A Spirulina probiotic fermentation composition for preventing and treating metabolic associated fatty liver disease, preparation method and application

Abstract

The present invention belongs to the technical field of microbial fermentation, and particularly relates to a Spirulina probiotic fermentation composition for preventing and treating metabolic associated fatty liver disease, a preparation method and an application. The present invention uses a combination of Lactobacillus acidophilus and Kluyveromyces marxianus to ferment Spirulina to obtain a fermentation composition for preventing and treating metabolic associated fatty liver disease; the Lactobacillus acidophilus was deposited at the China Center for Type Culture Collection on August 29, 2016, with the deposit address: Wuhan University, Wuhan, China, and the taxonomic name: Lactobacillus acidophilus KDB-03 ( Lactobacillus acidophilus KDB-03), deposit number: CCTCC NO: M 2016429. This fermentation composition can significantly increase the total flavonoid and total phenolic contents of Spirulina, enhance its antioxidant activity; significantly enhance the amino acid level in the liver of mice induced by a high-fat diet, significantly improve the metabolic profile of liver tissue, and relieve metabolic associated fatty liver disease induced by a high-fat diet; and can also effectively remove the fishy smell of Spirulina itself.

Description

Technical Field

[0001] The present invention belongs to the field of application of microbial fermentation technology, and specifically relates to a Spirulina probiotic fermentation composition for preventing and treating metabolic associated fatty liver disease. In particular, it also relates to the preparation method and application of the composition. Background Art

[0002] With the continuous improvement of urbanization and modernization, the lifestyle and diet structure of residents have changed greatly. Whether in Western countries or Asian countries, the prevalence of fatty liver is increasing day by day and has become an increasingly common and important public health problem globally. Among them, metabolic associated fatty liver disease (MAFLD), as the most common liver disease globally, is closely related to metabolic diseases such as type 2 diabetes, hyperlipidemia, and obesity, and has attracted much attention. According to relevant data statistics, the global prevalence of MAFLD has exceeded 25%, and the prevalence in the Asian region is about 27% or so, and the incidence is increasing day by day. The risk of liver cancer when MAFLD is combined with type 2 diabetes, hyperlipidemia, and hypertension will increase exponentially, seriously affecting the quality of life of patients.

[0003] MAFLD has become a serious public health crisis, posing a severe challenge to the medical and health system. So far, no specific drugs for treating MAFLD have been approved, and dietary nutrition regulation has become one of the effective ways to prevent and treat fatty liver.

[0004] Spirulina is rich in high-quality protein, fatty acids such as γ-linolenic acid, carotenoids, vitamins, and various trace elements such as iron, iodine, selenium, and zinc. Research shows that Spirulina has many physiological functions such as lipid-lowering and antioxidant effects, but its strong fishy smell seriously affects its product development. Probiotics are a class of active microorganisms beneficial to the host, and can biotransform and metabolize the original components of Spirulina into other components. However, due to strain specificity, there are certain differences in the beneficial effects of different probiotics fermenting Spirulina. Therefore, it is necessary to screen strains / combinations suitable for Spirulina fermentation.

[0005] In our previous studies, it was found that the content of spirulina protein and amino acids could be significantly increased and its antioxidant activity enhanced by fermentation with one of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus acidophilus, Bacillus coagulans, or in combination with Saccharomyces cerevisiae and Kluyveromyces marxianus (CN 114698714 A). However, the related strains / combinations more suitable for spirulina fermentation are still uncertain, and the effect of preventing and treating metabolic-related fatty liver disease is also unclear. In the prior art, there is a case of using a fermentation mixture containing spirulina for preventing grass carp fatty liver, but its components involve various traditional Chinese medicine ingredients, mainly Artemisia capillaris and Polygonum hydropiper, and the proportion of spirulina is relatively small, making it difficult to evaluate its efficacy in preventing grass carp fatty liver.

[0006] Currently, there are no reports or applications of using a spirulina-based fermented product for preventing and treating metabolic-related fatty liver disease. Based on this, the following invention was made. Summary of the Invention

[0007] The present invention belongs to the field of application of microbial fermentation technology, and specifically relates to a spirulina probiotic fermentation composition, a preparation method, and an application for preventing and treating metabolic-related fatty liver disease.

[0008] The spirulina probiotic fermentation composition provided by the present invention is specifically achieved by fermenting spirulina with a specific probiotic combination. After fermentation, the total flavonoid and total phenol contents of the obtained composition are significantly increased, the antioxidant activity is enhanced, and it can significantly improve the liver tissue metabolic disorder induced by a high-fat diet in animal experiments, effectively prevent and treat metabolic-related fatty liver disease, and at the same time can effectively overcome the fishy smell of spirulina itself.

