applications.

[0036] Based on the results of odor sensory evaluation and volatile flavor compound determination, 24 h was selected as the optimal harvesting time for the co-culture of Spirulina and Saccharomyces cerevisiae in dark light mud (Table 2-1 to Table 2-9, Figure 8 a and 8b).

[0037] Example 3: This example provides a method for measuring the biomass of Spirulina during its growth process;

[0038] The specific technical solution steps are as follows:

[0039] For the determination of Spirulina biomass, a variety of combined methods are used for accurate measurement. First, through OD 560 During the whole culture period of Spirulina, the OD value of aliquots of culture medium was measured by UV-visible spectrophotometer. 560The numerical value is measured and used as a basis for the preliminary judgment of the growth density of Spirulina. Secondly, the gradient dilution Spirulina cells are counted with the help of a hemocytometer and an optical microscope to intuitively obtain information on the number of cells and assist in the accurate calculation of the biomass. Thirdly, in order to accurately determine the dry weight, a certain volume (denoted as V) of aliquot culture medium is filtered using glass fiber filter paper (pore size 0.45 μm). After the filtration is completed, the filter paper is placed in an oven to dry. Then, the weight of the filter paper (denoted as M2) on which the cells are filtered after drying is subtracted from the weight of the original filter paper (denoted as M1). The difference is the dry weight of Spirulina in this part of the culture medium. Finally, the Spirulina biomass concentration (g / L) is calculated according to the following formula (1). Through the coordinated cooperation of the above multiple steps and multiple methods, a comprehensive and accurate measurement of the biomass of Spirulina during its growth process is achieved, providing reliable data support for Spirulina-related research and industrial applications.

[0040]

[0041] like Figure 1 As shown in a, under the optimal co-culture conditions, the biomass of both microorganisms increased with the extension of culture time. This indicates that at this inoculation ratio, the co-culture of the two microorganisms can achieve rapid proliferation. Acetic acid significantly increased the biomass yield of microalgae (P<0.05), while lactic acid had no such effect. The effect of acetic acid was similar to that of yeast extracellular solution ( Figure 5 a).

[0042] Example 4: This example provides a method for evaluating the color and pigment content of Spirulina algae powder.

[0043] The specific technical solution steps are as follows:

[0044] All sensory analyses were performed by a sensory panel of 15 experts, including 7 women and 8 men, who were trained in ISO5496:2006, ISO8586:2012 and GB / T39625-2020 (Chinese national standard).

[0045] The color of the microalgae was measured using a colorimeter equipped with the CIE color system. Results were evaluated in terms of L* (lightness), a* (+a, red; -a, green), and b* (+b, yellow; -b, blue).

[0046] Overall odor evaluation was performed by placing a 2-gram sample into an odorless, transparent SPME vial and allowing it to sit at room temperature for 30 minutes. Odor vocabulary was determined using a free-choice analysis (FCP) method. First, assessors elicited accurate odor descriptors as freely as possible to establish a corresponding microalgae odor vocabulary. They then discussed the odor attributes and reached consensus based on the 10 most frequently occurring descriptors. Finally, assessors assigned an overall odor rating for each microalgae sniff sample attribute on a linear scale of 0 to 10, describing odor intensity, based on the descriptive task discussed previously.

[0047] The results of odor sensory evaluation showed that the in situ light-induced co-cultivation of Spirulina and Saccharomyces cerevisiae could significantly improve the odor quality of the algae ( Figure 3 d). Among them, acetic acid significantly improved the aroma quality of microalgae (P<0.05), while lactic acid had no such effect. The effect of acetic acid was similar to that of yeast extracellular solution ( Figure 5 b). During the co-cultivation of Spirulina and Saccharomyces cerevisiae in a dark-light mud-like environment, the "floral and fruity aroma" of the algae was further released ( Figure 8 a and 8b). Compared with algal flour yeast fermentation, it has better aroma quality.

[0048] Color is a primary quality indicator of microalgae. Spirulina powder typically appears dark bluish-green and is characterized by a high phycocyanin content. However, due to the appetite-reducing effect of the blue color, the appearance of Spirulina biomass is unattractive in many food applications. Saccharomyces cerevisiae can be separated from co-cultures by centrifugation based on density differences. Freeze-dried Spirulina biomass co-cultured with in situ illumination exhibited a light green color. Co-cultivation induced an increase in L* (lightness), −a (green), and +b (yellowness) values in the microalgae biomass (P < 0.05), confirming the color shift from dark bluish-green in monoculture to light green in co-culture, suggesting a suitable approach to improve consumer product acceptability.

[0049] Example 5: This example provides a method for measuring the content of components in Spirulina powder.

[0050] The specific technical solution steps are as follows:

[0051] First, to determine the oil content in Spirulina powder: 20 mg of freeze-dried powder was accurately weighed and placed in a suitable container. 0.75 mL of a mixture of water, chloroform, and methanol in a volume ratio of 0.5:2:1 was added. The mixture was then shaken for 20 minutes to thoroughly mix the powder and the mixture. Next, the mixture was centrifuged at 10,000 rpm for 10 minutes to complete the initial separation. The extraction process was then repeated 3-5 times to ensure complete oil extraction. All chloroform layers were collected and combined, dried under vacuum to a constant weight, and the oil content of the algal cells was accurately calculated based on the weight difference and relevant calculation rules.

[0052] Next, to determine the content of chlorophyll a, chlorophyll b, and carotenoids, Spirulina powder was extracted using acetone (80% v / v) at a low temperature of 4°C. Extraction was continued until the powder was almost colorless within the tissue, indicating that the pigments had been essentially completely extracted. The extract was then measured using specialized instruments at wavelengths of 670nm, 646nm, and 470nm. Using specific pigment concentration calculation formulas (2-5) and combined with the measured data, the concentrations of chlorophyll a, chlorophyll b, and carotenoids were accurately calculated.

[0053] ΔA=A 实验 −A 空白 (2)

[0054] (3)

[0055] (4)

[0056] (5)

[0057] Furthermore, regarding the determination of the content of Spirulina algal protein (phycocyanin, allophycocyanin, and phycoerythrin), the biomass was ground using a high-speed, low-temperature tissue grinder for 30 minutes to fully disrupt the biomass structure. The ground biomass was then suspended in phosphate-buffered saline solution and stored in the dark at 4°C for 1 hour to allow for thorough mixing and stabilization. The algal protein content was then calculated using the corresponding formulas (6-8) combined with the measured data.

