Fermentation type high-astaxanthin microalgae compound for aquaculture and preparation method of fermentation type high-astaxanthin microalgae compound

Abstract

The invention discloses a fermentation type high astaxanthin microalgae compound for aquaculture and a preparation method thereof, and belongs to the technical field of aquaculture feeds, the fermentation type high astaxanthin microalgae compound comprises a fermentation product of haematococcus pluvialis subjected to stress induction culture, the haematococcus pluvialis is obtained by stress induction culture through a gradient nitrogen deficiency-pulse carbon supplementation method, and the haematococcus pluvialis is a high astaxanthin microalgae compound. The astaxanthin content in the dry weight is greater than or equal to 8.0 mg / g; the fermentation product is prepared by carrying out wall breaking on haematococcus pluvialis subjected to stress induction culture and then co-fermenting the haematococcus pluvialis and lactobacillus plantarum under an anaerobic condition; in the compound, the active titer of the phytocidin generated by the lactobacillus plantarum is greater than or equal to 600AU / g; after the compound is treated in simulated gastric juice with the pH value of 2.0 for 2 hours, the retention rate of astaxanthin is greater than or equal to 75%. The compound disclosed by the invention not only contains high-concentration astaxanthin, but also is rich in probiotic metabolites, so that the synergistic enhancement of oxidation resistance and immune regulation can be realized.

Description

[0001] A fermented high-astaxanthin microalgae complex for aquaculture and its preparation method Technical Field

[0003] This invention relates to the field of aquaculture feed technology, specifically to a fermented high-astaxanthin microalgae complex for aquaculture and its preparation method. Background Technology

[0005] Microalgae, rich in nutrients such as protein, polyunsaturated fatty acids, vitamins, and carotenoids, have become highly promising functional feed additives in aquaculture. Among them, Haematococcus pluvialis is one of the richest sources of astaxanthin in nature. Astaxanthin is a potent natural antioxidant with various physiological functions, including scavenging free radicals, enhancing immunity, and improving the body color of aquatic animals. It is of great significance for improving the disease resistance of farmed animals and the quality of farmed products.

[0006] Currently, the main method to increase the astaxanthin content in Haematococcus pluvialis is to apply environmental stress, such as strong light, nitrogen deficiency, and high salinity. For example, the literature (DelCampo et al., 2004) reported that nitrogen deficiency stress can significantly induce astaxanthin accumulation in Haematococcus pluvialis. However, single stress culture has problems such as unstable astaxanthin production and excessively rapid cell growth arrest. In addition, directly using high-astaxanthin algal powder as a feed additive has limited bioavailability of astaxanthin in the animal digestive tract, and the dense structure of the algal cell wall can hinder the release of other nutrients.

[0007] On the other hand, probiotic fermentation technology is widely used in the pretreatment of feed ingredients to degrade anti-nutritional factors, improve protein digestibility, and produce beneficial metabolites (such as bacteriocins and short-chain fatty acids). Some studies have attempted to ferment common microalgae to enhance their nutritional value. Summary of the Invention

[0009] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide a fermented high-astaxanthin microalgae complex for aquaculture. This complex not only contains a high concentration of astaxanthin, but is also rich in probiotic metabolites, which can achieve synergistic enhancement of antioxidant and immune regulation.

[0010] The second objective of this invention is to provide a method for preparing a fermented high-astaxanthin microalgae complex for aquaculture. This method innovatively combines the "gradient nitrogen deficiency-pulse carbon supplementation" culture process with the "microalgae-probiotic co-fermentation" process, thus solving the problems of difficult release of microalgae active ingredients and low bioavailability.

[0011] One of the objectives of this invention is achieved through the following technical solution:

[0012] A fermented high-astaxanthin microalgal complex for aquaculture, comprising fermentation products of Haematococcus pluvialis cultured under stress, wherein:

[0013] The Haematococcus pluvialis was obtained by stress-induced culture using a gradient nitrogen deficiency-pulse carbon supplementation method, and its astaxanthin content in dry weight was ≥8.0 mg / g;

[0014] The fermentation product is prepared by co-fermenting the stress-induced cultured Haematococcus pluvialis with Lactobacillus plantarum under anaerobic conditions after cell wall disruption.

[0015] In the complex, the plantaricin produced by the Lactobacillus plantarum has an activity potency ≥600AU / g;

[0016] After being treated in simulated gastric fluid at pH 2.0 for 2 hours, the complex retained ≥75% of its astaxanthin.