[0009] The specific technical solution of the present invention is as follows:

[0010] A spirulina probiotic fermentation composition for preventing and treating metabolic-related fatty liver disease, wherein the probiotics are Lactobacillus acidophilus and Kluyveromyces marxianus; the fermentation composition is prepared by fermenting spirulina with a combination of Lactobacillus acidophilus and Kluyveromyces marxianus.

[0011] Among them, Lactobacillus acidophilus was deposited at the China Center for Type Culture Collection on August 29, 2016. The deposit address is: Wuhan University, Wuhan, China. The taxonomic name is: Lactobacillus acidophilus KDB-03, and the deposit number is: CCTCC NO: M 2016429.

[0012] In particular, the scope that the present invention focuses on protecting also includes the spirulina fermented by the combination of Lactobacillus acidophilus and Kluyveromyces marxianus in the preparation of products with the efficacy of preventing and treating metabolic-related fatty liver disease.

[0013] A spirulina probiotic fermentation composition for preventing and treating metabolic associated fatty liver disease, and its preparation method comprises the following steps:

[0014] (1) Preparation of spirulina fermentation substrate: Fresh / dry spirulina is taken to prepare the fermentation substrate, and the substrate formula components include fresh / dry spirulina, carbon source (such as glucose or sucrose and other carbohydrate compounds), nitrogen source (such as peptone or yeast powder, etc.). It enters the fermentation tank through an ultra-high temperature instantaneous steam sterilization pipeline to obtain the spirulina fermentation substrate;

[0015] (2) Preparation of fermentation seed liquid: Lactobacillus and yeast are respectively spread on MRS / YPD solid plates for activation; after the activation is completed, single colonies are respectively picked into MRS / YPD liquid medium for cultivation. After subculture twice, the culture solution is centrifuged to remove the supernatant, and then the thalli are rinsed with phosphate buffer and resuspended to obtain the mother concentrated seed liquids of Lactobacillus and yeast respectively.

[0016] In the preliminary test of this step, the inventors used a variety of test strains, including: Lactobacillus plantarum, Lactobacillus casei, Lactobacillus acidophilus (commercially available), Lactobacillus acidophilus specially preserved in the present invention, Lactobacillus fermentum and other Lactobacillus strains, as well as Saccharomyces cerevisiae, Kluyveromyces marxianus and other yeasts.

[0017] The results show that fermentation can significantly improve the functional components and antioxidant capacity of spirulina. Among them, the single-strain fermentation has the best effect with Lactobacillus acidophilus specially preserved in the present invention, followed by Saccharomyces cerevisiae; in the double-strain fermentation, the combination of Lactobacillus acidophilus specially preserved in the present invention and Kluyveromyces marxianus for fermenting spirulina has the best effect, and the effect is very significant. Among them, the contents of total flavonoids and total phenols are increased by 46.8% and 65.7% respectively, and the DPPH free radical scavenging ability and ferric reducing ability are increased by 75.2% and 62.5% respectively.

[0018] In addition, the contents of amino acids and their derivatives, and organic acids in the double-strain fermentation product obtained after optimizing the fermentation process are greatly increased, and the compositions of lipids, nucleotides and their derivatives also change greatly. Compared with the high-fat control group, the weight gain of mice in the spirulina fermentation product treatment group decreased by about 10.4%, the liver tissue weight decreased by about 18.5%, the morphology and metabolic map of the liver tissue tended to the normal control level, and the body metabolic level was also more active, showing outstanding performance in relieving fatty liver caused by high-fat diet.

[0019] Especially, the double-strain combination screened in the present invention can greatly improve the fishy smell of spirulina through fermentation.

[0020] (3) Inoculate the two concentrated seed liquids in step (2) into the fermentation substrate for composite fermentation culture to obtain the spirulina fermentation product.

[0021] Preferably, in the matrix formulation of step (1), by mass percentage, the content of Spirulina dry matter is 0.5 - 4%, the content of carbon source is 0.5% - 5%, the content of nitrogen source is 0.1 - 5%, the ultra-high temperature instantaneous sterilization temperature is 115°C - 150°C, the sterilization time is 3 - 8 s, and the material pumping speed is 5 - 20 L / min.