[0058]

[0059]

[0060]

[0061] Finally, for further precise analysis of carotenoid content, the freeze-dried microalgae samples were placed at 4°C and ground using a tissue grinder for 10 cycles, each cycle lasting 60 seconds, to fully break up the algal powder. Subsequently, they were repeatedly extracted with methyl tert-butyl ether until the algal powder was almost colorless, ensuring sufficient carotenoid extraction. The samples were centrifuged at 6000 × g for 10 minutes to achieve solid-liquid separation. The extract was dried with nitrogen and then reconstituted in 500 μL of acetone. Finally, the reconstituted samples were analyzed by high-performance liquid chromatography to accurately determine the carotenoid content. Through this rigorous and meticulous series of steps, the precise content of each component of Spirulina powder can be measured, providing critical data support for many fields, such as spirulina product quality control and scientific research applications.

[0062] like Figure 2 As shown in b, under the optimal co-culture conditions, the lipid content of Spirulina increased with the extension of culture time, indicating that in situ light co-culture of microalgae promoted the increase of Spirulina lipid content.

[0063] There are three typical types of photosynthetic pigments in microalgae, namely chlorophyll, carotenoids and phycobiliproteins. The contents of chlorophyll a, chlorophyll b, carotenoids, phycocyanin, allophycocyanin and phycoerythrin in co-culture were significantly higher than those in single-culture microalgae (P<0.05), indicating that in situ light co-culture of Spirulina and Saccharomyces cerevisiae can induce the biosynthesis of the three typical photosynthetic pigments ( Figure 3 e and 3f). Compared with the single-culture microalgae, the chlorophyll a and carotenoid contents in the co-cultured algae increased by more than 2 times.

[0064] Similarly, to investigate whether organic acids produced by yeast are responsible for the improved quality of Spirulina in in situ light-treated co-culture, we investigated the effects of adding acetic acid, lactic acid, and yeast extracellular solution to the culture medium on pigment biosynthesis in Spirulina platensis ( Figure 5 c and 5d). Acetic acid significantly increased the chlorophyll and carotenoid contents of microalgae (P<0.05), while lactic acid had no such effect. The effect of acetic acid was similar to that of yeast extracellular solution. However, acetic acid had a negative effect on microalgae phycobiliprotein (P<0.05). Given that the addition of acetic acid during the culture period had no significant effect on the pH value of the culture medium (9.2-11.0) ( Figure 2 f), acetate appears to be the factor that plays a role in yeast-induced changes in the color appearance and aroma quality of Spirulina.

[0065] We further explored the types and specific contents of carotenoids during the in situ co-cultivation of microalgae. The results showed that the co-cultivation of Spirulina and Saccharomyces cerevisiae can significantly increase the content of β-carotene in the algae ( Figure 4). It complies with the requirement in the Technical Requirements for Spirulina Raw Materials that the content of β-carotene, a hallmark ingredient of edible-grade spirulina, must be ≥0.2g / kg.

[0066] Example 6: This example provides a method for determining the content of volatile flavor compounds in Spirulina algae powder.

[0067] The specific technical solution steps are as follows:

[0068] First, prepare the sample solution: Using saturated sodium chloride solution, prepare a 40 mg / mL salt solution of Spirulina powder. Measure 2 mL of each solution and carefully transfer it to a 20 mL extraction vial. Then, add 2-methyl-3-heptanone (100 μg / g, corresponding to a 20 μL volume) as an internal standard. This internal standard is used for subsequent data correction to ensure measurement accuracy.

[0069] Next, volatile compounds were enriched and analyzed: a sample vial containing the algal powder solution and internal standard was placed at a constant temperature of 60°C with continuous stirring at 300 rpm for 20 minutes to allow for the complete release of volatile compounds. Subsequently, a 50 / 30 μm extraction needle (model DVB / CAR / PDMS57348-U) was inserted for 30 minutes of headspace extraction to efficiently enrich the volatile compounds in the Spirulina powder. Prior to using the extraction needle, the extraction tip was heated at 250°C for 20 minutes at the gas injection port. This pretreatment step helps improve extraction efficiency. The enriched volatile compounds were analyzed using a gas chromatography-mass spectrometry system on an INNOWAX column (60 m length, 0.25 mm inner diameter, 0.25 μm film thickness). Helium was used as the carrier gas at a flow rate of 1.2 mL / min. During the initial GC-MS analysis, the system was maintained at a constant temperature of 40°C for 5 minutes, then heated to 240°C at a rate of 5°C / min, and then to 250°C at a rate of 10°C / min. First, the sample solution was prepared: a 40 mg / mL solution of Spirulina powder salt was accurately prepared using a saturated sodium chloride solution. 2 mL of each Spirulina powder solution was carefully transferred to a 20 mL extraction vial. 2-methyl-3-heptanone (100 μg / g, corresponding to a volume of 20 μL) was then added as an internal standard. This internal standard was used for subsequent data correction to ensure measurement accuracy.

[0070] Next, the volatile compounds were enriched: the sample extraction vial containing the algal powder solution and internal standard was placed at a constant temperature of 60°C with continuous stirring at 300 rpm for 20 minutes to promote the release of the volatile compounds. Subsequently, a 50 / 30 μm extraction needle (model DVB / CAR / PDMS57348-U) was inserted and headspace extraction was performed for 30 minutes to efficiently enrich the volatile compounds in the Spirulina powder. It is important to note that before using the extraction needle, the extraction tip must be heated at the gas injection port for 20 minutes at 250°C. This pretreatment step helps improve extraction efficiency.

[0071] Finally, volatile compounds were analyzed and detected. The enriched volatile compounds were analyzed using a gas chromatography-mass spectrometry system on an INNOWAX column (60 m length, 0.25 mm inner diameter, 0.25 μm film thickness). Helium was used as the carrier gas at a flow rate of 1.2 mL / min. The GC-MS analysis was initially maintained at 40°C for 5 minutes, then ramped to 240°C at a rate of 5°C / min, and then to 250°C at a rate of 10°C / min and held for 6 minutes. This temperature program effectively separated the different volatile compounds. The mass scan range was set at 29-550 Da, and the electron energy was 70 eV. Through this rigorous and standardized procedure, the content of volatile flavor compounds in Spirulina powder was accurately determined, providing strong data support for the research of spirulina flavor characteristics and product quality optimization.

[0072] In the present invention, the levels of several aldehydes, including valeraldehyde, hexanal, heptanal, (E)-2-heptenal, and octanal, increased significantly (P<0.05) during both in situ light-activated and sludge-induced dark-activated co-cultures of Spirulina and Saccharomyces cerevisiae (Tables 1-1 through 1-10 and 2-1 through 2-9). C5-C9 aldehydes impart a pleasant "fresh green / fruity" to "fatty green / fruity" aroma, and these C5-C8 aldehydes are believed to contribute to the "fruity" aroma imparted to Spirulina microalgae by yeast co-culture.

