Composition having hypoglycemic and / or hypolipidemic effects and preparation thereof

Abstract

The application discloses a composition with blood sugar and blood lipid reducing effects, and a preparation method thereof. The preparation method comprises the following steps: adding water to fenugreek seed powder and a compound yeast liquid to form a to-be-fermented liquid, wherein the compound yeast liquid is composed of lactobacillus plantarum seed liquid, lactobacillus casei seed liquid and saccharomyces cerevisiae seed liquid; sequentially performing anaerobic fermentation and aerobic fermentation on the to-be-fermented liquid to obtain a fermented liquid; performing purification and sterilization treatment on the fermented liquid to obtain a sterile fermented base liquid; performing hydrogen-rich fusion treatment on the sterile fermented base liquid to obtain a hydrogen-rich fermented base liquid; optionally adding auxiliary materials into the hydrogen-rich fermented base liquid; and sealing the hydrogen-rich fermented base liquid to obtain the composition. The composition prepared according to the application can significantly improve insulin resistance, reduce blood sugar and blood lipid levels, and inhibit lipid peroxidation, and is especially suitable for preparing a functional beverage for reducing blood sugar and blood lipid and improving metabolic syndrome, and the product has good stability and excellent taste.

Description

Technical Field

[0001] The present invention generally relates to a composition having hypoglycemic and / or hypolipidemic effects, and more particularly to a fenugreek seed-based composition or beverage. Background Technology

[0002] Currently, type 2 diabetes, hyperlipidemia, and metabolic syndrome have become chronic metabolic diseases worldwide. Their core pathological mechanisms involve insulin resistance, glucose and lipid metabolism disorders, gut microbiota imbalance, and excessive activation of oxidative stress. These diseases have a long course and numerous complications; long-term use of chemical hypoglycemic and lipid-lowering drugs can easily cause side effects such as liver and kidney damage and gastrointestinal discomfort.

[0003] Fenugreek seeds, a traditional food and medicine ingredient, are rich in active components such as trigonelline, 4-hydroxyisoleucine (4-HIL), dietary fiber, and saponins. Among them, 4-HIL can promote insulin secretion and improve insulin resistance, trigonelline can inhibit liver glycogenolysis and lower blood lipids, and dietary fiber can slow down carbohydrate absorption. However, the dissolution rate of active components in natural fenugreek seeds is low, and direct consumption can cause a bitter taste and bloating, thus severely limiting its practical application. Summary of the Invention

[0004] The purpose of this invention is to provide a fenugreek seed-based composition with hypoglycemic and / or hypolipidemic effects.

[0005] According to one aspect of the present invention, a method for preparing a composition having hypoglycemic and / or hypolipidemic effects is provided, comprising: Grind the fenugreek seeds into fenugreek seed powder; Prepare a compound yeast culture, which is composed of Lactobacillus plantarum seed culture, Lactobacillus casei seed culture and Saccharomyces cerevisiae seed culture in a live cell count ratio of (1~3):(1~3):1; Fenugreek seed powder and compound yeast liquid are mixed with water to form a fermentation liquid. The fermentation broth was subjected to anaerobic and aerobic fermentation sequentially to obtain the fermentation broth. The anaerobic fermentation temperature was 36℃~38℃ for 25~40h, and the aerobic fermentation temperature was 28℃~32℃ for 10~20h. The pH value of the fermentation broth was 4.0~4.5, and the total viable count was ≥1×10⁻⁶. 8 CFU / mL; After purification and sterilization of the fermentation broth, a sterile fermentation base liquid was obtained; Hydrogen-rich fermentation base liquid is obtained by hydrogen-rich fusion treatment of sterile fermentation base liquid; Additives may be added to the hydrogen-rich fermentation base liquid at your discretion; and The composition is obtained by sealing the hydrogen-rich fermentation base liquid.

[0006] According to the present invention, the hydrogen content in the hydrogen-rich fermentation base liquid is preferably 0.8~1.8 mg / L, more preferably 1~1.6 mg / L.

[0007] According to the present invention, the content of fenugreek seed powder in the fermentation liquid is preferably 50~90 g / L.