[0022] Preferably, in step (2), the activation conditions in the MRS / YPD solid plate are: culturing at 30 - 37°C for 24 - 48 h for activation; the culturing conditions of the colonies in the MRS / YPD liquid medium are: culturing at 30 - 37°C for 24 - 48 h.

[0023] Preferably, in step (3), the inoculation amount of the concentrated liquid is 0.1 - 6.0%, v / v, and the addition ratio is: Lactobacillus acidophilus concentrated seed liquid: Kluyveromyces marxianus concentrated seed liquid = 1 - 3:1 - 3.

[0024] Preferably, in step (3), the compound fermentation culture conditions are: fermentation temperature 28 - 36°C, fermentation time 24 - 48 h.

[0025] The Spirulina probiotic fermentation composition prepared by the above method can be applied to the preparation of products with the efficacy of preventing and treating metabolic-related fatty liver disease.

[0026] The above product can be a medicine; the product can be in solid, liquid, suspension, emulsion or other forms, and the product form is not limited.

[0027] The beneficial effects of the present invention are as follows:

[0028] 1. Using instantaneous steam sterilization can maximize the retention of the original quality of Spirulina. The combined fermentation of the screened Lactobacillus acidophilus and Kluyveromyces marxianus significantly improves the total flavonoid and total phenol contents and antioxidant activity in Spirulina. Compared with non-fermented Spirulina, the total flavonoid and total phenol contents are increased by 46.8% and 65.7% respectively, and the DPPH free radical scavenging ability and ferric reducing ability are increased by 75.2% and 62.5% respectively; compared with other combined fermentations, the total flavonoid content is 14.2 - 31.1% higher, the total phenol content is 22.5 - 66.2% higher, the DPPH free radical scavenging ability is 10.1 - 47.3% higher, and the FRAP content is increased by 1.0 - 20.8%;

[0029] 2. The combined fermentation of Lactobacillus acidophilus and Kluyveromyces marxianus in the present invention improves the original nutritional components of Spirulina. From the results of non-target metabolome LC-MS analysis, the contents of amino acids and their derivatives, and organic acids increase significantly after fermentation. The compositions of fatty acids, nucleotides and their derivatives also change greatly. Among them, the contents of amino acids and their derivatives and organic acids increase by 20.42% and 1.69% respectively, while long-chain polyunsaturated fatty acids, nucleotides and their derivatives decrease by 14.36% and 0.71% respectively.

[0030] 3. After taking the Spirulina fermented by the combined fermentation of Lactobacillus acidophilus and Kluyveromyces marxianus preserved in the present invention, various body indexes of fatty liver mice induced by high-fat diet can be significantly improved. Compared with the high-fat control group, the weight gain of mice in the Spirulina fermentation product treatment group decreases by about 10.4%, the liver tissue weight decreases by about 18.5%, and metabolites such as amino acids, bile acids, fatty acids and organic acids in the liver tissue morphology and liver tissue metabolites tend to be at the normal control level. The body metabolism level is also more active, showing outstanding performance in relieving fatty liver caused by high-fat diet and having good application effects in metabolic-related fatty liver diseases.

[0031] 4. The combined fermentation of Lactobacillus acidophilus and Kluyveromyces marxianus screened in the present invention improves the original flavor of Spirulina. From the detection results of gas-phase ion chromatography GC-IMS, the flavor substance map changes greatly before and after fermentation. The signal intensity of the main volatile flavor substances decreases significantly after fermentation. Among them, the proportion of substances with fishy and pungent smells such as aldehydes decreases significantly, effectively removing the fishy smell of Spirulina itself. The proportions of substances such as ketones, alcohols and furans increase significantly, endowing Spirulina with a unique fermentation flavor. From the sensory evaluation, the combined fermentation of the two achieves technical effects that cannot be achieved by the fermentation of other strains. Description of the Drawings

[0032] Figure 1 It is a diagram showing the influence of single factors on the amount of lactic acid bacteria and FRAP value in the double-bacteria fermentation of Spirulina in Example 2 of the present invention;

[0033] Figure 2 It is a response surface optimization diagram of the interaction of various factors on FRAP in Example 2 of the present invention;

[0034] Figure 3 It is the non-volatile compound metabolome analysis of unfermented Spirulina and fermented Spirulina in Example 3 of the present invention. Among them, Figure A is the heat map of non-volatile compounds, and Figure B is the diagram of the proportion of different compounds before and after fermentation;

[0035] Figure 4This is a graph showing the changes in indicators such as body weight, body composition, and metabolism of mice during intragastric administration of spirulina in Example 4 of the present invention. Among them, A: Body weight curve of mice aged 17 - 29 weeks; B: Weight gain curve of mice aged 17 - 29 weeks; C: State of mice before dissection; D: Body composition analysis of mice in the 13th week of the experiment; E: Oxygen consumption, carbon dioxide production, and respiratory quotient curves of mice in the 13th week of the experiment.