[0017] Furthermore, the gradient nitrogen deficiency-pulse carbon supplementation method includes:

[0018] Haematococcus pluvialis in the logarithmic growth phase was transferred from the total nutrient medium to the nitrogen-deficient medium;

[0019] The sodium nitrate concentration in the nitrogen-deficient culture medium was reduced in three steps: first, the concentration was reduced to 0.1 g / L and maintained for 24 hours; second, the concentration was reduced to 0.05 g / L and maintained for 24 hours; and third, the concentration was completely removed.

[0020] At 12 and 36 hours after complete nitrogen deficiency, sodium bicarbonate (NaHCO3) was instantaneously added to the culture system to achieve a final concentration of 2.0 g / L.

[0021] Furthermore, the co-fermentation conditions are as follows: the Haematococcus pluvialis pulp after cell wall disruption is mixed with the Lactobacillus plantarum at a weight ratio of 10:1, and fermented at 30°C under anaerobic conditions for 72 hours.

[0022] Furthermore, in the complex, the content of β-glucan with a molecular weight of 10-50 kDa is 150-200 mg / g dry weight, and the dissolution rate in simulated fish intestinal fluid is ≥70%.

[0023] Further, in step S3, the crude pigment is added to the cellulose nanocrystals (CNCs) colloid, wherein the mass ratio of the crude pigment to the cellulose nanocrystals is 1:1.

[0024] One of the objectives of this invention is achieved through the following technical solution:

[0025] A method for preparing a fermented microalgae complex high in astaxanthin includes the following steps:

[0026] S1. Haematococcus pluvialis was cultured using the gradient nitrogen deficiency-pulse carbon supplementation method to obtain algal powder with high astaxanthin content.

[0027] S2. The high-astaxanthin algal powder is subjected to cell wall breaking treatment.

[0028] S3. Mix the cell wall broken algae slurry with the plant lactobacillus and co-ferment under anaerobic conditions to obtain the fermentation product;

[0029] S4. The fermentation product is dried and pulverized to obtain the fermented high astaxanthin microalgae complex for aquaculture.

[0030] The third objective of this invention is to provide the use of a fermented high-astaxanthin microalgae complex for aquaculture in the preparation of an immune enhancer for aquatic animals.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] (1) The fermented high-astaxanthin microalgae complex for aquaculture provided by this invention addresses the issue that reactive oxygen species (ROS) are unavoidable byproducts of microbial metabolism, and excessive ROS can cause oxidative damage to cells. Haematococcus pluvialis accumulates large amounts of astaxanthin under gradient nitrogen deficiency-pulse carbon supplementation stress, and astaxanthin is a known potent antioxidant with the ability to scavenge ROS. Using high-astaxanthin algae powder as a substrate, it is used to protect probiotics in situ during co-fermentation. When high-astaxanthin algae are co-fermented with Lactobacillus plantarum, the astaxanthin released by the algae can effectively alleviate the oxidative stress on probiotics, thereby significantly improving fermentation efficiency. Meanwhile, the plant bactericides and short-chain fatty acids produced during fermentation directly regulate the intestinal flora and activate the immune system. Animal experiments have shown that Nile tilapia fed with the compound of this invention exhibited significantly higher levels of serum lysozyme and superoxide dismutase (SOD) activity than the control groups fed with high-astaxanthin algae powder alone, fermented ordinary algae powder alone, or a physical mixture of both, demonstrating a synergistic immune-enhancing effect.

[0033] (2) The fermented high-astaxanthin microalgae complex for aquaculture provided by this invention exhibits significantly higher cell wall damage in algal cells after stress pretreatment compared to ordinary algal cells, demonstrating that stress pretreatment loosens the cell wall structure, making it more easily digested by enzymes. This result proves that gradient nitrogen deficiency-pulse carbon supplementation culture not only accumulates astaxanthin but also produces a pretreatment effect on the cell wall. This effect has a significant synergistic effect with subsequent probiotic fermentation, greatly improving the release efficiency of intracellular nutrients. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments. It should be noted that, without conflict, the embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0036] Antioxidant mechanism of astaxanthin

[0037] Molecular structure determines function:

[0038] Astaxanthin is a ketocarotenoid whose molecular structure contains a long conjugated double bond and two oxygen-containing polar heads (ketone and hydroxyl groups). This unique structure enables it to:

[0039] Efficient quenching of singlet oxygen (¹O2): Converting high-energy singlet oxygen into harmless ground-state oxygen through physical means.

[0040] Free radical scavenging: Its polar head can capture free radicals (such as superoxide anion O2⁻• and hydroxyl radical •OH) in the aqueous and lipid phases inside and outside the cell membrane, interrupting the free radical chain reaction.

[0041] Stabilizes the cell membrane: It spans the cell membrane and protects both the inner and outer layers of the membrane from oxidative damage.

