Method for reducing fishy smell in chlorella

Abstract

The invention relates to the technical field of chlorella deodorization, in particular to a method for reducing fishy smell in chlorella. By regulating the concentration of inorganic selenium in the culture medium, growth of chlorella is not inhibited, selenium-enriched biological enhancement is realized, the content of typical marker hexanal with fishy smell and grass taste of chlorella is remarkably reduced, meanwhile, the content of methyl hexanoate with sweet fragrance and fruit fragrance is increased, and the content of chlorella is increased. The fishy smell reduction and aroma enhancement of the chlorella are fundamentally realized. According to the method, the flavor characteristic of the selenium-enriched chlorella is effectively improved, the application potential of the selenium-enriched chlorella in food and related fields is greatly promoted, and the selenium-enriched chlorella is easier to accept by consumers.

Description

Technical Field

[0001] The invention relates to the technical field of removing fishy smell from chlorella, and in particular to a method for reducing the fishy smell in chlorella. Background Art

[0002] Microalgae have attracted extensive attention due to their rapid reproduction, the fact that they do not require arable land for cultivation, are not restricted by time and space, and are rich in a variety of high-value bioactive substances, such as proteins, polysaccharides, lipids, amino acids, and minerals. So far, many countries have approved a variety of microalgae for human consumption. Spirulina, Haematococcus pluvialis, and Chlorella are the three major types of edible microalgae. Chlorella is rich in protein, carotenoids, polysaccharides, and vitamins, and has antioxidant, anti-inflammatory, and immune-enhancing activities. Its protein content exceeds 50% of its dry weight, and it is considered to be a promising and sustainable new plant-based protein source. Volatile flavor compounds have an important influence on food flavor, and they play a key role in food evaluation. Recent studies have reported that the types of volatile compounds in edible microalgae are species-specific, mainly composed of aldehydes, sulfides, terpenes, ketones, and alcohols.

[0003] However, the fishy substances that are unique to microalgae also exist in Chlorella pyrenoidosa, and these fishy substances greatly limit its consumer acceptability in food. In order to reduce the odor interference of microalgae in food industry applications, it is crucial to reduce unpleasant tastes. For example, Spirulina is fermented with lactic acid bacteria and Bacillus to improve its sensory acceptance. Chlorella is genetically modified using mutagenesis technology to obtain a chlorophyll-deficient mutant with better flavor. Despite this, there is still the problem of incomplete removal of odor substances, namely fishy smell. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for reducing the fishy smell in Chlorella, which can effectively reduce the content of hexanal, a typical compound with fishy and grassy smells in Chlorella, and increase the content of methyl hexanoate with sweet and fruity aroma, thereby achieving the purpose of effectively reducing the fishy smell.

[0005] A method for reducing the fishy smell in Chlorella comprises the following steps:

[0006] The inoculum of Chlorella is inoculated into the mBG-11 medium containing inorganic selenium, and the culture is carried out under light for 10 to 16 days. The inorganic selenium is used to reduce the content of acetaldehyde in Chlorella and increase the content of methyl hexanoate. The precipitate is centrifuged, washed, and dried to remove the fishy smell of Chlorella.

[0007] In the culture medium, the concentration of inorganic selenium is 0.25 mg / L to 4 mg / L, calculated as selenium element.

[0008] The present invention reduces the content of hexanal, a typical marker of fishy and grassy smells of Chlorella, by controlling the concentration of inorganic selenium. At the same time, the content of methyl hexanoate with sweet and fruity aroma is increased, thereby fundamentally achieving the purpose of reducing the fishy smell and increasing the aroma of Chlorella.

[0009] In another preferred embodiment, the inoculation amount of Chlorella is based on the optical density value OD of Chlorella in mBG-11 medium. 750 Determine that the OD 750 It is 0.45~0.75.

[0010] In another preferred embodiment, the inorganic selenium is sodium selenite.

[0011] In another preferred embodiment, the illumination density of the illumination culture is 100 μmol photon / m 2 / s~150μmol photon / m 2 / s.

[0012] In another preferred embodiment, the light culture process further comprises introducing compressed air, wherein the compressed air contains CO 2 It accounts for 1% of the air; the compressed air is controlled by two partial pressure flow meters, and the ventilation volume of the compressed air is 20L / h.

[0013] In another preferred embodiment, the method further comprises activating the Chlorella before inoculating the Chlorella into the mBG-11 medium containing inorganic selenium;

[0014] The specific process of chlorella activation is as follows:

[0015] Chlorella stored in a triangular flask was inoculated into mBG-11 medium and cultured at 60 μmol photon / m 2 / s~80μmol photon / m 2 The algae are cultured under a light density of 1.500 nm / s. When the algae are cultured to the logarithmic phase, i.e., 5 to 7 days, the algae cells are collected by centrifugation and washed to complete the activation of Chlorella.

[0016] In another preferred embodiment, the centrifugal rotation speed is 4000 rpm to 6000 rpm and the time is 5 min to 15 min.

[0017] In another preferred embodiment, the Chlorella is Chlorella pyrenoidosa.