[0036] Figure 5 This is a graph showing the HE staining of liver tissues of mice in different treatment groups in Example 4 of the present invention.

[0037] Figure 6 This is a heat map of differential metabolites in the liver of mice in Example 4 of the present invention.

[0038] Figure 7 This is a box plot showing the contents of glycine (A), lysine (B), leucine (C), serine (D), 2 - methyl - 4 - pentenoic acid (E), myristic acid (F), glycylleucine (G), and p - hydroxybenzylacetic acid (H) of differential metabolites in the liver of mice in Example 4 of the present invention.

[0039] Figure 8 This is a radar chart of the sensory evaluation of spirulina fermented by different strains / combinations in Example 5 of the present invention.

[0040] Figure 9 This is a Gallery Plot fingerprint spectrum of flavor substances of spirulina fermented by Lactobacillus acidophilus and Kluyveromyces marxianus screened and preserved in Example 5 of the present invention. Detailed implementation manners

[0041] In order to enable those skilled in the art to better understand the present invention, the present invention will be further elaborated below in combination with specific implementation manners. The following examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0042] Test bacteria: For the convenience of representation in the figures, each strain is abbreviated as follows: Lactobacillus plantarum LP, Lactobacillus casei BC, Lactobacillus acidophilus LA1 (isolated strain from common commercially available products), Lactobacillus acidophilus LA (specially preserved strain of the present invention), Lactobacillus fermentum LC, Saccharomyces cerevisiae SC, Kluyveromyces marxianus KM, a total of 7 kinds.

[0043] Example 1 (Screening of fermentation strains / combinations)

[0044] A spirulina probiotic fermentation combination for preventing and treating metabolic - associated fatty liver disease is prepared as follows:

[0045] (1) Preparation of spirulina fermentation substrate: Take fresh / dry spirulina to prepare the fermentation substrate, and the substrate formula components include fresh / dry spirulina, carbon source, and nitrogen source.

[0046] Specifically: the dry matter content of spirulina is 1%, the carbon source content is 2%, the nitrogen source content is 1%, the ultra-high temperature instantaneous sterilization temperature is 135 °C, the sterilization time is 5 s, and the material pumping speed is 10 L / min;

[0047] (2) Preparation of the fermentation seed liquid: The 7 test bacteria are respectively spread on MRS / YPD solid plates for activation. Among them, lactic acid bacteria are cultured at 37 °C for 48 h, and yeasts are cultured at 30 °C for 24 h; after activation, single colonies are respectively picked into MRS / YPD liquid medium and cultured at 30 - 37 °C for 24 h. After subculturing twice, they are amplified and cultured. After 24 h, the culture solution is centrifuged to remove the supernatant, and then the bacteria are washed with phosphate buffer and resuspended to obtain concentrated seed liquids of 7 test bacteria respectively;

[0048] Lactic acid bacteria use MRS medium; yeasts use YPD medium;

[0049] Among them, MRS medium: beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, peptone 10.0 g. For solid medium, another 18 g of agar powder is added, made up to 1 L with distilled water, and sterilized at 115 °C for 20 min in an autoclave;

[0050] YPD medium: glucose 20 g / L, peptone 20 g / L, yeast extract powder 10 g / L, distilled water 1000 mL. For solid medium, another 18 g of agar powder is added, and sterilized at 115 °C for 20 min in an autoclave;

[0051] (3) Respectively inoculate 1% of the 7 single-bacterium concentrated seed liquids in step (2) into separate fermentation substrates (v / v), ferment and culture at 35 °C for 24 h. Take part of the fermentation broth for coating and counting, and the other part is used to measure the antioxidant activity. The test results are shown in Table 1; and screening of composite bacteria fermentation is carried out with antioxidant activity as the index, the inoculation amount of bacteria is 1% (v / v), the prepared fermentation broth is centrifuged to take the supernatant to obtain the spirulina fermentation product.