[0042] Operating environment:

[0043] Although astaxanthin itself is fat-soluble, it is usually stored in liposomes or oil droplets in microalgal cells in an esterified form. When algal cells are disrupted or partially degraded, these astaxanthin-rich lipid particles are released into the fermentation broth, forming tiny hydrophobic regions that become "reactive oxygen species (ROS) scavenging hotspots."

[0044] I. Cultivation of Haematococcus pluvialis with high astaxanthin (gradient nitrogen deficiency-pulse carbon supplementation method)

[0045] Algal strain activation: The preserved Haematococcus pluvialis algal strain was inoculated into SE medium and cultured in shake flasks under conditions of 2500 lux light intensity, 25℃ temperature, and air containing 1% CO2 until it entered the logarithmic growth phase.

[0046] Stress-induced:

[0047] Step 1 (Transfer and Initial Nitrogen Reduction): Collect the algal culture in the logarithmic growth phase by centrifugation, wash with sterile water, and resuspend in nitrogen-deficient SE medium to achieve an initial algal density of 1.0 × 10⁵ cells / mL. The sodium nitrate concentration in the nitrogen-deficient SE medium is 0.1 g / L during this stage, and the culture is carried out for 24 hours.

[0048] Step 2 (further nitrogen reduction): After 24 hours, the sodium nitrate concentration in the culture medium was further reduced to 0.05 g / L, and the culture was continued for another 24 hours.

[0049] Step 3 (Complete Nitrogen Deficiency): After another 24 hours, completely remove sodium nitrate from the culture medium to enter a state of complete nitrogen deficiency.

[0050] Pulsed carbon supplementation: At 12 hours and 36 hours after complete nitrogen deficiency, solid sodium bicarbonate (NaHCO3) was instantaneously added to the culture system to achieve a final concentration of 2.0 g / L.

[0051] Harvest: After 7 days of complete nitrogen deficiency, the algal cells turned deep red, indicating a large accumulation of astaxanthin. At this point, the algal solution was centrifuged, the algal sludge was collected, frozen at -80℃, and then freeze-dried under vacuum to obtain high-astaxanthin Haematococcus pluvialis powder (referred to as Haematococcus pluvialis cultured under stress). The astaxanthin content was determined by high-performance liquid chromatography (HPLC).

[0052] II. Preparation of Fermented High-Astaxanthin Microalgae Complex (Co-fermentation Method)

[0053] Cell wall breaking treatment: The stress algae powder obtained above is subjected to cell wall breaking treatment using a ball mill and passed through a 100-mesh sieve to obtain cell wall broken algae powder.

[0054] Strain activation: The preserved Lactobacillus plantarum was inoculated into MRS liquid medium and cultured under anaerobic conditions at 37°C for 24 hours to obtain seed culture.

[0055] Co-fermentation:

[0056] Algae powder with broken cell walls is mixed with sterile water at a ratio of 1:10 (w / v) to make algae slurry;

[0057] The Lactobacillus plantarum seed solution was inoculated into the algal slurry at a weight ratio of 1:10 (bacterial solution: algal slurry);

[0058] The mixture was transferred to a fermenter and fermented at 30°C under anaerobic conditions for 72 hours.

[0059] Post-processing: After fermentation, the fermentation broth was vacuum dried at 50°C, then pulverized and passed through an 80-mesh sieve to obtain the fermented high astaxanthin microalgae complex of the present invention.

[0060] III. Detection Methods for Key Indicators

[0061] Astaxanthin content: determined by high performance liquid chromatography (HPLC) in accordance with national standard GB31645-2018.

[0062] Plantaricin activity potency: The agarwell diffusion assay was used. Staphylococcus aureus was used as the indicator bacterium, and a standard plantaricin was used as a control. The diameter of the inhibition zone of the sample was measured, and its activity units (AU / g) were calculated.

[0063] Viable bacteria count: The count was performed using the MRS agar plate method after anaerobic incubation at 37°C for 48 hours.

[0064] Example 1

[0065] The preparation method of the fermented high-astaxanthin microalgae complex for aquaculture provided in this embodiment includes the following steps:

[0066] S1. Haematococcus pluvialis was cultured using the gradient nitrogen deficiency-pulse carbon supplementation method to obtain algal powder with high astaxanthin content.

[0067] S2. The high-astaxanthin algal powder is subjected to cell wall breaking treatment.

[0068] S3. Mix the cell wall broken algae slurry with the plant lactobacillus and co-ferment under anaerobic conditions to obtain the fermentation product;

[0069] S4. The fermentation product is dried and pulverized to obtain the fermented high-astaxanthin microalgae complex for aquaculture. The fermented high-astaxanthin microalgae complex of the present invention is prepared according to the steps in "Specific Embodiments".