[0018] In another preferred embodiment, the drying temperature is 50°C to 60°C.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention significantly reduces the content of hexanal, a typical marker of fishy and grassy smells in the algae, by regulating the concentration of inorganic selenium in the culture medium without inhibiting the growth of Chlorella. At the same time, the method of the present invention can also increase the content of methyl hexanoate with sweet and fruity aroma, fundamentally reducing the fishy smell and increasing the aroma of Chlorella. The method of the present invention effectively improves the flavor characteristics of selenium-enriched Chlorella, greatly promotes its application potential in food and related fields, and makes it more acceptable to consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention is a flow chart of the method for reducing fishy smell in Chlorella.

[0022] Figure 2 The PCA score graph and radar graph of the electronic nose response data of Chlorella under different selenium concentration treatments; A is the PCA score graph and B is the radar graph.

[0023] Figure 3 The differences between the two-dimensional topographic map and the comparative topographic map of HS-GC-IMS analysis of Chlorella volatiles under different selenium concentration treatments; among them, A is the two-dimensional topographic map of HS-GC-IMS analysis, and B is the difference between the comparative topographic maps of HS-GC-IMS analysis.

[0024] Figure 4 Fingerprint analysis of Chlorella volatiles under different selenium concentration treatments.

[0025] Figure 5 This is a graph showing the quantity of volatile compounds in Chlorella under different selenium concentrations based on HS-SPME-GC-MS analysis.

[0026] Figure 6 The total concentration of volatile compounds in Chlorella under different selenium concentrations was analyzed based on HS-SPME-GC-MS.

[0027] Figure 7 Cluster analysis heat map of volatile compounds of Chlorella under different selenium concentration treatments based on HS-SPME-GC-MS analysis.

[0028] Figure 8 OPLS-DA score diagram of the main flavor components.

[0029] Fig. 9 OPLS-DA alternative test model diagram for the main flavor components.

[0030] Fig.10 VIP map of the main flavor components.

[0031] Fig.11 Bioplot of the main flavor components. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the embodiments of the present invention, the materials and equipment used can be purchased on the market unless otherwise specified.

[0034] The rapid oxidation of polyunsaturated fatty acids is the main factor causing off-flavor in algae products. Selenium is one of the trace nutrients necessary for life activities, and it has antioxidant function at a certain dose. Studies have found that adding selenium to plants can improve their volatile flavor. For example, selenium-enriched treatment effectively promoted the synthesis of volatile aroma compounds in broccoli florets. Foliar spraying of glucosamine selenium increased the levels of volatile compounds such as floral and honey aroma in summer and autumn tea soups. However, the research on selenium biofortification in algae tends to change nutritional quality, such as total selenium content and organic selenium forms, but there are no reports on the effect of selenium on the characteristic flavor substances of algae. In addition, there are few reports on the analysis of volatile compounds in protein-core Chlorella, and the same microalgae synthesizes different volatile compounds under different culture conditions. Therefore, it is necessary to study the effect of the addition of selenium and its different addition doses on the volatile compounds of protein-core Chlorella.

[0035] like Figure 1 As shown, the present invention obtains the fingerprint characteristics of protein-core Chlorella and its volatile compounds with different selenium contents by applying different inorganic selenium concentrations, and the total selenium content of Chlorella is determined by atomic fluorescence spectrometry. The volatile compounds of Chlorella are analyzed by electronic nose, headspace-gas chromatography ion mobility spectrometry and headspace-solid phase microextraction-gas chromatography-mass spectrometry. In addition, the multivariate statistical analysis method is used to identify the differential volatile components and key aroma substances. The profile of volatile compounds of selenium-rich protein-core Chlorella is reported for the first time, which provides a new idea for improving the flavor of microalgae.

[0036] A method for reducing the fishy smell in Chlorella is described in detail below.

[0037] Chlorella pyrenoidosa (code FACHB-5) in the following examples was purchased from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences.

[0038] Each liter of mBG-11 medium contains the following ingredients:

[0039] 40g / L K2 HPO 4 , 75 g / L MgSO 4 7H 2 O, 36 g / L CaCl 2 ·2H 2 O, 20 g / L Na 2 CO 3 , 2.288 g / L H 3 BO 3 , 1.448 g / L MnCl 2 ·4H 2 O, 0.1726 g / L ZnSO 4 7H 2 O, 0.312 g / L NaMoO 4 ·2H 2 O, 0.0632 g / L CuSO 4 ·5H 2 O, 0.03932 g / L Co(NO 3 ) 2 6H 2 O, 3.15 g / L FeCl 3 6H 2 O, 4.36 g / L Na 2 EDTA·2H 2 O, 6g / L C 6 H 8 O 7 , the rest is solvent, and the solvent is deionized water.

[0040] 1. Sample processing

[0041] Preparation of Chlorella:

[0042] The sterile Chlorella vulgaris stored in a conical flask was inoculated into a glass column photobioreactor containing 1.2 mL of BG-11 culture medium, and compressed air was introduced at a flow rate of 20 L / h through two partial pressure flow meters. 2 The glass column photobioreactor has an inner diameter of 6 cm and a length of 60 cm. The light density is 60 μmol photon / m 2 / s.