[0052] The ratio of the composite bacteria fermentation combination is lactic acid bacteria: yeast = 1:1, and the specific combinations are as follows:

[0053] Lactobacillus acidophilus LA + Kluyveromyces marxianus KM, namely LA + KM;

[0054] Lactobacillus acidophilus LA + Saccharomyces cerevisiae SC, namely LA + SC;

[0055] Lactobacillus acidophilus LA1 + Saccharomyces cerevisiae SC, namely LA1 + SC;

[0056] Lactobacillus acidophilus LA1 + Kluyveromyces marxianus KM, namely LA1 + KM;

[0057] Lactobacillus fermentum LC + Saccharomyces cerevisiae SC, namely LC + SC;

[0058] Lactobacillus fermentum LC + Kluyveromyces marxianus KM, namely LC + KM;

[0059] The test results are shown in Table 1.

[0060] Table 1 Fermentation effect of probiotic-fermented spirulina

[0061] Serial number Item Total flavonoids Total phenols DPPH free radical scavenging ability Iron reducing ability 1 Unfermented sample (CK) 50.37±1.24 38.64±2.35 38.54±3.07 62.3±1.34 2 Lactobacillus plantarum (LP) 55.32±0.42 41.24±0.13 47.78±1.06 69.74±0.53 3 Lactobacillus casei (BC) 53.70±0.52 39.21±0.33 38.53±0.45 74.25±1.11 4 Lactobacillus acidophilus (LA1) 44.21±0.35 40.13±0.19 51.88±0.51 71.69±1.52 5 Lactobacillus acidophilus (LA) 58.53±0.45 43.51±0.1 61.74±1.42 90.27±3.82 6 Lactobacillus fermentum (LC) 55.86±0.45 43.54±0.24 49.56±0.87 64.32±0.76 7 Saccharomyces cerevisiae (SC) 56.18±0.45 67.59±1.17 58.62±0.79 97.57±4.59 8 Kluyveromyces marxianus (KM) 55.27±3.23 59.93±0.1 55.34±1.34 95.14±1.91 9 LA + SC 56.61±2.3 54.23±0.44 49.61±0.79 100.41±0.57 10 LA + KM 73.94±0.6 64.02±0.93 67.51±1.57 101.22±3.25 11 LA1 + SC 58.26±0.79 38.45±0.65 56.43±0.24 88.26±1.57 12 LA1 + KM 65.11±0.97 53.67±0.58 60.67±1.92 100.86±1.48 13 LC + SC 66.77±0.53 55.06±0.05 49.28±0.63 101.08±0.76 14 LC + KM 65.27±1.23 55.33±0.24 63.63±1.26 101.62±1.15

[0062] As can be seen from Table 1, among the lactic acid bacteria, Lactobacillus acidophilus LA has the best effect on fermenting spirulina, and the contents of total flavonoids, total phenols and antioxidant capacity are all significantly improved. Lactobacillus fermentum LC also has a strong promoting effect on the contents of total flavonoids, total phenols and DPPH free radical scavenging ability.

[0063] Moreover, it can be seen from Table 1 that the effects of the two yeasts on fermenting spirulina are both good. Therefore, Lactobacillus acidophilus LA and Lactobacillus fermentum LC are selected to be combined with yeasts (SC&KM) in pairs. The results show that the combination of Lactobacillus acidophilus LA and Kluyveromyces marxianus KM has the best fermentation effect. Compared with non-fermented spirulina, the contents of total flavonoids and total phenols are increased by 46.8% and 65.7% respectively, and the DPPH free radical scavenging ability and ferric reducing ability are increased by 75.2% and 62.5% respectively.

[0064] Thus, it can be seen that the combination of Lactobacillus acidophilus LA and Kluyveromyces marxianus KM is the optimal strain combination for fermenting spirulina.

[0065] Example 2 (Optimization of the process for fermenting spirulina with two strains)

[0066] The single factor and response surface methods were used to optimize the best process for fermenting spirulina with two strains.

[0067] The effects of single factors such as the inoculation amount of microorganisms (1%, 2%, 4%, 6%), inoculation ratio (LA:KM = 3:1, 2:1, 1:1, 1:2, 1:3), fermentation temperature (28 °C, 30 °C, 32 °C, 34 °C, 36 °C) on the amount of lactic acid bacteria and ferric reducing antioxidant power (FRAP) value during the fermentation process of spirulina were investigated respectively. The results are shown in Figure 1 。

[0068] Figure 1 Figure showing the effects of single factors on the amount of lactic acid bacteria and FRAP value during the fermentation of spirulina with Lactobacillus acidophilus LA and Kluyveromyces marxianus KM

[0069] Single-factor experiments showed that: when the inoculation amount was 4%, the inoculation ratio of Lactobacillus acidophilus LA to Kluyveromyces marxianus KM was 1:2, and the fermentation temperature of the two strains was 34°C, the amount of lactic acid bacteria and the FRAP value could reach the optimum.