[0073] During the in situ light-induced co-culture of Spirulina and Saccharomyces cerevisiae and the dark-induced co-culture of slurry, the contents of norisoprene compounds such as trans-β-ionone, epoxy-β-ionone and dihydroactinolide in microalgae increased significantly (P<0.05) (Tables 1-1 to 1-10, Tables 2-1 to 2-9 and Figure 8c). Isoprenes are a diverse class of aromatic compounds that, to some extent, explain the pleasant "floral" and "fruity" microalgae aromas imparted by co-cultivation with yeast. These results indicate that Spirulina powder produced through Saccharomyces cerevisiae-Spirulina co-cultivation exhibits a strong overall fruity and floral aroma.

[0074] Tables 1-1 to 1-10 show the changes in volatile flavor compounds in the algae during in situ illumination of Spirulina and Saccharomyces cerevisiae co-culture (mg / kg)

[0075] Table 1-1

[0076] Ketone compound name Component RI CAS Number control group In situ co-culture group 1 2-Butanone 2-Butanone 894 78-93-3 0.305±0.031 0.061±0.002*** 2 2,3-Butanedione 2,3-Butanedione 971 431-03-8 0.286±0.016 0.352±0.015* 3 2-Methyl-3-heptanone 2-methyl-3-Heptanone 1161 13019-20-0 0.489±0.009 10.25±0.204*** 4 2-Heptanone 2-Heptanone 1177 110-43-0 1.82±0.024 2.851±0.042*** 5 3-Octanone 3-Octanone 1249 106-68-3 0.896±0.005 0.167±0.003*** 6 6-Methyl-2-heptanone 6-methyl-2-Heptanone 1232 928-68-7 0.285±0.012 0.166±0.003*** 7 2-Octanone 2-Octanone 1281 111-13-7 1.155±0.028 0.365±0.037*** 8 Hydroxyacetone 1-hydroxy-2-Propanone 1292 116-09-6 0.283±0.006 ND 9 2,2,6-Trimethylcyclohexanone 2,2,6-trimethyl-Cyclohexanone 1314 2408-37-9 0.964±0.008 1.126±0.06* 10 6-Methyl-5-hepten-2-one 6-methyl-5-Hepten-2-one 1332 110-93-0 1.525±0.029 0.761±0.068*** 11 2-Methyl-2-hepten-4-one 2-methyl-2-Hepten-4-one 1357 22319-24-0 0.901±0.015 0.305±0.004*** 12 2-Nonanone 2-Nonanone 1384 821-55-6 2.039±0.008 0.119±0.001*** 13 3-Octen-2-one 3-Octen-2-one 1402 1669-44-9 ND 0.87±0*** 14 2-Decanone 2-Decanone 1489 693-54-9 0.591±0.009 0.591±0.001 15 1-(2-Methyl-1-cyclopentenyl)ethanone 1-(2-methyl-1-cyclopenten-1-yl)-Ethanone 1589 3168-90-9 0.299±0.001 0.241±0.007*** 16 4-Hydroxy-4-methylcyclohexanone 4-hydroxy-4-methyl-Cyclohexanone 1601 17429-02-6 25.174±1.775 50.598±0.329*** 17 acetophenone Acetophenone 1648 98-86-2 0.21±0.015 0.167±0.022 18 3-Acetyl-2-octanone 3-Acetyl-2-octanone 1663 27970-50-9 1.067±0.017 1.744±0.019*** 19 4-Oxoisophorone 2,6,6-Trimethyl-2-cyclohexene-1,4-dione 1691 1125-21-9 0.999±0.001 0.576±0.005*** 20 p-Methylacetophenone 1-(4-methylphenyl)-Ethanone 1775 122-00-9 ND 0.216±0.003*** 21 Geranyl acetone Geranylacetone 1849 3796-70-1 0.807±0.137 1.701±0.04*** 22 beta-ionone trans-β-Ionone 1939 79-77-6 11.847±0.125 29.688±0.255*** 23 Ionone oxide epoxy-β-ionone 1993 23267-57-4 4.212±0.01 13.234±0.191*** 24 3,4-Dehydroionone 3,4-Dehydro-β-ionone 1999 1203-08-3 ND 0.235±0.004*** 25 Phyton 6,10,14-trimethyl-2-Pentadecanone 2121 502-69-2 1.017±0.058 3.106±0.077*** 26 Oxyionone (E)-4-Oxo-β-ionone 2460 27185-77-9 0.136±0.004 0.296±0.003*** 27 2-Pyrrolidone 2-Pyrrolidinone 2027 616-45-5 1.55±0.008 0.467±0.027*** 28 3-Ethyl-4-methyl-pyrrole-2,5-dione 3-ethyl-4-methyl-1H-Pyrrole-2,5-dione 2251 20189-42-8 9.652±0.042 21.381±0.066*** 29 Menthol ketone ether Piperitenoneoxide 2144 35178-55-3 ND 0.647±0.006***

[0077] Table 1-2

[0078] Name of aldehyde compound Component RI CAS Number control group In situ co-culture group 1 acetaldehyde Acetaldehyde 691 75-07-0 0.293±0.011 0.272±0.018 2 2-Methylbutanal 2-methyl-Butanal 907 96-17-3 0.812±0.01 0.127±0.005*** 3 Isovaleraldehyde 3-methyl-Butanal 911 590-86-3 1.3±0.081 0.293±0.05*** 4 Valeraldehyde Pentanal 972 110-62-3 0.41±0.008 1.256±0.045*** 5 Hexanal Hexanal 1075 66-25-1 7.415±0.094 14.336±0.275*** 6 Heptanal Heptanal 1180 111-71-7 0.9±0.008 1.416±0.069*** 7 Octanal Octanal 1283 124-13-0 ND 0.531±0.009*** 8 trans-2-heptenal (E)-2-Heptenal 1320 18829-55-5 ND 0.199±0*** 9 Nonanal Nonanal 1388 124-19-6 0.622±0.001 3.47±0.22*** 10 trans-2-octenal (E)-2-Octenal 1425 2548-87-0 0.319±0.001 0.601±0.002*** 11 3-Furfural 3-Furaldehyde 1454 498-60-2 0.117±0.002 0.127±0.002** 12 trans-2-nonanal (E)-2-Nonenal 1533 18829-56-6 0.251±0.007 1.04±0.032*** 13 BETA-Cyclocitral β-Cyclocitral 1621 432-25-7 5.31±0.09 5.515±0.004* 14 Safranal Safranal 1645 116-26-7 0.271±0.009 0.259±0.001 15 3-Ethylbenzaldehyde 3-ethyl-Benzaldehyde 1707 34246-54-3 0.054±0.002 ND 16 (E,Z)-2,4-Decadienal (E,Z)-2,4-Decadienal 1810 25152-83-4 ND 0.165±0.012***