[0043] Chlorella was inoculated in mBG-11 medium containing different concentrations of sodium selenite, wherein the inoculation amount of Chlorella was based on the optical density value OD of Chlorella in mBG-11 medium. 750 OK, OD750 Calculated by selenium, different concentrations of sodium selenite were prepared, and the concentrations of sodium selenite were 0.25 mg / L, 1 mg / L, and 4 mg / L, respectively. The cells were cultured in the above-mentioned glass column photobioreactor and stirred with compressed air. The CO in the compressed air was 2 1% of air. Three replicates were set for each sodium selenite concentration treatment, and the culture without sodium selenite was used as the control group. 2 / s fluorescence density for 12 days; after the culture, the algae cells were collected by centrifugation and washed three times with sterile deionized water to remove the sodium selenite on the surface of the Chlorella cells to obtain algae mud. The algae mud was dried in a 50℃ oven to constant weight and stored at -20℃.

[0044] 2. Determination of biomass and total selenium content

[0045] Take 5 mL of algae mud and place it on a 0.22 μm nitrocellulose filter membrane and weigh it as W. 1 , filter to obtain the algae-containing filter membrane, place it in an oven at 105°C for 10 hours, and after the filter membrane is cooled in a dryer for 30 minutes, accurately weigh it and record it as W 2 , and calculate the biomass concentration according to the formula, the calculation formula for biomass concentration is: Biomass (g / L) = (W 2 -W 1 )×200.

[0046] The Chlorella vulgaris on the dried cellulose nitrate filter was hung to obtain sample powder. 0.05g of sample powder was taken into a polytetrafluoroethylene-coated digestion tube. Three parallels were prepared for each sample. 7mL of nitric acid was added to each tube. After the tube cover was tightly closed, it was placed symmetrically in a microwave digestion instrument to digest until the mixed solution was colorless and transparent. After the digestion was completed, the digestion tube with the sample was moved into a fume hood, the lid was opened, and the temperature-controlled electric heating acid removal instrument was placed. The acid was removed at 180°C until about 1mL of the digestion solution remained, and then 5mL of 50% HCl was added to continue to remove the acid to 1mL. After the acid removal was completed, the sample in the digestion tube was poured into a screw-mouth centrifuge tube, fixed to 10mL with 10% HCl, and the solution was diluted 1000 times before the total selenium content was detected. The selenium content was determined by hydride generation atomic fluorescence spectrometry, and the standard curve was drawn with a 10μg / mL selenium standard stock solution diluted with ultrapure water.

[0047] 3. Electronic nose analysis

[0048] The sample analysis was performed using a cNose electronic nose system consisting of 28 metal oxide semiconductor sensors, wherein the cNose electronic nose system was purchased from Shanghai Baosheng Industrial Development Co., Ltd.

[0049] Weigh 0.2g of sample powder into a 50mL headspace glass bottle and equilibrate at 25℃ for 2h. Test conditions: cleaning time 120s, injection time 60s, flow rate 1L / min. Repeat 4 times for each sample, and describe the aroma characteristics by the corresponding response value of the sensor. A combination of 18 MOS sensors is used, including S1: short-chain alkanes, smoke, S2: carbon-containing substances, alcohol, smoke, S3: hydrogen, S4: sulfide, S5: nitrogen-containing substances, ammonia, S6: aldehydes and ketones, S7: short-chain alkane combustible gases, S8: liquefied gas, S9: alkanes, alcohols, ketones, S10: hydrogen-containing substances, S11: alkanes, liquefied gas, S12: organic solvents, methane, S13: short-chain alkanes, S14: methane, gas, smoke, S15: ammonia, amines, S16; hydrogen sulfide, S17: hydrogen-containing substances, S18: organic solvents, aromatic hydrocarbons, aliphatic hydrocarbons, halogenated hydrocarbons, ethers, esters, glycol derivatives, acetonitrile, pyridine, and phenol.

[0050] 4. HS-GC-IMS analysis

[0051] The samples were analyzed using HS-GC-IMS equipped with an autosampler, 490 Micro Gas Chromatograph (Agilent Technologies, CA, USA) and a drift chamber.

[0052] Weigh 0.2 g of sample powder into a 20 mL headspace glass injection bottle and incubate at 40°C for 20 min. Then, at 85°C (splitless mode), 500 μL of headspace sample was automatically injected into the injector using a syringe. A capillary column of FS-SE-54-CB-1, 15 m×0.53 mm×1.0 μm was used, the column temperature was 60°C, and the carrier gas was nitrogen (purity>99.9%). The linear pressure program was: 2 mL / min for 2 min, linearly increased to 10 mL / min from 2 to 10 min, and linearly increased to 150 mL / min from 10 to 20 min. The ionized volatiles were transferred to a drift tube (9.8 cm) and operated at a nitrogen flow rate of 150 mL / min and a constant pressure of 45°C (5 kV). The retention index (RI) of volatiles was calculated by automatic mass spectrometry deconvolution identification system using C4-C9 ketone (Sinopharm Chemical Reagent, Beijing, China) as external standard. Volatiles were then identified based on the RI and drift time of the standards in the GC-IMS library.