[0070] Figure 2 Figure 4 is the response surface optimization diagram of the interaction of various factors on FRAP. Through further optimization of the response surface, the optimal fermentation process was obtained.

[0071] According to the regression equation, the optimal fermentation conditions predicted by the regression model were an inoculation amount of 4.73%, an inoculation ratio of Lactobacillus acidophilus LA to Kluyveromyces marxianus KM of 1:2.17, and a fermentation temperature of 32.541°C. At this time, the maximum predicted FRAP value was 207.501 μmol·L -1 .

[0072] Considering the actual operation requirements, the optimal fermentation conditions were determined as an inoculation amount of 4.7%, an inoculation ratio of Lactobacillus acidophilus LA to Kluyveromyces marxianus KM of 1:2, and a fermentation temperature of 32.5°C. To verify the reliability of the model and the predicted maximum value of 207.501 μmol·L -1 , 5 parallel verification tests were carried out with the above optimal medium formula, and the average FRAP value was 206.96 μmol·L -1 , which was very close to the predicted value, indicating that the model could accurately reflect the relationship between the changes of various factors and the antioxidant activity. The design of this model was reliable. Under these conditions, Spirulina was fermented, and the obtained fermentation products were used for component analysis and animal experiments.

[0073] Example 3 (Analysis of the composition of nutrients in fermented Spirulina)

[0074] Non-target metabolomics (LC-MS) was used to analyze the metabolites before and after fermentation of Lactobacillus acidophilus LA and Kluyveromyces marxianus KM. The results are shown in Figure 3 .

[0075] Figure 3 In Figure 5, Figure A is the heat map of non-volatile compounds, and Figure B is the proportion of different types of compounds before and after fermentation.

[0076] As shown in Figure A of Figure 3 , after fermentation, the calorific values of amino acid and organic acid components were significantly higher than those before fermentation, and there were also obvious differences in other components such as lipids and nucleotides; Figure 3As shown in Figure B, after fermentation, the contents of amino acids and their derivatives, organic acids, and phospholipids increased by 20.42%, 1.69%, and 0.17%, respectively; free fatty acids, glycerides, long-chain polyunsaturated fatty acids (LCP), lysophosphatidylcholine (LPE), nucleotides and their derivatives, carbohydrates, and vitamin compounds decreased by 0.74%, 0.82, 14.36%, 1.30%, 0.71%, 2.83%, and 1.54%, respectively. Thus, it can be seen that fermentation significantly changed the nutrient composition of Spirulina.

[0077] Example 4 (Efficacy Test of Spirulina Fermentum in Preventing and Treating Metabolic Associated Fatty Liver Disease)

[0078] Mice with fatty liver were induced by a high-fat diet for 10 weeks. On this basis, the Spirulina fermentum obtained under the optimal fermentation process conditions was administered by gavage, and the weight gain of the mice was continuously monitored. The following 3 groups were set up for the experiment:

[0079] Low-fat control group: Normal mice + corresponding dose of physiological saline;

[0080] High-fat control group: MAFLD mice + corresponding dose of physiological saline;

[0081] Intervention group: MAFLD mice + mice administered with the product of Spirulina fermented by Lactobacillus acidophilus LA and Kluyveromyces marxianus KM

[0082] 10 mice were selected from each group. After 10 weeks of gavage, the mice were dissected, and physiological indexes and liver tissue sections were observed respectively. The results showed that the Spirulina fermentum could significantly reduce the weight gain of mice, improve blood lipid and liver tissue fat accumulation. The changes in indexes such as animal weight, body composition, and metabolism during the period of gavage with Spirulina are shown in Figure 4 , where A: Body weight curve of mice aged 17 - 29 weeks; B: Weight gain curve of mice aged 17 - 29 weeks; C: State of mice before dissection; D: Body composition analysis of mice at the 13th week of the experiment; E: Curves of oxygen consumption, carbon dioxide production, and respiratory quotient of mice at the 13th week of the experiment.