[0008] According to the present invention, the added excipients may include steviol glycosides, citric acid, xanthan gum, and hawthorn juice. For example, the weight percentage of the excipients may be 1% to 5% of the total weight of the composition.

[0009] According to the present invention, the hydrogen-rich fusion treatment can adopt a closed pressurized hydrogen dissolution mode with a pressure of 0.3~0.5 MPa, a temperature of 20~25℃, a hydrogen dissolution time of 40~60 min, and a hydrogen purity of ≥99.99%.

[0010] According to another aspect of the present invention, a composition having hypoglycemic and / or hypolipidemic effects prepared according to the above method is also provided.

[0011] The composition according to the present invention can be in the form of a liquid beverage, preferably with a pH value of 4 to 4.5.

[0012] The composition according to the present invention preferably comprises: trigonelline 50-120 mg / L; 4-hydroxyisoleucine 30-80 mg / L; γ-aminobutyric acid 20-50 mg / L; and hydrogen 0.8-1.8 mg / L.

[0013] This invention significantly improves the dissolution rate and bioavailability of hypoglycemic / lipid-lowering active ingredients in fenugreek seeds through a specific compound fermentation method, while degrading bitter substances and improving the product's taste. In addition, this invention also regulates glucose and lipid metabolism and reduces oxidative stress through a dual synergistic system—fermentation active ingredients and hydrogen—and further improves the gut microbiota.

[0014] Animal experiments and in vitro cell experiments have verified that the composition prepared according to the present invention can significantly improve insulin resistance, reduce blood glucose and lipid levels, and inhibit lipid peroxidation. It is especially suitable for preparing functional beverages that can help lower blood sugar and lipids and improve metabolic syndrome. The product has good stability and excellent taste. Attached Figure Description

[0015] Figure 1A and Figure 1B The bar charts show the comparison of fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) levels in rats from each experimental group after 8 weeks of intervention. Figure 2A and Figure 2B The figures show a comparative analysis of fasting insulin (FINS) concentration and insulin sensitivity index (ISI) in rats of different experimental groups after 8 weeks of intervention. Figure 3 A bar chart showing the comparison of serum total cholesterol (TC) and triglyceride (TG) concentrations in rats from different experimental groups after 8 weeks of intervention; Figure 4 This is a comparative analysis of serum low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) concentrations in rats from different experimental groups after 8 weeks of intervention. Figure 5 A bar chart comparing the activity of superoxide dismutase (SOD) in liver tissue of rats in different groups after 8 weeks of intervention; and Figure 6 A bar chart comparing the levels of malondialdehyde (MDA), a lipid peroxidation product, in the liver tissue of rats in different experimental groups after 8 weeks of intervention. Detailed Implementation

[0016] Example 1: Preparation of a daily conditioning hypoglycemic / lipid-lowering beverage Raw material preparation: 10 kg fenugreek seeds, Lactobacillus plantarum seed solution (live bacteria count 1×10⁻⁶) 9 CFU / mL), Lactobacillus casei seed culture (live count 1×10⁻⁶) 9 CFU / mL), Saccharomyces cerevisiae seed culture (live count 1×10⁻⁶) 9 Ingredients: CFU / mL, 50 g steviol glycosides, 100 g citric acid, 40 g xanthan gum, 2 L hawthorn juice, 99.995% high-purity hydrogen, and 140 L purified water.

[0017] Lactobacillus plantarum (CICC 20315), Lactobacillus casei (CICC 6111), and Saccharomyces cerevisiae (CICC 1203) were all purchased from the China Industrial Microbial Culture Collection Center; the corresponding seed culture was a high-concentration liquid culture of live bacteria obtained by activating and expanding the above strains respectively.

[0018] Fenugreek seed pretreatment: Fenugreek seeds were cleaned of impurities, rinsed with water 3 times, baked at 55℃ for 2.5 h, pulverized and passed through a 70-mesh sieve to obtain fenugreek seed powder; the fenugreek seed powder was placed at 92℃ for 12 min to inactivate enzymes, and then sterilized by high-pressure steam at 121℃ and 0.1 MPa for 18 min for later use.

[0019] Prepare the compound yeast culture: Mix the seed cultures of Lactobacillus plantarum, Lactobacillus casei, and Saccharomyces cerevisiae in a volume ratio of 2:2:1, and set aside.