[0079] Table 1-3

[0080] Name of alcohol compound Component RI CAS Number control group In situ co-culture group 1 ethanol Ethanol 925 64-17-5 1.854±0.082 0.376±0.011*** 2 n-Butanol 1-Butanol 1137 71-36-3 12.617±0.177 3.464±0.216*** 3 1-Pentanol 1-Pentanol 1241 71-41-0 1.573±0.006 ND 4 1-Hexanol 1-Hexanol 1341 111-27-3 0.763±0.121 0.397±0.064* 5 Mushroom alcohol 1-Octen-3-ol 1437 3391-86-4 5.504±0.085 4.424±0.101*** 6 1-Heptanol 1-Heptanol 1442 111-70-6 0.442±0.011 ND 7 2-Ethylhexanol 2-ethyl-1-Hexanol 1476 104-76-7 2.418±0.043 4.569±0.767* 8 1-octanol 1-Octanol 1544 111-87-5 0.565±0.012 0.147±0.022*** 9 Nonyl alcohol 1-Nonanol 1646 143-08-8 0.52±0.008 0.164±0.02*** 10 Benzyl alcohol Benzylalcohol 1862 100-51-6 1.818±0.08 1.357±0.039*** 11 Phenylethyl alcohol PhenylethylAlcohol 1898 60-12-8 0.196±0.003 0.217±0.011 12 Decyl alcohol 1-Decanol 1952 112-30-1 ND 0.156±0.003*** 13 Phytol Phytol 2575 150-86-7 ND 0.259±0.009***

[0081] Table 1-4

[0082] Names of nitrogen-containing compounds Component RI CAS Number control group In situ co-culture group 1 Pyrazine Pyrazine 1203 290-37-9 0.09±0.002 ND 2 2-Methylpyrazine 2-methyl-Pyrazine 1259 109-08-0 1.181±0.066 0.162±0.007*** 3 2,5-Dimethylpyrazine 2,5-dimethyl-Pyrazine 1316 123-32-0 1.784±0.005 0.321±0.001*** 4 2,6-Dimethylpyrazine 2,6-dimethyl-Pyrazine 1322 108-50-9 0.298±0.002 ND 5 2-Ethyl-6-methylpyrazine 2-ethyl-6-methyl-Pyrazine 1379 13925-03-6 0.117±0.003 ND 6 Pyrrole Pyrrole 1501 109-97-7 0.072±0.01 ND 7 2-Acetylpyrrole 1-(1H-pyrrol-2-yl)-Ethanone 1958 1072-83-9 0.078±0.005 0.17±0*** 8 Trimethylamine N,N-dimethyl-Methylamine 642 75-50-3 2.41±0.325 5.367±0.165***

[0083] Table 1-5

[0084] Name of phenolic compound Component RI CAS Number control group In situ co-culture group 1 3-Methyl-4-isopropylphenol 3-Methyl-4-isopropylphenol 1410 3228-02-2 1.258±0.002 0.736±0.013*** 2 2,6-di-tert-butyl-4-methylphenol ButylatedHydroxytoluene 1903 128-37-0 0.057±0.002 0.5±0.016*** 3 phenol Phenol 1986 108-95-2 0.204±0.003 15.146±0.012*** 4 4-tert-Butylphenol p-tert-butyl-Phenol 2267 98-54-4 0.144±0.001 1.358±0.001*** 5 2,4-di-tert-butylphenol 2,4-Di-tert-butylphenol 2286 96-76-4 0.369±0.017 1.104±0.078***

[0085] Table 1-6

[0086] Names of sulfur-containing compounds Component RI CAS Number control group In situ co-culture group 1 Methyl mercaptan Methanethiol 677 74-93-1 0.069±0.008 ND 2 dimethyl disulfide Dimethyldisulfide 1063 624-92-0 0.3±0.021 ND 3 dimethyl trisulfide Dimethyltrisulfide 1375 3658-80-8 0.114±0.005 ND 4 dimethyl sulfoxide DimethylSulfoxide 1568 67-68-5 0.069±0 ND

[0087] Table 1-7

[0088] Acid compound name Component RI CAS Number control group In situ co-culture group 1 Acetic acid Aceticacid 1435 64-19-7 1.319±0.001 0.589±0.001*** 2 bitter Octanoic acid 2037 124-07-2 0.774±0.032 0.408±0.058*** 3 Decanoic acid n-Decanoicacid 2248 334-48-5 ND 0.264±0.003***

[0089] Table 1-8

[0090] Furan compound name Component RI CAS Number control group In situ co-culture group 1 3-Methylfuran 3-methyl-Furan 857 930-27-8 0.102±0.009 0.073±0.005* 2 2-n-pentylfuran 2-pentyl-Furan 1224 3777-69-3 0.898±0.026 2.368±0.169***

[0091] Table 1-9

[0092] Ester compound name Component RI CAS Number control group In situ co-culture group 1 Dihydroactin Dihydroactinidiolide 2358 17092-92-1 4.982±0.014 16.727±0.223***

[0093] Table 1-10

[0094] Name of alkane compound Component RI CAS Number control group In situ co-culture group 1 Decane Decane 996 124-18-5 0.84±0.041 0.279±0.001*** 2 Undecane Undecane 1089 1120-21-4 4.533±0.78 0.284±0.003*** 3 Acetylcyclohexene 1-(1-cyclohexen-1-yl)-Ethanone 1115 932-66-1 ND 0.221±0.001*** 4 5-Ethyldecane 5-Ethyldecane 1129 17302-36-2 0.551±0.025 ND 5 Limonene D-Limonene 1191 5989-27-5 ND 0.078±0.006*** 6 Dodecane Dodecane 1196 112-40-3 12.567±0.079 1.954±0.038*** 7 Styrene Styrene 1251 100-42-5 1.153±0.027 ND 8 Tridecane Tridecane 1297 629-50-5 6.323±0.1 5.507±0.087*** 9 2-Methyltridecane 2-methyl-Tridecane 1353 1560-96-9 0.401±0.007 0.409±0.001 10 3-Methyltridecane 3-methyl-Tridecane 1363 6418-41-3 1.939±0.003 2.193±0.005*** 11 Tetradecane Tetradecane 1395 629-59-4 2.979±0.517 5.455±0.118*** 12 Pentadecane Pentadecane 1495 629-62-9 49.193±0.109 30.276±5.551** 13 Hexadecane Hexadecane 1595 544-76-3 39.117±1.442 24.063±0.051*** 14 Heptadecane Heptadecane 1705 629-78-7 421.347±19.469 260.721±4.31*** 15 Octadecane Octadecane 1797 593-45-3 1.331±0.017 ND 16 3-Heptadecene (Z)-3-Heptadecene 1717 1000141-67-3 36.228±1.801 12.014±0.011***

[0095] Note: ND means not detected. Significant differences compared with single culture samples are marked with "*", "**" and "***" at P < 0.05, P < 0.01 and P < 0.005, respectively.