[0083] From Figure 4 (A & B & C), it can be seen that the body weight of mice administered with the Spirulina fermentation product was lower than that of the high-fat control group, and the weight increase rate was significantly reduced, with a decrease range of 10.4 - 49.3%; the body composition analysis in Figure D showed that the Spirulina fermentation product treatment could reduce the body fat and body fluid content of mice, but the difference was not significant, and the lean meat content was basically the same; Figure E showed that compared with the high-fat group, the mice in the Spirulina fermentation product treatment group had an obvious tendency of increased oxygen consumption and carbon dioxide production, and the respiratory quotient changed insignificantly, indicating that the Spirulina fermentation product could increase the activity of mice. It is suggested that the lipid-lowering effect of the Spirulina group may be related to the increased activity and enhanced metabolism of mice.

[0084] In addition, the HE staining results of the liver tissues of mice in different treatment groups are shown in Figure 5 , where RC represents mice fed with normal diet and gavaged with normal saline; HFD represents mice fed with high-fat diet and gavaged with normal saline; SPI represents mice fed with high-fat diet and gavaged with Spirulina fermentum.

[0085] It can be seen from Figure 5 that the hepatocytes of mice fed with normal diet are intact, evenly distributed, with clear cell contours, clearly visible nucleus colors, no fat vacuoles and inflammatory cell aggregation. In contrast, the high-fat diet control group led to severe lipid accumulation in the liver tissue, with a large number of fat vacuoles in the cells, significantly enlarged hepatocyte volume and the cell nucleus displaced to the cell edge by lipid droplets. Inflammatory infiltration can be seen in the enlarged local image. In the mice gavaged with Spirulina fermentum, the white fat vacuoles in the hepatocytes were significantly reduced, the lipid droplets became smaller, the hepatocyte volume decreased, no inflammatory reaction was observed, and the histological characteristics of the liver basically returned to the normal control state, with the liver tissue weight decreased by about 18.5%.

[0086] Thus, it can be seen that the Spirulina fermentation product can significantly inhibit hepatic steatosis and greatly alleviate liver injury caused by high-fat diet.

[0087] Figure 6 is the heat map of differential metabolites in mouse liver. It can be seen from the figure that a total of 79 differential metabolites were detected in the mouse liver. The differential metabolites can be divided into 10 categories, including amino acids, benzoic acid, bile acids, carbohydrates, carnitines, fatty acids, indoles, organic acids, polypeptides and short-chain fatty acids. Compared with the normal group, the contents of amino acids, benzoic acid, bile acids, carnitine, fatty acids and polypeptides in the liver of high-fat group mice were significantly down-regulated, indicating that under the influence of high-fat diet, the normal functions of amino acid synthesis and bile synthesis in the liver of high-fat group mice were severely damaged.

[0088] Moreover, the contents of amino acids, benzoic acid, bile acids, carnitine, fatty acids and polypeptides in the liver metabolites of the Spirulina fermentum intervention group mice were significantly restored to normal levels.

[0089] Figure 7 is the box plot of the contents of differential metabolites glycine (A), lysine (B), leucine (C), serine (D), 2-methyl-4-pentenoic acid (E), myristic acid (F), glycylleucine (G) and p-hydroxyphenylacetic acid (H) in mouse liver.

[0090] Figure 7It was shown that, compared with the high-fat group, amino acids such as glycine and lysine, fatty acids 2-methyl-4-pentenoic acid and myristic acid, polypeptide glycyl-leucine and p-hydroxyphenylacetic acid were significantly increased in the Spirulina ferment extract intervention group; the recovery of the contents of amino acids such as glycine and lysine, glycyl-leucine and unsaturated fatty acid 2-methyl-4-pentenoic acid in the intervention group indicated that the Spirulina ferment extract could significantly restore the normal function of the liver.

[0091] Example 5 (Sensory Evaluation and Flavor Component Analysis of Spirulina Probiotic Fermentation Composition)

[0092] To clarify that the composition obtained by fermenting Spirulina with Lactobacillus acidophilus LA + Kluyveromyces marxianus KM has a good flavor and taste, 12 volunteers (6 males and 6 females, aged 18 - 40 years old) were organized for sensory evaluation. The sensory evaluation criteria are shown in Table 2, and the sensory evaluation of fermented Spirulina is shown in Figure 8 .

[0093] Pour the Spirulina fermentation broth into tasting cups and randomly number them, and conduct a comprehensive score from aspects such as aroma, taste, appearance color, and acceptability.

[0094] Table 2 Sensory Evaluation Criteria Table of Spirulina Fermentation Broth

[0095]

[0096] It can be seen from Figure 8 that unfermented Spirulina scored the lowest in terms of aroma, taste, appearance color, and acceptability. The fermentation with Lactobacillus acidophilus LA and Kluyveromyces marxianus KM would significantly improve its sensory characteristics. Among them, the scores of each sensory index of Spirulina after double-strain fermentation were the highest and the overall flavor was the best.