[0020] Pretreated fenugreek seed powder was mixed with 140 L of purified water and stirred evenly to prepare a fermentation substrate. Then, a compound yeast solution was inoculated at a volume ratio of 4% to form the fermentation broth. The broth was then placed at 37℃ for anaerobic fermentation for 30 h, followed by aerobic fermentation at 30℃ for 15 h. Fermentation continued until the final pH value was within the range of 4.0–4.5, and the total viable cell count was ≥1×10⁻⁶. 8 CFU / mL was used to obtain the fenugreek seed fermentation broth.

[0021] Post-treatment of fermentation broth: The fermentation broth was coarsely filtered through a 200-mesh filter and then centrifuged at 4500 r / min for 18 min to remove bacterial residues; 0.08% chitosan was added, and the mixture was allowed to stand at room temperature for 12 h to clarify. Subsequently, it was finely filtered through a 0.45 μm ceramic membrane, irradiated with ultraviolet light for 30 min, and then pasteurized at 65℃ for 30 min to obtain sterile fenugreek seed fermentation base broth.

[0022] Hydrogen-rich fusion: The fermentation base liquid is transferred to a sealed hydrogen-rich preparation tank, and high-purity hydrogen gas is introduced. The pressure is controlled at 0.4 MPa and the temperature at 22℃. Hydrogen dissolution is carried out for 50 minutes. The hydrogen concentration is monitored online until it reaches 1.1 mg / L, thus obtaining a hydrogen-rich fermentation base liquid. Online detection is achieved using a hydrogen microelectrode online detection method. The hydrogen microelectrode probe is directly inserted into the fermentation base liquid in the sealed hydrogen-rich preparation tank. The microelectrode is connected to an online concentration monitor. After the detection signal stabilizes, the hydrogen dissolved concentration in the fermentation base liquid is read and displayed in real time. When the monitored value reaches 1.1 mg / L, it is determined that the hydrogen has reached the preset saturation concentration, and the hydrogen dissolution operation is immediately stopped, completing the hydrogen-rich fusion process.

[0023] Preparation and filling: Add the above-mentioned steviol glycosides, citric acid, xanthan gum and hawthorn juice to the hydrogen-rich fermentation base liquid, stir at 25℃ for 25 min until uniform, and adjust the pH value to 4.2; use aseptic cold filling process to fill into PET-aluminum foil composite bottles at a speed of 30 mL / s, 300 mL per bottle, and screw cap on to seal.

[0024] Quality test results: The composition contains 68 mg / L trigonelline, 4-hydroxyisoleucine (4-HIL) 42 mg / L, γ-aminobutyric acid (GABA) 26 mg / L, and H2 1.05 mg / L. The microbial limits meet the GB 7101 standard, which means it is a qualified daily conditioning beverage.

[0025] Example 2: Preparation of a fortified intervention-type hypoglycemic / lipid-lowering beverage Raw material preparation: 15 kg fenugreek seeds, Lactobacillus plantarum seed liquid (live bacteria count 1×10⁻⁶) 9 CFU / mL), Lactobacillus casei seed culture (live count 1×10⁻⁶) 9 CFU / mL), Saccharomyces cerevisiae seed culture (live count 1×10⁻⁶) 9Ingredients: CFU / mL, 60 g steviol glycosides, 120 g citric acid, 50 g xanthan gum, 3 L hawthorn juice, 99.995% high-purity hydrogen, 210 L purified water.

[0026] Fenugreek seed pretreatment: Same as in Example 1.

[0027] Prepare the compound yeast culture: Same as in Example 1.

[0028] Pretreated fenugreek seed powder was mixed with 210 L of purified water and stirred evenly to prepare a fermentation substrate. Then, a compound yeast solution was inoculated at a volume ratio of 5% to form the fermentation broth. The broth was then placed at 37℃ for anaerobic fermentation for 36 h, followed by aerobic fermentation at 30℃ for 18 h. Fermentation continued until the final pH value was within the range of 4.0–4.5 and the total viable cell count was ≥1×10⁻⁶. 8 CFU / mL was used to obtain the fenugreek seed fermentation broth.