[0096] Tables 2-1 to 2-9 show the changes in volatile flavor compounds in the algae during the co-culture of Spirulina and Saccharomyces cerevisiae (mg / kg)

[0097] Table 2-1

[0098] Ketone compound name Component RI CAS Number 0h 12h 24h 36h 1 2-Butanone 881 78-93-3 6.25±0.93b 7.5±0.07c 5.92±0.46b 4.78±0.38a 2 2,3-Butanedione 960 431-03-8 22.32±0.32d 15.57±0.33c 11±1b 9.18±0.18a 3 3-Butyl-cyclopentanone 1101 57283-81-5 10.69±0.01b ND 10.51±0.51b 10.74±0.31b 4 6-Methyl-7-octenone 1139 35215-49-7 23.52±0.14b 25.55±1.05c 21.27±0.16a 23.12±1.09b 5 2-Heptanone 1164 110-43-0 18.08±0.08b 18.19±0.87b 14.24±0a 339.64±2.09c 6 6-Methyl-2-heptanone 1220 928-68-7 67.33±1.82b 74.48±0.58c 64.03±0.86a 62.45±0.22a 7 3-Octanone 1236 106-68-3 18.41±0.19c 11.1±0.04b 7.53±1.66a 8.25±1.39a 8 2-Octanone 1267 111-13-7 44.06±0.14c 32.07±2.2ab 23.91±3.24bc 38.66±8.49a 9 Mushroom Ketone 1283 4312-99-6 12.29±1.58a 14.33±0.84b 14.07±0.07ab 18.09±0.97c 10 2,2,6-Trimethylcyclohexanone 1299 2408-37-9 150.42±9a 153.83±1.34a 165.37±23.16a 162.17±5.15a 13 2-Nonanone 1370 821-55-6 18.46±0.17c 9±0.3a ND ND 14 Isophorone 1384 78-59-1 236.51±1.7ab 218.39±47.8ab 207.08±44.48a 276.46±20.13b 15 3-Octen-2-one 1387 1669-44-9 12.7±0a 17.22±0.24a 17.74±5.35a 50.32±0.32b 16 3,4,4-Trimethylcyclohexenone 1429 17299-41-1 19.49±0.31a 21.51±0.16b 21.82±1.04b 23.68±0.34c 17 2,4,6-Trimethylcyclohexane-1,3-dione 1504 20990-16-3 62.59±2.41b 62.01±0.62b 58.19±1.26a 64.23±2.78b 18 3-Butylcycloheptanone 1520 1000164-85-6 809.68±11.06b 802.61±1.65b 736.52±24.44a 776.74±30.76b 20 3-Acetyl-2-octanone 1646 27970-50-9 ND 22.28±0.08b 27.34±0.34c 35.21±0.52d 21 4-Oxoisophorone 1673 1125-21-9 14.9±0.53a 17.45±0.84c 16.23±0.42b 18.25±0.24c 22 p-Methylacetophenone 1733 122-00-9 2.73±0.3a 3.34±0.22b 2.42±0.03a 2.5±0.09a 23 BETA-Dihydroionone 1811 17283-81-7 ND 4.63±0.48c 3.75±0.25b 5.87±0.39d 24 Geranyl acetone 1829 3796-70-1 66.56±4.03b 60.01±1.81a 55.8±0.88a 66.3±4.38b 26 beta-ionone 1917 79-77-6 1054.62±40.54a 1158.5±28.89b 1226.96±19.42c 1393.64±47.65d 27 Ionone oxide 1971 23267-57-4 628.3±19.71a 647.92±14.94a 637.14±18.58a 749.58±10.47b 28 3,4-Dehydroionone 1976 1203-08-3 9.06±0.69a 10.72±0.63b 10.41±0.54b 14.89±0.84c 29 Phyton 2110 502-69-2 12.47±0.03b ND 23.87±0.67c 27.51±3.86c 30 Menthol ketone ether 2120 35178-55-3 21.58±0.75a 23.22±1.03b 25.12±0.38c 35.27±1.05d 31 BETA-Dihydroionone 2183 17283-81-7 ND 4.63±0.48c 3.75±0.25b 5.87±0.39d 32 2'-Hydroxy-5'-methoxyacetophenone 2226 705-15-7 67.81±6.97c 56.35±4.64b ND ND 33 Oxyionone 2435 27185-77-9 10.93±0.95a 11.14±0.2a 10.2±0.22a 14.35±0.93b 34 6-Methyl-6-nitro-2-heptanone 1841 142963-25-5 248.49±4.09a 336.93±5.24b 371.87±10.68c 437.66±1.92d

[0099] Table 2-2

[0100] Name of alcohol compound Component RI CAS Number 0h 12h 24h 36h 1 Methyl mercaptan 671 74-93-1 2.76±0.24a 3.31±0.02b 3.27±0.23b 3.71±0.05c 3 Isobutanol 1080 78-83-1 7.82±0.06c 7.59±0.29c 6.65±0.75b ND 4 n-Butanol 1129 71-36-3 68.73±3.25c 73.38±1.98c 53.26±1.61b 46.68±3a 5 Isoamyl alcohol 1187 123-51-3 149.39±15.4ab 196.57±4.97c 157.56±11.77b 127.96±12.12a 6 1-Pentanol 1228 71-41-0 61±4.11a 72.28±0.62b 58.41±0.57a 55.33±5.54a 7 1-Hexanol 1328 111-27-3 433.48±12.41c 324.18±0.95b 241.68±6.19a 232.97±12.81a 8 Mushroom alcohol 1422 3391-86-4 669.76±0.75a 909.43±6.44b 908.16±58.4b 877.45±43.66b 9 1-Heptanol 1427 111-70-6 82.79±3.53c 66.34±0b 51.96±4.04a 55.63±3.34a 10 2-Ethylhexanol 1461 104-76-7 99.6±1.99c 87.8±0.44b 79.89±8.24ab 73.69±0.63a 11 Terpineol 1514 29803-82-5 295.94±3.32a 394.3±1.18c 365.15±15.15b 405.81±15.07c 12 1-octanol 1529 111-87-5 75.63±2.09c 70.79±3.72bc 60.65±0.37a 66.87±5.31ab 13 3,4-Dimethylcyclohexanol 1582 5715-23-1 1454.7±16.04a 1661.17±24.32b 1797.71±191.56b 1761.84±15.07b 14 Nonyl alcohol 1630 143-08-8 110.84±4.93d 48.37±4.43c 25.47±3.98b ND 15 3,5-Dimethylcyclohexanol 1764 5441-52-1 125.1±3.17a 163.19±2.93b 172.23±6c 180.92±3.45d 16 Benzyl alcohol 1840 100-51-6 19.09±0.75c 18.45±0.28c 14.82±0.73a 16.84±1.13b 17 Phenylethyl alcohol 1877 60-12-8 47.67±0a 68.11±1.1b 116.46±4.02c 100.13±1.7d 18 Decyl alcohol 1931 112-30-1 39.76±1.02c 21.12±1.39b 17.47±0.47a 15.94±2.18a 19 Phenoxyethanol 2102 122-99-6 17.82±0.33c 9.14±1.13b 7.55±0.28a 8.34±1.02ab 24 Phytol 2560 150-86-7 13.4±0.42c 7.5±0b 1.56±0.24a 13.66±1.65c