[0097] Taking unfermented Spirulina as a control, gas chromatography-ion mobility spectrometry (GC-IMS) was used to detect the flavor component composition of the double-strain fermentation of Lactobacillus acidophilus LA and Kluyveromyces marxianus KM. The Gallery Plot fingerprint spectrum of the flavor components of the composition is shown in Figure 9 .

[0098] Figure 9It can be seen that ketones and aldehydes such as 2,3-butanedione, 3-hydroxy-2-butanone, 2-octanone, 2-pentanone, 2-pentylfuran, hexanol, 2-methylbutanol, and 2-methylpropanol have the highest contents in the fermented sample (EF); while aldehydes such as E-2-heptenal, furfural, 2-methylbutanal, E-2-hexenal, butanal, benzaldehyde, heptanal, E-2-octenal, pentanal, and nonanal have the highest contents in the unfermented sample (UEF). Aldehydes generally have a certain pungent taste and are also the main components of the fishy smell produced by algae or freshwater fish. It can be seen that the fishy smell substances are greatly reduced after fermentation, while the specific aromatic compounds unique to fermentation increase significantly. The specific changes of different components can be seen in Table 3.

[0099] Table 3 Volatile substances in the GC-IMS identification sample information

[0100]

[0101] Note: UEF, unfermented spirulina; EF, fermented spirulina.

Claims

1. A Spirulina probiotic fermentation composition for preventing and treating metabolic associated fatty liver disease, characterized in that, The probiotics are Lactobacillus acidophilus and Kluyveromyces marxianus; the fermentation composition is obtained by combining Lactobacillus acidophilus and Kluyveromyces marxianus to ferment spirulina; among them, Lactobacillus acidophilus was deposited at the China Center for Type Culture Collection on August 29, 2016. The deposit address is Wuhan University, Wuhan, China. The taxonomic name is Lactobacillus acidophilus( Lactobacillus acidophilus ) KDB-03, and the deposit number is CCTCC NO: M 2016429; The preparation method of the fermentation composition is as follows: (1) Preparation of Spirulina fermentation substrate: Take fresh / dry Spirulina to prepare the fermentation substrate. The matrix formula components include fresh / dry Spirulina, carbon source, and nitrogen source, and enter the fermentation tank through an ultra-high temperature instantaneous steam sterilization pipeline to obtain the Spirulina fermentation substrate. By mass percentage, the dry matter content of Spirulina is 0.5 - 4%, the carbon source content is 0.5% - 5%, and the nitrogen source content is 0.1 - 5%; The ultra-high temperature instantaneous steam sterilization is carried out before the material enters the sterilized fermentation tank. The ultra-high temperature instantaneous sterilization temperature is 115°C - 150°C, the sterilization time is 3 - 8 s, and the material pumping speed is 5 - 20 L / min; (2) Preparation of fermentation seed liquid: Coat Lactobacillus acidophilus and Kluyveromyces marxianus on MRS / YPD solid plates for activation. After the activation is completed, pick single colonies into MRS / YPD liquid medium for cultivation. After subculturing twice, carry out enlarged cultivation. After 20 - 30 h, centrifuge the culture solution to remove the supernatant, and then wash the bacteria with phosphate buffer and resuspend to obtain concentrated seed liquids of Lactobacillus acidophilus and Kluyveromyces marxianus respectively; The activation conditions on the MRS / YPD solid plate are: cultivate at 30 - 37°C for 24 - 48 h; The cultivation conditions of the colonies in the MRS / YPD liquid medium are: cultivate at 30 - 37°C for 24 - 48 h; (3) Inoculate the two concentrated seed liquids in step (2) into the fermentation substrate prepared in (1) for compound fermentation cultivation to obtain the Spirulina fermentation product. Among them, the inoculation amount of the concentrated liquid is 0.1 - 6%, and the addition ratio is: concentrated seed liquid of Lactobacillus acidophilus: concentrated seed liquid of Kluyveromyces marxianus = 1 - 3:1 - 3. The compound fermentation cultivation conditions are: fermentation temperature 28 - 37°C, fermentation time 24 - 48 h.

2. Use of a Spirulina probiotic fermentation composition for preventing and treating metabolic associated fatty liver disease according to claim 1 in the preparation of drugs with the efficacy of preventing and treating metabolic associated fatty liver disease.

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