[0053] 5. HS-SPME-GC-MS analysis

[0054] 1) Extraction of volatile compounds

[0055] The sample volatiles were collected using headspace solid phase microextraction (HS-SPME): 2 cm increased to 50 / 30 μm DVB / Carboxen / PDMS, Supelco, Bellafonte.

[0056] Weigh 0.2g of sample powder into a 20mL headspace vial with a PTFE / Si septum cap, and add 0.5g of NaCl to improve the detection sensitivity of the SPME extraction technique. Subsequently, add 5μL of 0.84mg / mL 2-methyl-3-heptanone as an internal standard mixture of neutral / alkaline compounds and 0.83mg / mL methyl nonanoate as an internal standard mixture of ester compounds, mix well and seal. Pre-equilibrate the sample vial at 40°C for 10min. Then, insert the SPME needle into the vial and expose it to the headspace extraction for 25min. After completion, the sample is immediately injected into the GC-MS and desorbed at 250°C for 3 minutes.

[0057] 2) Gas chromatography-mass spectrometry

[0058] The analysis of volatile compounds was performed on a TRACE1300 gas chromatograph (GC) and an ISQ 3000 mass spectrometer (MS) (Thermo Fisher Scientific, Waltham, MA, USA). An HP-5 chromatographic column (30 m × 0.25 mm × 0.25 μm) was used for separation of volatiles. The temperature program was as follows: column temperature 40 °C, maintained for 3 min; increased to 150 °C at 4 °C / min, maintained for 2 min; increased to 250 °C at 8 °C / min, maintained for 6 min. The carrier gas was high-purity helium (>99.999%) with a flow rate of 1 mL / min. The injection port temperature was 250 °C, and the injection was non-split. The mass selective detector used the positron ionization mode (70 eV), ion source temperature: 230 °C, interface temperature: 250 °C; mass scanning range: 40 amu-450 amu.

[0059] 3) Qualitative and quantitative analysis Qualitative analysis

[0060] The MS spectra of volatile compounds were matched with the data in the NIST 11 library, and the retention index (RI) was calculated using C7-C40 normal alkanes (Sigma-Aldrich Co., Ltd, Shanghai, China) for qualitative analysis. The calculation formula is shown in Equation (1), where compounds with both positive and negative matching degrees less than 800 were excluded from the analysis.

[0061]

[0062] n is the carbon number of the normal alkane before the component to be measured, RT X is the retention time of the component to be analyzed, RT nis the retention time of the n-alkanes preceding the component to be analyzed, RT n+1 is the retention time of the n-alkanes following the component to be analyzed.

[0063] Quantitative analysis: The quantitative analysis was performed based on the content of the internal standard mixture of 2-methyl-3-heptanone and methyl nonanoate. The calculation formula is shown in formula (2).

[0064]

[0065] Among them, C i is the concentration of volatile substances (μg / kg), C S is the internal standard concentration (mg / L), V S is the volume of the internal standard (μL), m o is the sample mass (g), A i is the peak area of ​​volatile compounds, A S is the internal standard peak area.

[0066] 4) Evaluation of key volatile compounds The key volatile substances were analyzed by calculating the odor activity value (OAV), and the calculation formula is shown in formula (3).

[0067]

[0068] Among them, T i It is the odor threshold of volatile substances in water.

[0069] 5) Data analysis

[0070] SPSS26.0 software was used for one-way analysis of variance and Duncan's test for statistical analysis. All results were expressed as mean ± standard deviation (n = 3), and P < 0.05 was considered statistically significant. MetaboAnalyst5.0 was used for principal component analysis (PCA). SIMCA 14.1 was used for orthogonal partial least squares analysis (OPLS-DA). Origin 2021 software was used for drawing.

[0071] 7. Results Analysis

[0072] 1) Effect of selenium concentration on biomass and total selenium content of Chlorella vulgaris

[0073] Different sodium selenite concentrations significantly affected the growth and total selenium content of Chlorella pyrenoidosa. 2 SeO 3With the increase of concentration, the total selenium concentration of protein nucleus Chlorella increased significantly. It is worth noting that under the treatment of 4.00 mg / L, that is, high sodium selenite concentration, although the total selenium content of protein nucleus Chlorella reached a peak of 357.88±7.61μg / g, which was 5.57 and 1.47 times that of the 0.25 mg / L, low selenium concentration and 1.00 mg / L, medium selenium concentration treatment groups, respectively, its growth was significantly inhibited, indicating that exposure to high selenium concentrations will also cause excessive selenium absorption by the algae and cause poisoning. In summary, the algae powder obtained from the 0.25 mg / L, 1 mg / L and 4 mg / L selenium treatment groups were marked as low selenium group, medium selenium group and high selenium group, respectively, for the following aroma analysis.