[0029] Post-treatment of fermentation broth: Same as in Example 1.

[0030] Hydrogen-rich fusion: The fermentation base liquid is transferred to a closed hydrogen-rich preparation tank, high-purity hydrogen is introduced, the pressure is controlled at 0.45 MPa and the temperature at 22℃, hydrogen is dissolved for 55 min, and the hydrogen concentration is detected online to reach 1.5 mg / L to obtain hydrogen-rich fermentation base liquid.

[0031] Preparation and filling: Same as in Example 1.

[0032] Quality test results: The composition contains trigonelline 105 mg / L, 4-HIL 68 mg / L, GABA 42 mg / L, and hydrogen 1.48 mg / L. The microbial limits meet the GB 7101 standard, which means it is a qualified fortified intervention beverage.

[0033] Comparative Example 1: Preparation of hydrogen-free beverages Everything else is the same as in Example 1; the difference is that the hydrogen-rich fusion step is omitted. Comparative Example 2: Preparation of a Simple Hydrogen-Rich Beverage Raw material preparation: 140 L purified water, 50 g steviol glycosides, 100 g citric acid, 40 g xanthan gum, 2 L hawthorn juice, and high-purity hydrogen (99.995%).

[0034] After the purified water and excipients are mixed evenly, they are transferred to a sealed hydrogen-rich preparation tank. High-purity hydrogen gas is introduced, and the pressure is controlled at 0.4 MPa and the temperature at 22°C. The hydrogen is dissolved for 50 minutes, and the hydrogen concentration reaches 1.1 mg / L. The mixture is then aseptically filled and sealed to obtain a hydrogen-rich beverage that contains only hydrogen gas and no fermented components.

[0035] Animal experiments 1. Experimental Animals and Model Establishment: Eighty male SPF-grade SD rats, weighing 220-250 g, were selected and randomly divided into four groups (n=13-14 each): a blank control group, a model group, a comparative example group 1, a comparative example group 2, an example example group 1, and an example example group 2. Except for the blank control group, the other groups were fed a high-sugar, high-fat diet for 4 weeks, combined with intraperitoneal injection of streptozotocin (STZ, 35 mg / kg) to establish a type 2 diabetes mellitus complicated with hyperlipidemia. The criteria for successful modeling were: fasting blood glucose ≥11.1 mmol / L, total cholesterol (TC) ≥5.0 mmol / L, and total triglycerides (TG) ≥1.8 mmol / L.

[0036] 2. Dosing regimen: After successful modeling, Comparative Example 1 was administered the hydrogen-free beverage obtained from Comparative Example 1 by gavage, Comparative Example 2 was administered the simple hydrogen-rich beverage obtained from Comparative Example 2 by gavage, Example 1 was administered the combined beverage prepared in Example 1 by gavage, Example 2 was administered the combined beverage prepared in Example 2 by gavage, and the blank control group and model group were administered the same volume of purified water by gavage. The administration was once daily, 10 mL / kg each time, for 8 consecutive weeks. During this period, all groups except the blank control group (model group and each administration group) continued to be given a high-sugar and high-fat diet.

[0037] 3. Detection indicators and methods: (1) Glucose metabolism indicators: Fasting blood glucose (FBG) was measured by tail vein blood collection at 0, 4 and 8 weeks of intervention; at 8 weeks of intervention, blood was collected by enucleation and glycated hemoglobin (HbA1c) and fasting insulin (FINS) were measured by enzyme-linked immunosorbent assay (ELISA) and insulin sensitivity index (ISI=ln [1 / (FBG×FINS)]).

[0038] (2) Lipid metabolism indicators: After 8 weeks of intervention, serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were detected using a fully automated biochemical analyzer.

[0039] (3) Oxidative stress indicators: After 8 weeks of intervention, rat liver tissue was taken, homogenized, and the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were detected.