[0101] Table 2-3

[0102] Ester compound name Component RI CAS Number 0h 12h 24h 36h 1 Methyl acetate 808 79-20-9 6.11±1.04c 6.7±0c 3.6±0.68b ND 2 Ethyl acetate 865 141-78-6 17.82±2.45b 28.96±1.32c 15.71±1.89ab 13.88±0.24a 3 Methyl propionate 888 554-12-1 2.31±0.29c 1.95±0.07b ND ND 4 Methyl butyrate 967 623-42-7 5.99±0.56c 6.16±0.16c 5.28±0.28b ND 5 Ethyl butyrate 1020 105-54-4 ND 15.65±1.46b 26.51±2.52c 23.71±2.67c 6 Methyl crotonate 1088 18707-60-3 25.57±1.01c 25.82±4.04c 15.93±0.1b 11.11±2.42a 7 Isopropyl valerate 1116 18362-97-5 24.48±1.96a 28.88±0.27a 34.95±2.12b 36.88±5.23b 8 Butyl butyrate 1196 109-21-7 ND ND 4.1±0.1b 3.93±0.38b 9 Ethyl hexanoate 1214 123-66-0 ND 4.12±0.07b 5.44±0.9c 6.52±0.51d 10 Hexyl acetate 1253 142-92-7 7.82±0.06c 7.27±0.16b ND ND 11 Vinyl hexanoate 1303 3050-69-9 40.32±0.38a 70.82±0.91c 58.54±2.14b 41.94±1.94a 12 Mushroomyl acetate 1357 77149-68-9 ND 4.8±0.05a 4.9±0a 29.78±24.22b 13 Methyl octanoate 1368 111-11-5 35.3±0.39b 13.47±0.31a 9.47±2.24a 29.03±6.95b 14 Methyl S-3-hydroxybutyrate 1455 53562-86-0 717.8±13.19c 925.32±68.79d 644.95±20.02b 525.35±22.23a 15 Methyl 2-hydroxy-2-methylpropionate 1459 1000289-09-5 60.78±0.4b 65.93±1.19c 58.47±3.73ab 54.83±3.14a 16 Ethyl 3-hydroxybutyrate 1491 5405-41-4 341.86±3.09a 626.29±5.32c 484.14±37.99b 333.79±15.33a 17 Methyl decanoate 1574 110-42-9 13.15±0.15c ND ND 9.48±1.44b 18 Ethyl benzoate 1645 93-89-0 1.5±0.1b 1.74±0.14c ND ND 19 Fructose 1778 1648615 20.34±0.9b 20.67±1.09b ND ND 20 Ionyl acetate 1805 58430-94-7 5.59±0.11a 9.55±1.79b 13.02±2.74c 14.2±1c 21 Methyl palmitate 2192 112-39-0 144.26±13.92c 35.86±9.03a 73.15±14.37b 127.57±20.02c 22 Methyl palmitoleate 2218 1120-25-8 65.98±2.11c ND ND 26.39±4.72b 23 Ethyl palmitate 2231 628-97-7 ND ND 94.07±0.21b 207.25±0.82c 24 Dihydroactin 2332 17092-92-1 651.96±14.23a 694.64±31.36a 656.7±7.35a 925.38±33.69b

[0103] Table 2-4

[0104] Name of aldehyde compound Component RI CAS Number 0h 12h 24h 36h 1 acetaldehyde 684 75-07-0 11.16±1b 9.89±0.29ab 9.86±0.83ab 9.07±0.78a 2 2-Methylbutanal 898 96-17-3 2.68±0.52a 5.58±0.1c 5.31±0.91bc 4.4±0.27b 3 Isovaleraldehyde 902 590-86-3 15.45±2.31a 23.76±0.43b 22.55±3.07b 23.82±0.73b 4 Valeraldehyde 961 110-62-3 22.13±2.99a 33.77±0.67b 48.04±1.89c 43.31±5.75c 5 Crotonaldehyde 1025 4170-30-3 5.46±0.45b 3.87±0.68a 3.6±0.4a 3.53±0.37a 6 Hexanal 1063 66-25-1 313.71±14.82a 429.89±3.42b 620.14±87.42c 481.46±7.9b 7 Heptanal 1166 111-71-7 111.64±0.04b 144.95±0.6c 158.08±17.17c ND 8 Octanal 1271 124-13-0 37.13±0.13a 42.74±3.74c 38.53±0.94ab 41.98±1.35bc 9 trans-2-heptenal 1306 18829-55-5 34.1±1.09a 52.46±0.71b 49.45±1.87b 50.48±4.5b 10 Nonanal 1374 124-19-6 403.38±5.92b 360.89±9.29a 353.07±8.49a 387.91±10.96b 12 trans-2-octenal 1410 2548-87-0 93.29±0.64a 120.3±1.15b 120.39±1.83b 124.64±9.19b 13 3-Furfural 1437 498-60-2 16.89±2.02a 25.55±0.75c 22.01±0.44b 23.39±1.05bc 14 3-Isopropylbenzaldehyde 1438 34246-57-6 7.15±0.15d 5.68±0.07c 3.95±0.05b 3.36±0.16a 17 5-Methylfuraldehyde 1531 620-02-0 ND 3.79±0.07c 3.46±0.03b 3.95±0.04d 18 BETA-Cyclocitral 1603 432-25-7 842.3±19.75a 944.97±9.67b 949.09±37.23b 982.92±37.07b 19 Safranal 1626 116-26-7 62.69±1.35a 88.67±1.26b 108.54±3.84c 121.48±9.46d 20 Salicylaldehyde 1657 90-02-8 4.33±0.28a 5.28±0.01b 4.3±0.08a 9.11±0.16c 22 trans-2,4-decadienal 1743 25152-84-5 20.51±0.51b 15.23±0.23a 47±0c 77.99±0.01d

[0105] Table 2-5

[0106] Acid compound name Component RI CAS Number 0h 12h 24h 36h 1 Acetic acid 1421 64-19-7 62.14±6.06a 204.07±17.78d 110.36±3.04c 81.79±6.92b 2 Isobutyric acid 1536 79-31-2 ND 10.59±0.52b 20.88±1.06c ND 3 Butyric acid 1596 107-92-6 24.55±2.85a 39.99±1.92d 35.52±2.15c 29.8±1.14b 4 Isovalerate 1638 503-74-2 46.58±5.36a 146.07±0.42c 179.46±23.11d 95.07±22.69b 5 Valeric acid 1704 109-52-4 10.61±0.68a 13.69±0.95b 9.63±0.44a 10.84±0.46a 6 4-Methylvaleric acid 1768 646-07-1 5.06±0.06c 4.5±0b 4.34±0.34b ND 7 Hexanoic acid 1808 142-62-1 144.19±3.45b 168.35±1.45c 131.23±4.87a 172.5±5.64c 8 bitter 2020 124-07-2 69.16±8.6c 36.29±0.01a 25.63±3.3a 49.19±8.57b 9 Nonanoic acid 2128 112-05-0 26.33±1.46ab 24.24±3.07a 29.19±3.34bc 32.34±0.17c 10 Decanoic acid 2232 334-48-5 52.35±9.39b 51.36±8.34b 56.18±10.87b ND