[0074] Table 1 Total selenium content and biomass of Chlorella pyrenoidosa under different selenium concentration treatment conditions

[0075] Sodium selenite concentration (mg / LSe) Total selenium content (μg / g) Biomass (g / L) 0.00 <![CDATA[0.00±0.00 d ]]> <![CDATA[2.17±0.32 c ]]> 0.25 <![CDATA[64.30±2.06 c ]]> <![CDATA[2.53±2.17 c ]]> 1.00 <![CDATA[243.45±2.12 b ]]> <![CDATA[1.72±2.18 b ]]> 4.00 <![CDATA[357.88±7.61 a ]]> <![CDATA[1.39±0.06 a ]]>

[0076] 2) Electronic nose results

[0077] In the electronic nose, the sensor array is responsible for adsorbing and responding to odor molecules, generating corresponding electrical signals, and the analog-to-digital converter is responsible for processing and converting the electrical signals. Finally, the data is analyzed through machine learning algorithms. Principal component analysis (PCA) is very useful for data exploration due to its unsupervised nature and is widely used to process and interpret high-dimensional data in many fields. PCA was used to explore the sensor response values ​​of Chlorella vulgaris with different selenium contents. Figure 2 As shown in A, there are three samples in the PCA score graph. The first two components PC1 and PC2 account for 81.4% and 9.3% respectively, explaining 90.7% of the variance. The sample space area shows that the distance between the control group and the low selenium group is closer, and the distance between the medium selenium group and the high selenium group is closer, indicating that the flavor components between the two groups are closer. In the electronic nose radar graph of Chlorella with different selenium contents, Figure 2 As shown in B, there are differences in the sensor response values ​​between the samples, indicating that the aroma distribution between them is different. The response values ​​of S1 (short-chain alkanes, smoke), S2 (carbon-containing substances, alcohol, smoke), S5 (nitrogen-containing substances, ammonia), S14 (methane, gas, smoke), S15 (ammonia, amines), S16 (hydrogen sulfide), and S18 (organic solvents, aromatic hydrocarbons, aliphatic hydrocarbons, halogenated hydrocarbons, ethers, esters, diol derivatives, acetonitrile, pyridine, and phenol) in the medium-selenium group and the high-selenium group of Chlorella protonema increased significantly. Among them, the response value of Chlorella protonema in the high-selenium group was significantly higher than that of the other groups, indicating that the high-selenium group produced richer flavor substances. However, the response values ​​of S1 (short-chain alkanes, smoke), S5 (nitrogen-containing substances, ammonia), and S15 (ammonia, amines) in Chlorella protonema in the low-selenium group were lower than those in the control group. These data show that the addition of selenium and the different addition amounts have a significant effect on the volatile flavor substances of Chlorella protonema.

[0078] 3) Identification of volatile compounds

[0079] HS-GC-IMS was used to determine the volatile compounds of four types of selenium-containing Chlorella pyrenoidosa, and the ion mobility spectra were obtained as shown in Figure 3 A. The ordinate represents the retention time (RT) of GC, the abscissa represents the drift time of IMS, the red vertical line at the ion migration time of 1.0 on the abscissa is the reactive ion peak (RIP), and the points on the right side of the RIP peak represent the detected volatile compounds. The redder the point, the stronger the signal intensity. Although most of the signals appear at drift time 1.0-2.0 (RIP relative), the HS-GC-IMS spectrum shows that there are differences in the volatile compounds of the four test samples, such as Figure 3 As shown in A. Taking the control group as the reference, the differences of the other three components can be clearly distinguished after deduction, such as Figure 3 As shown in Figure B, the colored dots show the signal intensity of each compound detected by IMS, and the white, blue, and red colors after background subtraction represent the same concentration of the compound, which is lower or higher than the reference. Compared with the control group, a large number of red and blue signals were observed in the low, medium, and high selenium groups, indicating that the volatile substances of the four types of selenium content in Chlorella vulgaris were quite different. A total of 45 signals were detected in the four samples, of which 14 signals could not be identified due to the lack of relevant IMS information in the GC-IMS library. In particular, 7 volatile compounds appeared in the form of monomers and dimers, which is because monomer ions and neutral molecules may form auxiliary substances in the drift zone, resulting in multiple signals (monomers, dimers, trimers, and polymers) for a single compound. Therefore, HS-GC-IMS identified a total of 24 volatile compounds in the four samples, including 2 terpenes, 8 aldehydes, 7 alcohols, 4 esters, 2 ketones, and 1 other compound. The detailed information of the volatile compounds is shown in Table 2.