[0040] 4. Experimental Results: The results showed that all indicators in the blank control group remained at normal physiological levels; after treatment with a high-sugar, high-fat diet combined with STZ, the glucose and lipid metabolism and oxidative stress indicators in the model group were significantly abnormal, indicating successful modeling. After 8 weeks of intervention, the indicators in each treatment group improved to varying degrees, with the best improvement observed in Example 2 group, followed by Example 1 group. Both groups were significantly better than Comparative Example 1 and Comparative Example 2 groups (P<0.01), demonstrating the synergistic effect of fermentation components and hydrogen. Table 1 shows the comparison of glucose metabolism indicators in each group of rats (8 weeks of intervention); Table 2 shows the comparison of lipid metabolism indicators in each group of rats (8 weeks of intervention); Table 3 shows the comparison of liver oxidative stress indicators in each group of rats (8 weeks of intervention).

[0041]

[0042]

[0043]

[0044] Figure 1A and Figure 1B The bar charts show the comparison of fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) levels in rats from each experimental group after 8 weeks of intervention. Figure 2A and Figure 2B The figures show a comparative analysis of fasting insulin (FINS) concentration and insulin sensitivity index (ISI) in rats of different experimental groups after 8 weeks of intervention. Figure 3 A bar chart showing the comparison of serum total cholesterol (TC) and triglyceride (TG) concentrations in rats from different experimental groups after 8 weeks of intervention; Figure 4 This is a comparative analysis of serum low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) concentrations in rats from different experimental groups after 8 weeks of intervention. Figure 5 A bar chart comparing the activity of superoxide dismutase (SOD) in liver tissue of rats in different groups after 8 weeks of intervention; and Figure 6 A bar chart comparing the levels of malondialdehyde (MDA), a lipid peroxidation product, in the liver tissue of rats in different experimental groups after 8 weeks of intervention.

[0045] The above results indicate that the beverage compositions prepared according to Examples 1 and 2 are significantly superior to Comparative Example 1 and Comparative Example 2 in terms of lowering blood sugar, lowering blood lipids, improving insulin sensitivity, antioxidation, and liver protection (P<0.01), demonstrating a significant synergistic effect between the fermentation active ingredients and hydrogen. Example 2 showed the best performance in lowering blood sugar, lowering blood lipids, and antioxidation, while Example 1 also exhibited good intervention effects. Furthermore, both groups showed better HDL-C enhancement than the single-component groups (fermented fenugreek seed base without hydrogen and hydrogen-rich water without fenugreek seed fermentation components), demonstrating a unique synergistic advantage.

[0046] In vitro cell experiments 1. Experimental groups: Blank control group: cells were not induced to differentiate and no test sample was applied; Model group: cells were induced to differentiate adipogenically but no test sample was applied; Comparative example 1: cells were induced to differentiate adipogenically and were also treated with the Comparative example 1 sample; Comparative example 2: cells were induced to differentiate adipogenically and were also treated with the Comparative example 2 sample; Example 2: cells were induced to differentiate adipogenically and were also treated with the Example 2 beverage.

[0047] 2. Experimental methods: 3T3-L1 preadipocytes were induced to differentiate into mature adipocytes. During the induction process, each drug group was given the corresponding drug (fermentation base, hydrogen-rich water, and hydrogen-rich fermentation beverage were all diluted 10 times). After differentiation, the lipid accumulation in adipocytes was detected by Oil Red O staining and the intracellular TG content was detected by enzymatic method.

[0048] 3. The experimental results are shown in Table 4, which provides a comparison of lipid accumulation-related indicators in 3T3-L1 adipocytes of each group.

[0049]

[0050] The above results indicate that the lipid content in the undifferentiated adipocytes of the blank control group was extremely low; after induction of differentiation, the model group showed significant lipid accumulation and a significant increase in TG content; both comparative groups 1 and 2 could inhibit lipid accumulation and TG production to some extent, but the effect was limited; while the lipid accumulation area inhibition rate of the second example group reached 65.3%, and the TG content decreased to (28.6 ± 3.5) mg / g prot, which was significantly better than the first two groups (P<0.01), confirming that the fermentation components and hydrogen in the beverage composition have a significant synergistic effect in inhibiting adipocyte differentiation and reducing lipid accumulation (which is far beyond the simple additive effect).