[0107] Table 2-6

[0108] Name of phenolic compound Component RI CAS Number 0h 12h 24h 36h 1 3-Methyl-4-isopropylphenol 1393 485076 101.55±0.42d 95.67±1.3b 87.82±0.4a 99.18±1.41c 2 4-Ethylguaiacol 1494 2785-89-9 53.26±5.34a 61.93±0.68b 59.65±5.79ab 78.94±3.37c 3 2,6-di-tert-butyl-4-methylphenol 1882 128-37-0 2.29±0.08b 1.73±0.12a 2.19±0.19b 2.57±0.16c 5 Thymol Thymol 2167 89-83-8 16.49±0.91b 24.57±0.03c 7.54±0.46a 15.91±3.01b 6 3-tert-Butylphenol 2241 585-34-2 50.5±1.08c 11.56±0.42b 7.76±0.52a 11.82±0.22b 7 2,4-di-tert-butylphenol 2261 96-76-4 68.07±3.77b 57.9±1.23a 65.19±0.48b 91±1.45c

[0109] Table 2-7

[0110] Names of nitrogen-containing compounds Component RI CAS Number 0h 12h 24h 36h 1 2-Methylpyrazine 1247 109-08-0 10.07±0.05c 10.65±1.37c 7.84±0.7b 6.21±0.05a 2 2,5-Dimethylpyrazine 1303 123-32-0 61.27±0.23c ND 39.49±1.28b 40.11±0.11b 3 2,6-Dimethylpyrazine 1309 108-50-9 8.1±1b 8.68±0.21b 6.68±0.32a 5.93±1.03a 4 2-Ethyl-6-methylpyrazine 1365 13925-03-6 6.46±0.61b 5.77±0.19ab 6.08±0.59ab 5.44±0.04a 5 2,6-Diethylpyrazine 1424 13067-27-1 52.9±0.65d 42.86±0.27c 22.95±1.93b 18.69±0.49a 6 2-Butylpyridine 1555 5058-19-5 ND ND ND 7.79±0.24b

[0111] Table 2-8

[0112] Furan compound name Component RI CAS Number 0h 12h 24h 36h 1 2-Methylfuran 845 534-22-5 3.51±0.48a 5.14±0.22b 7.48±1.57c 4.81±0.05ab 2 3-Methylfuran 872 930-27-8 6.7±0.89a 9.99±0.26b 10.97±1.17b 10.74±0.11b 3 2-n-Butylfuran 1108 4466-24-4 3.23±0.14a 4.67±0.38c 3.72±0.24b 3.8±0.09b 4 2-n-pentylfuran 1208 3777-69-3 34.57±1.77a 36.1±0.12a 34.06±0.69a 40.08±1.35b 5 5-Methyl-2-acetylfuran 1465 1193-79-9 4.96±0.33a 7.47±0.47b 7.81±0.25b 4.69±0.07a 6 2,5-diformylfuran 1540 823-82-5 60.84±1.08a 82.97±0.9c 75.44±0.53b 75.19±4.31b

[0113] Table 2-9

[0114] Name of alkane compound Component RI CAS Number 0h 12h 24h 36h 1 2-Carene 1037 554-61-0 2.77±0a 3.95±0.1c 3.55±0.15b 3.8±0c 2 Undecane 1073 1120-21-4 12.72±0.02a 15.04±1.82a 12.77±3.26a 15.18±3.82a 3 Terpinene 1124 99-86-5 31.67±1.44a 44.04±0.22bc 40.66±4.19b 47.1±0.82c 4 Dodecane 1180 112-40-3 46.99±0.28a 47.04±6.86a 61.27±8.43b 86.64±1.48c 5 Tridecane 1284 629-50-5 44.52±3.7b 57.59±14.24b 49.81±0.07b 15.68±2.19a 6 Pentadecane 1485 629-62-9 406.58±52.7a 347.35±31.02a 518.28±105.98ab 642.19±124.78b 7 Heptadecane 1688 629-78-7 6970.16±676.56a 6563.78±293.18a 10196.88±1475.35b 9877.05±1613.16b 8 3-Heptadecene 1700 1000141-67-3 396.97±45.95a 348.5±22.93a 579.39±122.33b 636.6±133.6b 9 6,9-heptadecadiene 1737 81265-03-4 47.03±7.15a 35.98±3.49a 83.71±3.71b 81.2±20.23b

[0115] Note: ND means not detected, different letters in the same row indicate significant differences (p<0.05).

[0116] Example 7: This example provides a method for determining aroma precursor cleavage enzymes during the co-cultivation process of Spirulina.

[0117] The specific technical solution steps are as follows:

[0118] First, sample collection and pretreatment were performed: Spirulina platensis and Saccharomyces cerevisiae were separated using a dialysis tubing cellulose membrane (pore size 20 kDa, dimensions 76 mm × 49 mm; purchased from Sigma-Aldrich, USA). Yeast was inoculated into the dialysis tubing cellulose membrane during logarithmic phase and then cultured in Spirulina medium for 18 hours. Microalgae cells were then collected for genome sequencing and transcriptome analysis.

[0119] For genomic DNA related operations:

[0120] Extraction and purity test: Total DNA was extracted from Spirulina platensis cells using QIAamp DNA microkit (Qiagen, California, USA). DNA integrity and potential RNA / protein contamination were analyzed by agarose gel electrophoresis. DNA was analyzed by NanoDrop One spectrophotometer (ThermoScientific, Massachusetts, USA) based on OD 260 / OD 280 The DNA purity was determined by the ratio of 4:1 to 1:1; the DNA concentration was quantified using a Qubit 3.0 fluorometer (ThermoScientific, Massachusetts, USA).

[0121] Library Construction and Sequencing: 2.5 μg of qualified DNA was used for library construction, a process that involved a series of steps, including magnetic bead purification, fragmentation and end-repair, barcode tagging, sample pooling, and sequencing adapter ligation. The constructed library was loaded onto an R9.4 sequencing chip and sequenced on a PromethION sequencer (Oxford Nanopore Technologies, Oxford, UK) for 48–72 hours. Raw data were quality-controlled to remove low-quality and short reads. High-accuracy Illumina data (Q30 >85%) were assembled using Unicycler 0.4.9 software to construct a high-quality genome framework. This was then linked to the Nanopore data to form a complete genome map. The assembled genome was then corrected using Pilon 1.23 to obtain a more accurate genome.