[0080] Table 2 Volatile compounds of four samples based on HS-GC-IMS

[0081]

[0082]

[0083]

[0084]

[0085] 4) HS-GC-IMS fingerprint comparison

[0086] The Gallery Plot plugin was used to generate volatile fingerprints of four types of selenium-containing Chlorella to better understand the differences between samples with different selenium contents. Figure 4 As shown, each row represents the signal peaks of all volatile compounds detected in a sample, and each column represents the signal peaks of a compound in different samples. The brightness of the signal peak color indicates the level of the compound, and the brighter the color, the higher the content. M and D in the brackets after the substance name represent the monomer and dimer of the substance, respectively. The high-selenium group Chlorella vulgaris has the most volatile compounds, with a total of 28 compounds detected and 17 identified. Twenty volatile compounds were detected in the medium-selenium group samples, 14 compounds were identified, and 17 volatile compounds were detected in the low-selenium group samples, 13 compounds were identified. The control group samples had the least volatiles among the four tested samples, with a total of 17 compounds detected, and five compounds could not be identified due to the lack of relevant IMS information in the GC-IMS library. Figure 4 Figure a shows 9 compounds observed in all 4 samples, mainly 5 aldehydes, 2 ketones, and 2 alcohols, including butyraldehyde (pungent, cocoa, musty, bready), hexanal (green, grassy, ​​leafy, fruity), isopentanal (ethereal, aldehydic, chocolate, peach), (Z)-4-heptenal (oily, green, milky, creamy), (E)-2-pentenal (green, fatty, oily, fruity), 2,3-butanedione (sweet, creamy, buttery, pungent), 2-butanone (acetone, ethereal, fruity, camphoreous), (E)-2-hexen-1-ol (fresh, green, fruity, herbal), and (Z)-2-penten-1-ol (green, phenolic, nasturtium, ethereal). Figure 4 In b, three compounds unique to the control group and the low-selenium group were identified, namely 3-methyl-2-butyraldehyde, benzaldehyde, and (Z)-2-hexen-1-ol, and two compounds unique to the medium-selenium group and the high-selenium group, namely isopropyl butyrate and limonene. The control group and the low-selenium group showed similar signal peak distributions in the fingerprint, which corresponded to the electronic nose results, confirming that the two samples had similar volatile components, and the same was true for the medium-selenium group and the high-selenium group. Figure 4As shown in Figure c, some compounds have higher peak volumes in Chlorella pyrenoidosa with specific selenium contents. The peak volume of heptanal was higher in the control group, the peak volumes of isovaleraldehyde, isopentenal, and isopentanol were higher in the low-selenium group, the peak volumes of ethyl acetate, 1,4-cineole, and 1-octen-3-ol were higher in the medium-selenium group, and the peak volumes of 1-penten-3-ol, butanol, ethyl 3-hydroxybutyrate, isopentanol, 1-octanol, and 3-ethylpyridine were higher in the high-selenium group, which are their typical volatiles. It is worth noting that 14 separated volatile compounds were not identified due to the limitations of the GC-IMS database. Therefore, GC-MS was used to further clarify the aroma characteristics of Chlorella pyrenoidosa with different selenium contents.

[0087] 5) HS-SPME-GC-MS method for identification of volatile organic compounds

[0088] A total of 34 volatile compounds were detected from four kinds of pyrenoidosa powders with different selenium contents by HS-SPME-GC-MS, including 5 ketones, 6 aldehydes, 1 alcohol, 14 esters, 4 alkanes, 2 alkenes, 1 furan and 1 ether, as shown in Table 3. The four kinds of pyrenoidosa powders with different selenium contents have similar VOC categories such as Figure 5 As shown, there were 28 VOCs in the control group, including 4 ketones, 6 aldehydes, 1 alcohol, 11 esters, 4 alkanes, 1 olefins, and 1 furan; there were 27 VOCs in the low selenium group, including 4 ketones, 5 aldehydes, 1 alcohol, 11 esters, 4 alkanes, 1 olefins, and 1 furan; there were 29 VOCs in the medium selenium group, including 4 ketones, 4 aldehydes, 1 alcohol, 14 esters, 4 alkanes, 1 olefins, and 1 ether; there were 28 VOCs in the high selenium group, including 5 ketones, 4 aldehydes, 1 alcohol, 11 esters, 4 alkanes, 2 olefins, and 1 ether.

[0089] Table 3 Compositions and concentrations of volatile compounds of four samples identified based on HS-SPME-GC-MS

[0090]

[0091]

[0092]

[0093] Note: nf means not installed, and nd means not detected.

[0094] According to the semi-quantitative analysis results of HS-SPME-GC-MS, ester compounds are the most abundant volatile compounds in the four groups, followed by aldehydes, ketones, etc. Figure 6Among the ester compounds, the highest content was methyl hexanoate (29305.376-36421.417 μg / kg), and its content showed a trend of increasing first and then decreasing with the increase of selenium content, reaching a peak of 36421.417±1140.055 μg / kg in the medium selenium group, as shown in Table 3 and Figure 7 As shown. In addition to methyl hexanoate, there are other ester compounds with high contents, namely methyl heptanoate 3828.561-6472.728 μg / kg, methyl octanoate 3599.786-4280.244 μg / kg and methyl palmitate 794.053-986.050 μg / kg. Among the aldehyde compounds, the highest content is hexanal 3772.403-4157.359 μg / kg. The content of hexanal gradually decreases with the increase of selenium content. In addition, benzaldehyde was only detected in the control group, which corresponds to its signal peak distribution in GC-IMS. (E,E)-2,4-heptadienal was only present in the control group and the low selenium group. Among the ketone compounds, (E,E)-3,5-octadien-2-one had the highest content of 763.267-1432.517 μg / kg, and had a herbal flavor. Its content generally showed an increasing trend with the increase of selenium content. α-ionone and β-ionone had the highest content in the medium selenium group, at 114.469 μg / kg and 156.434 μg / kg respectively, and were common ketone compounds in microalgae.