[0051] In summary, the composition or beverage prepared according to the present invention can significantly reduce fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) levels in model rats, increase insulin sensitivity index (ISI), and simultaneously reduce total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), while increasing high-density lipoprotein cholesterol (HDL-C). In vitro 3T3-L1 adipocyte models and HepG2 hepatocyte models have further confirmed that it can inhibit adipocyte differentiation, reduce lipid accumulation, and inhibit hepatocyte gluconeogenesis. Its hypoglycemic and lipid-lowering effects are significantly better than those of simple fenugreek seed fermented beverages or simple hydrogen-rich beverages.

[0052] The compositions prepared according to the present invention can be used as functional beverages for patients with type 2 diabetes, hyperlipidemia, high-risk groups for metabolic syndrome, and middle-aged and elderly people with abnormal glucose and lipid metabolism. For example, the above-mentioned daily conditioning beverage is recommended to be consumed 1-2 bottles (300 mL per bottle) daily, suitable for people with mild glucose and lipid metabolism disorders and for daily prevention. The above-mentioned enhanced intervention beverage is recommended to be consumed 2 bottles (300 mL per bottle) daily, divided into two doses in the morning and evening, suitable for people with mild to moderate type 2 diabetes and hyperlipidemia as an adjunct treatment. It can be stored at room temperature away from light. Within 12 months, the content of the core active ingredients decreases by ≤10%, and the hydrogen concentration is maintained at ≥0.5 mg / L.

[0053] This invention utilizes a specific compound fermentation method, leveraging the metabolic activity of specific microorganisms such as probiotics, to degrade anti-nutritional factors in fenugreek seeds, thereby increasing the dissolution rate of active ingredients. Simultaneously, it produces secondary bioactive substances such as γ-aminobutyric acid (GABA) and short-chain fatty acids, enhancing bioavailability and improving product taste. Through hydrogen-rich fusion, hydrogen can penetrate cell membranes to eliminate harmful free radicals, reducing oxidative stress damage caused by metabolic disorders. This synergistic effect with the fermented active ingredients of fenugreek seeds further regulates the intestinal microecology.

[0054] Unlike existing fenugreek seed-related beverages obtained through simple extraction processes, the composition or beverage prepared according to the method of the present invention has obvious and significant effects and excellent taste, making it particularly suitable for industrial-scale production.

Claims

1. A method for preparing a composition having hypoglycemic and / or hypolipidemic effects, comprising: Grind the fenugreek seeds into fenugreek seed powder; Prepare a compound yeast culture, which is composed of Lactobacillus plantarum seed culture, Lactobacillus casei seed culture and Saccharomyces cerevisiae seed culture in a live cell count ratio of (1~3):(1~3):1; Fenugreek seed powder and compound yeast liquid are mixed with water to form a fermentation liquid. The fermentation broth was subjected to anaerobic and aerobic fermentation sequentially to obtain the fermentation broth. The anaerobic fermentation temperature was 36℃~38℃ for 25~40h, and the aerobic fermentation temperature was 28℃~32℃ for 10~20h. The pH value of the fermentation broth was 4.0~4.5, and the total viable count was ≥1×10⁻⁶. 8 CFU / mL; After purification and sterilization of the fermentation broth, a sterile fermentation base liquid was obtained; Hydrogen-rich fermentation base liquid is obtained by hydrogen-rich fusion treatment of sterile fermentation base liquid; Additives may be added to the hydrogen-rich fermentation base liquid at your discretion; and The composition is obtained by sealing the hydrogen-rich fermentation base liquid.

2. The preparation method according to claim 1, wherein the hydrogen content of the hydrogen-rich fermentation base liquid is 1~1.6 mg / L.

3. The preparation method according to claim 1, wherein the content of fenugreek seed powder in the fermentation liquid is 50~90 g / L.

4. The preparation method according to claim 1, wherein the added excipients include steviol glycosides, citric acid, xanthan gum and hawthorn juice.

5. A composition having hypoglycemic and / or hypolipidemic effects, prepared by the method according to any one of claims 1-4.

6. The composition according to claim 5, wherein it is in the form of a liquid beverage and has a pH value of 4 to 4.

5.

7. The composition according to claim 6, comprising: Trigonelline 50~120 mg / L; 4-hydroxyisoleucine 30~80 mg / L; γ-aminobutyric acid 20~50 mg / L; hydrogen 0.8~1.8 mg / L.

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