[0122] Gene Prediction and Functional Annotation: Protein-coding genes were predicted using Prodigal 2.6.3; tRNA genes were predicted using tRNAscan-SE 2.0; and rRNA gene analysis was performed using Barrnap 0.7. For genomic functional annotation, predicted gene sequences were aligned to the GO and KEGG functional databases using BLAST. The circlizeR package was used to visualize genomic components and their relationships.

[0123] For transcriptome RNA related operations:

[0124] Extraction and quality assessment: Total RNA was extracted from Spirulina platensis cells using QIAamp RNA minikit (Qiagen, California, USA). 260 / OD 280The RNA purity was determined by the ratio of 40:1, and the RNA integrity was determined by Agilent 2100 bioanalyzer (Agilent Technologies, California, USA).

[0125] Library Construction and Sequencing: After quality assessment of the total RNA samples, cDNA libraries were constructed using the TruSeq RNA Sample Prep Kit (Illumina, California, USA). Sequencing was then performed using the Sequencing by Synthesis (SBS) method on a second-generation high-throughput sequencing platform. Prior to assembly, stringent read length filtering was performed to ensure data quality.

[0126] Transcriptome Analysis: The resulting high-quality cleanreads were used for de novo transcriptome assembly. CPC2 was applied to identify unigenes with coding potential, and BLAST was used to align the Nr, Nt, Swissprot, KEGG, KOG, Pfam, and GO databases to obtain annotation information for the new genes. Read counts were normalized to FPKM (fragments per kilobase of transcript per million mapped fragments) values for further analysis. Through in-depth mining and analysis of genomic and transcriptomic data, genes related to aroma precursor cleavage enzymes were identified, enabling the determination of aroma precursor cleavage enzymes during Spirulina co-cultivation. This provides key data support for subsequent in-depth research on the mechanism of Spirulina aroma formation and the improvement of product flavor quality.

[0127] Genome sequencing and transcriptome analysis were performed to reveal the transcriptional responses of Spirulina platensis to yeast co-culture and acetate addition. Figure 6 As shown, the Spirulina platensis FACHB-902 strain used in this study has a single circular chromosome of 6.44 Mb, a total sequence length of 6,437,174 bp, an average G+C content of 44.9%, and no plasmid DNA sequences were observed. Figure 7 a shows the enriched KEGG pathways and gene counts related to pigment synthesis and degradation in the transcriptome. The study found that chlorophyll biosynthesis is closely related to "porphyrin and chlorophyll metabolism", "photosynthesis", "carbon metabolism", "nitrogen metabolism", and "magnesium metabolism". Phycobiliprotein biosynthesis is related to "nitrogen metabolism", "amino acid metabolism", and "photosynthesis". Carotenoid biosynthesis is closely related to "carotenoid biosynthesis", "carbon metabolism", "ABC transporters", and "photosynthesis". Lipid biosynthesis is closely linked to "fatty acid biosynthesis", "fatty acid metabolism", and "glycerophospholipid metabolism". Aroma production is related to "fatty acid degradation" and "carotenoid biosynthesis".

[0128] In this study, nine genes involved in acetate-derived carotenoid and fatty acid biosynthesis were enriched in the Spirulina platensis transcriptome ( Figure 7 h), and their fragments per kilobase of transcript per million mapped fragments (FPKM) values were significantly increased by yeast co-cultivation and acetate addition ( Figure 7 b and 7f).

[0129] In the present invention, five chlorophyll biosynthesis genes ( Figure 7 h), and their FPKM values were significantly increased by yeast co-culture and acetate addition ( Figure 7 e). Therefore, acetate produced by yeast may promote the significant increase in the content of chlorophyll a and chlorophyll b in co-cultured Spirulina by enhancing the biosynthesis of phytol side chains in chlorophyll.

[0130] In cyanobacteria, chlorophyll biosynthesis competes with phycobiliprotein biosynthesis for the common substrate protoporphyrin IX. Figure 7 (d) Two phycobiliprotein biosynthesis genes enriched in the Spirulina platensis transcriptome, according to their FPKM values, were transcriptionally repressed upon yeast co-cultivation and acetate addition, consistent with the slightly decreased phycobiliprotein levels in Spirulina platensis following yeast co-cultivation and acetate addition. This may be due to acetate-induced increases in chlorophyll biosynthesis, which limits the availability of protoporphyrin IX.

[0131] In the present invention, eight genes involved in the enzymatic degradation of carotenoids and polyunsaturated fatty acids were enriched in the Spirulina platensis transcriptome ( Figure 7 h), and their FPKM values were significantly increased by yeast co-culture and acetate addition ( Figure 7 c and 7g). Therefore, the reactive oxygen species generated by acetate produced by yeast may induce the enzymatic degradation of carotenoids and polyunsaturated fatty acids to produce norisoprenoids and C5-C9 aldehydes, thereby giving Spirulina the pleasant microalgae aroma such as "floral" and "fruity".

[0132] The above embodiments illustrate and describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention and are not intended to limit the scope of the present invention in any way. Various changes and modifications may be made to the present invention without departing from the scope of the present invention, and all such changes and modifications fall within the scope of the claims.

Claims

1. A pre-harvest physiological management method for improving the color and odor quality of Spirulina, characterized in that: A method combining in situ light co-culture and mud dark light co-culture was used; The in-situ illumination co-cultivation method is to add glucose and acetic acid to the culture medium and inoculate cerevisiae when the spirulina grows to the late logarithmic growth stage or the stable stage; The specific steps include: (1) When Spirulina is grown in Zarrouk's minimal medium to the late logarithmic growth phase or the stationary phase, 3-7 g / L glucose and 0.5-1.5 g / L acetic acid are added to the medium, and Saccharomyces cerevisiae is inoculated at 0.1%-10% of the Spirulina biomass. The culture is then carried out under light and aeration conditions for 12-36 h. (2) The co-culture solution is filtered through 400 mesh to remove 80%-90% of the culture medium to obtain a mixed slurry of Spirulina and Saccharomyces cerevisiae. The co-culture is continued for 12-36 hours in a dark environment.

2. The pre-harvest physiological management method for improving the color and odor quality of Spirulina according to claim 1, characterized in that: In the step (1), the amount of glucose added is 5 g / L, the amount of acetic acid added is 1 g / L, and the amount of brewer's yeast inoculated is 1% of the Spirulina biomass.

3. The pre-harvest physiological management method for improving the color and odor quality of Spirulina according to claim 1, characterized in that: The total culture time in step (1) is 24 h.

4. The pre-harvest physiological management method for improving the color and odor quality of Spirulina according to claim 1, characterized in that: The total incubation time in step (2) is 24 h.

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