[0095] 6) Identification of key volatile compounds

[0096] The formation of the odor profile of Chlorella pyrenoidosa is the result of the interaction of multiple substances, which is not only related to the concentration but also to the odor threshold. In general, compounds with OAV ≥ 1 are considered to be the main contributors to the flavor. In order to understand the contribution of each substance to the overall aroma, the OAV values ​​of 20 volatile substances were calculated with reference to the odor thresholds reported in the literature. As shown in Table 4, Chlorella pyrenoidosa with four selenium contents contained a total of 17 key odor compounds, including 6 aldehydes, 4 ketones, 1 alcohol, 4 esters, 1 olefin and 1 furan. Aldehyde and ketone compounds have low odor thresholds and are the main source substances of the volatile flavor of Chlorella pyrenoidosa. Among them, β-ionone was identified as the most critical odor compound due to its extremely low odor threshold, contributing violet and raspberry aroma to Chlorella pyrenoidosa, and had the highest OAV value of 22347.725 in the medium selenium group. At the same time, hexanal contributed the main grassy and fatty flavors to Chlorella pyrenoidosa due to its high content and low threshold, and its odor activity was the weakest in the medium selenium group. In addition, methyl heptanoate showed aromatic, waxy and apple aromas, and was the main source of the pleasant odor of Chlorella pyrenoidosa. It is worth noting that some compounds only exist in specific selenium content components, and whether their OAV value is ≥1 determines whether they can be used as potential differential volatile substances to distinguish Chlorella pyrenoidosa with different selenium contents. For example, benzaldehyde (almond, burnt sugar) was only identified in the control group, (E,E)-2,4-heptadienal (nut, fat) and 2-n-pentylfuran (greenbean, butter) were only present in the control group and the low selenium group, and α-cubane (wood, spice) was only present in the high selenium group. Because these substances had OAV>1 in the identified components, they became the main contributors to the flavor of this component. However, the high selenium group lacked the aroma of red wine compared with other components because methyl decanoate, a substance with a low threshold, was not identified. In addition, although (Z)-2-penten-1-ol could be detected in all selenium-containing Chlorella, due to the different concentrations, it had obvious plastic and rubber smells in the control and high-selenium groups, and only played a modifying role in the low-selenium and medium-selenium groups.

[0097] Table 4 Odor thresholds, odor activity values ​​and odor descriptions of volatile compounds identified in the four samples

[0098]

[0099]

[0100]

[0101] Note: nf means not installed.

[0102] 7) Identification of volatile compounds

[0103] Orthogonal partial least squares discriminant analysis (OPLS-DA) is a supervised logistic regression model that can more accurately reflect the differences between samples and classify samples according to the correlation of differential substances. This model has been widely used in the screening and identification of metabolomics differences. In order to further explore the differences in volatile substances between the four types of selenium-containing pyrenoid algae, OPLS-DA analysis was performed on the 17 key odor compounds identified. Figure 8 As shown, the independent variable fit index (R 2 X) is 0.994, and the dependent variable fitting index (R 2 Y) is 0.996, and the model prediction index (Q 2 ) is 0.976, R 2 and Q 2 If the value is greater than 0.5, the model fitting result is acceptable. After 200 permutation tests, Fig. 9 As shown in the figure, the intercept of the Q2 regression line and the vertical axis is less than zero, indicating that the model does not have overfitting. It is believed that the model results can be used to analyze the differential volatile substances in Chlorella with different selenium contents. The variable importance projection value (VIP) is usually used to evaluate the importance of variables in the OPLS-DA model. It is generally believed that variables with VIP>1 have a higher contribution rate to the distinction of samples. Fig.10 As shown in the figure, based on VIP>1 and P<0.05, six different volatile compounds were screened out, namely benzaldehyde, α-ionone, β-cyclocitral, (E,E)-2,4-heptadienal, hexanal, and methyl hexanoate, which can be used as key aroma compounds to distinguish Chlorella vulgaris with different selenium contents. Fig.11 As shown in the figure, the four samples of Chlorella vulgaris with different selenium contents are distributed in four quadrants, among which the compounds represented by green dots are the key aroma substances with different differences in the samples in the same quadrant, that is, the content of the substance in this sample is higher than that in other samples or it only exists in this sample. Hexanal and benzaldehyde are the key aroma substances with different differences in the control group, α-ionone and methyl hexanoate are the key aroma substances with different differences in the medium selenium group, (E,E)-2,4-heptadienal and β-cyclocitral can be used as the key aroma substances with different differences in the low selenium group and the high selenium group, respectively. Based on the above key aroma substances with different differences, the four samples of Chlorella vulgaris with different selenium contents can be effectively distinguished.