[0070] Astaxanthin content in the stressed algal powder: determined by HPLC to be 9.2 mg / g dry weight.

[0071] Final complex: The potency of the plant bactericide was 720 AU / g; after 2 hours of treatment with simulated gastric juice, the astaxanthin retention rate was 78%; and the β-glucan dissolution rate was 75%.

[0072] Experiment Example 1: Verification of Synergistic Immune Enhancement Effect (Animal Experiment)

[0073] Experimental subjects: healthy Nile tilapia (average weight 50±5g), randomly divided into 4 groups, with 3 replicates in each group and 30 fish in each replicate.

[0074] Experimental Design:

[0075] Control group A: basal feed.

[0076] Control group B: Basic diet + 0.5% ordinary algae powder (unstressed culture, astaxanthin content 1.5mg / g).

[0077] Control group C: Basic feed + 0.5% fermented common algae powder (fermented using common algae powder according to the method in Example 1).

[0078] Control group D: Basic feed + 0.5% physical mixture (0.25% high astaxanthin algae powder and 0.25% fermented ordinary algae powder are physically mixed).

[0079] Experimental group E: Basic feed + 0.5% of the compound of this invention (i.e., the product of Example 1).

[0080] Feeding and management: Feed twice daily for a 4-week breeding cycle. Maintain water temperature at 28±1°C during this period.

[0081] Detection indicators: After the experiment, blood samples were randomly collected from 5 fish in each replicate to measure the activities of lysozyme (LZM) and superoxide dismutase (SOD) in the serum, which were used as immune indicators. The experimental results are shown in Table 1.

[0082] Table 1. Serum lysozyme (LZM) activity and superoxide dismutase (SOD) activity

[0083]

[0084] Results analysis: The lysozyme and SOD activities in experimental group E were significantly higher than those in all control groups (P<0.01). Their activities were not only far higher than those in the single-function groups (B, C), but also significantly higher than those in the combined-function group (D). This strongly demonstrates that the complex of this invention has a synergistic immune-enhancing effect.

[0085] Experiment Example 2: Verification of Fermentation Efficiency (Comparison of Viable Cell Count)

[0086] Experimental design: High-astaxanthin algal powder and ordinary algal powder were used as substrates and co-fermented in the same way (the method is the same as in Example 1). At the end of 72 hours of fermentation, the number of viable bacteria in the fermentation product was measured.

[0087] Experimental results:

[0088] Using ordinary algae powder as a substrate: the viable bacteria count is 1.8 × 10⁹ CFU / g; using high astaxanthin algae powder as a substrate (in this invention): the viable bacteria count is 2.5 × 10⁹ CFU / g.

[0089] Results analysis: Using high-astaxanthin algal powder as a substrate, the final viable bacteria count increased by 38.9%, which indicates that astaxanthin protects probiotics and promotes fermentation efficiency.

[0090] Experimental Example 3

[0091] Verification of cell wall degradation efficiency and nutrient release

[0092] Experimental objective: To verify whether Haematococcus pluvialis pretreated with gradient nitrogen deficiency-pulse carbon supplementation is more easily degraded during co-fermentation than ordinary algae, thereby releasing more soluble nutrients.

[0093] Experimental Design:

[0094] Sample preparation:

[0095] Sample A: Common Haematococcus pluvialis powder (not subjected to stress culture, astaxanthin content approximately 1.5 mg / g).

[0096] Sample B: High-astaxanthin Haematococcus pluvialis powder (cultured according to the method in Example 1, using a gradient nitrogen deficiency-pulse carbon supplementation method).

[0097] Fermentation treatment:

[0098] Sample A and Sample B were subjected to cell wall disruption treatment (the method is the same as in Example 1).

[0099] Equal amounts (dry weight) of the cell wall-broken algae slurry from A and B were taken and fermented according to the co-fermentation conditions of Example 1 (anaerobic fermentation with Lactobacillus plantarum at 30°C for 72 hours).

[0100] Meanwhile, two unfermented control groups were set up: namely, the cell wall broken algae slurry of sample A and sample B, which were not inoculated with bacteria and were left to stand for 72 hours under the same conditions.

[0101] Testing indicators:

[0102] Soluble polysaccharide content: The soluble polysaccharide content (calculated as glucose equivalent, mg / g) in the fermentation broth and control supernatant was determined using the phenol-sulfuric acid method. The results are shown in Tables 2 and 3.

[0103] Soluble protein content: The soluble protein content (mg / g) in the fermentation broth and control group supernatant was determined using the BCA method. The results are shown in Tables 2 and 3.