[0104] In the present invention, HS-GC-IMS and HS-SPME-GC-MS were used to detect the volatile substances of 4 kinds of protein-core Chlorella with different selenium contents. These two detection techniques identified 24 and 34 volatile compounds, respectively, mainly including esters, aldehydes, ketones and alcohols. These results are consistent with the categories of volatile substances of the genus Chlorella reported previously. Esters are the most abundant compounds in protein-core Chlorella. They are usually formed by esterification reaction between alcohol and acid, and play an important role in the pleasant flavor of protein-core Chlorella. HS-GC-IMS detected 4 esters, among which are the most abundant volatile substances, which can provide fragrance, which is related to the overall fragrance of ester substances identified by HS-GC-IMS, namely sweet and fruity fragrance. HS-SPME-GC-MS detected a total of 14 ester substances, which is the most diverse category among all detected VOCs. Among them, methyl hexanoate is the compound with the highest ester content, which can provide sweet and fruity fragrance, pineapple. At the same time, methyl heptanoate contributes the main sweet and apple aroma to Chlorella pyrenoidosa because of its low odor threshold. In addition, the total content of ester compounds in Chlorella pyrenoidosa in the selenium-enriched group increased significantly compared with the control group, and the content was the highest in the medium selenium group, which was 1.17 times that of the control group, as shown in Table 3, indicating that the addition of selenium increased the level of volatile flavors such as sweet and fruity aromas in Chlorella pyrenoidosa. Aldehydes may come from the self-oxidation or enzymatic degradation of polyunsaturated fatty acids (PUFA). Chlorella mainly contains shorter-chain PUFAs such as α-linolenic acid. Generally, unsaturated aldehydes are associated with fishy smells, and because of their low odor threshold, they are the main source of unpleasant flavors in Chlorella pyrenoidosa. HS-GC-IMS detected 8 aldehydes, HS-SPME-GC-MS detected 6 aldehydes, and only hexanal and benzaldehyde were detected at the same time. Hexanal was present in all four samples and at a high content, which is consistent with the previous report that hexanal is a biomarker for Chlorella pyrenoidosa. Benzaldehyde is a key volatile in algae, and has been reported in Spirulina platensis, Haematococcus rain, and Chlamydomonas reinhardtii. The total content of aldehydes was significantly reduced in the selenium-enriched group, and the lowest content was in the medium selenium group, which was reduced by about 28% compared with the control group. This shows that an appropriate amount of selenium may reduce the production of aldehydes by inhibiting the oxidation of PUFAs, thereby improving the volatile flavor of Chlorella pyrenoidosa.

[0105] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Obviously, various changes and modifications may be made to the present invention by those skilled in the art without departing from the spirit and scope of the present invention. Thus, the present invention is intended to include such changes and modifications if they fall within the scope of the technical equivalents of the present invention.

Claims

1. A method for reducing the fishy smell in Chlorella, characterized in that: The following steps are involved: The chlorella was inoculated into the mBG-11 culture medium containing inorganic selenium, and the culture was carried out under light for 10 to 16 days. The inorganic selenium was used to reduce the content of acetaldehyde in the chlorella and increase the content of methyl hexanoate. The precipitate was centrifuged, washed, and dried to remove the fishy smell of the chlorella. In the culture medium, the concentration of inorganic selenium is 0.25 mg / L to 4 mg / L, calculated as selenium element.

2. The method for reducing the fishy smell in Chlorella according to claim 1, characterized in that: The inoculation amount of the Chlorella inoculation is based on the optical density value OD of Chlorella in mBG-11 medium. 750 Determine that the OD 750 It is 0.45~0.

75.

3. The method for reducing the fishy smell in Chlorella according to claim 1, characterized in that: The inorganic selenium is sodium selenite.

4. The method for reducing the fishy smell in Chlorella according to claim 1, characterized in that: The illumination density of the light culture is 100 μmol photon / m 2 / s~150μmol photon / m 2 / s.

5. The method for reducing the fishy smell in Chlorella according to claim 1, characterized in that: The method further comprises activating the Chlorella before inoculating the Chlorella into the mBG-11 culture medium containing inorganic selenium; The specific process of chlorella activation is as follows: Chlorella was inoculated into mBG-11 medium and cultured at 60 μmol photon / m 2 / s~80μmol photon / m 2 / s light density to the logarithmic phase, the Chlorella cells were collected by centrifugation and washed to complete the activation of Chlorella.

6. The method for reducing the fishy smell in Chlorella according to claim 5, characterized in that: The centrifugal speed is 4000 rpm to 6000 rpm, and the time is 5 min to 15 min.

7. The method for reducing the fishy smell in Chlorella according to claim 1, characterized in that: The chlorella is pyrenoid chlorella.

8. The method for reducing the fishy smell in Chlorella according to claim 1, characterized in that: The drying temperature is 50°C to 60°C.

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