[0104] Scanning electron microscopy (SEM) observation: The residues of samples A and B after 72 hours of fermentation were dried and sputter-coated with gold. The degree of cell wall damage and microstructural changes were observed using scanning electron microscopy.

[0105] Table 2. Release of soluble nutrients under different treatments

[0106]

[0107] Table 3. Increase rate of soluble substance release

[0108]

[0109] SEM observation results:

[0110] Fermented ordinary algae powder residue: The cell fragments are relatively large, and the outline of the cell wall is still visible. The surface is relatively smooth, and the degradation is incomplete.

[0111] Fermented high-astaxanthin algal powder residue: cell fragments are small and severely broken, cell walls have a large number of holes and cracks, and the structure is loose and "honeycomb-like", indicating that the cell walls have been deeply degraded.

[0112] Results Analysis: Compared with fermented ordinary algal powder, fermented high-astaxanthin algal powder showed approximately 51% and 56% higher contents of soluble polysaccharides and soluble proteins, respectively. SEM images clearly showed that the cell walls of algal cells pretreated with stress were significantly more damaged after fermentation than those of ordinary algal cells, demonstrating that stress pretreatment loosens the cell wall structure, making it more easily digested by enzymes. These results demonstrate that gradient nitrogen deficiency-pulsed carbon supplementation culture not only accumulates astaxanthin but also produces a pretreatment effect on the cell wall. This effect has a significant synergistic effect with subsequent probiotic fermentation, greatly improving the efficiency of nutrient release from the cells.

[0113] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A fermented high-astaxanthin microalgae complex for aquaculture, characterized in that, It contains fermentation products of Haematococcus pluvialis cultured under stress, including: The Haematococcus pluvialis was obtained by stress-induced culture using a gradient nitrogen deficiency-pulse carbon supplementation method, and its astaxanthin content in dry weight was ≥8.0 mg / g; The fermentation product is prepared by co-fermenting Haematococcus pluvialis, which has been cultured under stress, with Lactobacillus plantarum under anaerobic conditions after cell wall disruption. In the complex, the activity potency of the plant bactericide produced by the Lactobacillus plantarum is ≥600AU / g; After being treated in simulated gastric fluid at pH 2.0 for 2 hours, the astaxanthin retention rate of the complex was ≥75%.

2. The fermented high-astaxanthin microalgae complex for aquaculture according to claim 1, characterized in that, The gradient nitrogen deficiency-pulse carbon supplementation method includes: Haematococcus pluvialis in the logarithmic growth phase was transferred from the total nutrient medium to the nitrogen-deficient medium; The sodium nitrate concentration in the nitrogen-deficient culture medium was reduced in three steps: first, the concentration was reduced to 0.1 g / L and maintained for 24 hours; second, the concentration was reduced to 0.05 g / L and maintained for 24 hours; and third, the concentration was completely removed. At 12 and 36 hours after complete nitrogen deficiency, sodium bicarbonate was instantaneously added to the culture system to achieve a final concentration of 2.0 g / L.

3. A fermented high-astaxanthin microalgae complex for aquaculture according to claim 1 or 2, characterized in that, The co-fermentation conditions are as follows: the Haematococcus pluvialis pulp after cell wall disruption is mixed with the Lactobacillus plantarum at a weight ratio of 10:1, and fermented at 30°C under anaerobic conditions for 72 hours.

4. A fermented high-astaxanthin microalgae complex for aquaculture according to any one of claims 1 to 3, characterized in that, The complex contains 150-200 mg / g dry weight of β-glucan with a molecular weight of 10-50 kDa, and has a dissolution rate of ≥70% in simulated fish intestinal fluid.

5. A method for preparing a fermented high-astaxanthin microalgae complex for aquaculture as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Haematococcus pluvialis was cultured using the gradient nitrogen deficiency-pulse carbon supplementation method to obtain algal powder with high astaxanthin content. S2. The high-astaxanthin algal powder is subjected to cell wall breaking treatment. S3. Mix the cell wall broken algae slurry with the plant lactobacillus and co-ferment under anaerobic conditions to obtain the fermentation product; S4. The fermentation product is dried and pulverized to obtain the fermented high astaxanthin microalgae complex for aquaculture.

6. The use of a fermented high-astaxanthin microalgae complex for aquaculture as described in any one of claims 1 to 4 in the preparation of an immune enhancer for aquatic animals.

7. An aquaculture feed, characterized in that, The fermented high-astaxanthin microalgae complex comprising any one of claims 1 to 4 is added at an amount of 0.5% to 3.0% of the total weight of the feed.

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