Microalgal beverage additive and method of making

Abstract

The present application belongs to the field of biological feed, and particularly relates to a kind of microalgae beverage additive and a preparation method thereof; the steps are as follows: firstly, inoculate microalgae in logarithmic phase in microalgae culture medium for culture, obtain microalgae culture solution after culture; then, the microalgae culture solution is ultrasonically treated, obtain the microalgae culture solution after ultrasonic treatment, and fill CO2 in the culture solution, obtain CO2 mixed culture solution; finally, add inulin, fructooligosaccharide and psyllium husk powder in the CO2 mixed culture solution, mix uniformly, obtain the microalgae beverage additive. The present application can retain various nutrients to the greatest extent; at the same time, assisted by ultrasonic treatment, it is beneficial to the exudation of intracellular substances, combined with the addition of CO2 to improve the taste of the beverage, and provide conditions for the survival of active microalgae. The preparation method is simple, green and environmentally friendly; the prepared microalgae beverage additive can be added with daily drinking water, promote the growth of dairy cows and improve the quality of cow milk, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological feed, specifically relating to a microalgae beverage additive and its preparation method. Background Technology

[0002] Microalgae, as photosynthetic plants, are rich in valuable nutrients such as proteins, lipids, algal polysaccharides, β-carotene, and various inorganic elements. Microalgae have the advantages of short growth cycles and rapid proliferation; moreover, as aquatic plants, they have low dependence on soil.

[0003] Chlorella is a single-celled freshwater algae with advantages such as high protein, low fat, low sugar, low calories, and rich content of vitamins and minerals. Micrococcus pluvialis is a single-celled marine algae rich in EPA, β-carotene, etc., and can accumulate large amounts of EPA in a very short time, exhibiting high ecological and economic value.

[0004] Currently, microalgae has been studied as a feed additive, as exemplified by patent CN107494985A. This involves preparing feed pellets and then adhering microalgae powder to the surface of the pellets or encapsulating the pellets with algae powder to create microalgae feed. However, the equipment required for drying and pulverizing microalgae is expensive, and high-temperature processing can destroy nutrients such as protein and β-carotene, reducing the nutritional value of the microalgae. Furthermore, algae powder presents inconveniences in use, such as its small size and light weight, making it susceptible to environmental influences when used outdoors. Additionally, the processing of microalgae powder involves numerous and cumbersome steps, requiring significant equipment investment and energy consumption, failing to fully meet the demands for simple and convenient production. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a microalgae beverage additive that uses active microalgae to retain various nutrients to the greatest extent and is fully digestible and usable by dairy cows. This microalgae beverage additive can be consumed by dairy cows for a long time, improving milk quality and enhancing the physical condition of calves.

[0006] To achieve the above technical objectives, the present invention adopts the following solution:

[0007] A method for preparing a microalgae beverage additive, specifically including the following steps:

[0008] (1) Microalgae culture: First, microalgae in the logarithmic growth phase are inoculated in a microalgae culture medium and cultured. After the culture is completed, a microalgae culture solution is obtained. The culture conditions are: light intensity of 3000-5000 Lux, temperature of 16-30℃, and culture time of 7-14 days.

[0009] (2) Ultrasonic treatment: The microalgae culture medium obtained in step (1) is ultrasonically treated at 100-300W for 5-30 minutes under certain temperature conditions to obtain the ultrasonically treated microalgae culture medium; the ultrasonic treatment damages the cell wall of the microalgae, and the internal substances of the cells seep out while the microalgae still maintain their activity.

[0010] (3) Introducing CO2: Introduce 2-4 times the volume of the microalgae culture medium with a concentration of 0.5%-5% CO2 into the ultrasonically treated microalgae culture medium to obtain a CO2 mixed culture medium;

[0011] (4) Adding additives: Based on the mass of the CO2 mixed culture medium, add 0.5%-1.5% of inulin, 0.25%-0.5% of fructooligosaccharides and 2%-5% of plantain husk powder to the CO2 mixed culture medium, mix evenly, and you will get the microalgae beverage additive.

[0012] Preferably, the microalgae mentioned in step (1) include *Microcystis aeruginosa* and *Chlorella vulgaris*; wherein the microalgae culture medium corresponding to *Microcystis aeruginosa* is composed of artificial seawater or seawater and f / 2 culture nutrient components; the f / 2 culture nutrient components are NaNO3 75 mg / L, NaH2PO4·H2O 5 mg / L, Na2SiO3·9H2O 30 mg / L, FeCl3·6H2O 3.15 mg / L, and Na2EDTA·2H2O 4.36 mg / L;

[0013] The microalgal culture medium for Chlorella consists of: CO(NH2)2 300 mg / L, KH2PO4 60 mg / L, MgSO4·7H2O 60 mg / L, FeSO4·7H2O 1 mg / L, Na2SiO3·9H2O 4 mg / L, CaCl2·2H2O2 2 mg / L, MgCl2·6H2O 1 mg / L, and distilled water to a final volume of 1 L.

[0014] Preferably, the inoculum amount of microalgae in step (1) is 0.2 g / L.

[0015] Preferably, the culture temperature in step (1) is 22.4℃ and the culture time is 12 days.

[0016] Preferably, the temperature mentioned in step (2) is 20-25℃.

[0017] Preferably, in step (2), the ultrasonic power is 100W and the ultrasonic time is 5min; the ultrasonic treatment method is ultrasonic for 5s, intermittent for 5s, and so on.

[0018] Preferably, in step (3), the CO2 concentration is 3% and the filling volume is twice the volume of the microalgae culture medium.

[0019] Preferably, the amount of additives used in step (4) is: 1% inulin, 0.5% fructooligosaccharide and 5% plantain husk powder by mass of the CO2 mixed culture medium.

[0020] The microalgae beverage additive prepared by the above method is used as or in the preparation of animal feed additives.

[0021] The microalgae beverage additive is used to feed dairy cows to promote their growth and improve the quality of their milk. The specific operation is as follows: the microalgae beverage additive is added to the drinking water used to feed the dairy cows, and the amount added is 1% to 5% by volume of the drinking water.

[0022] The beneficial effects of this invention are:

[0023] (1) This invention provides a method for preparing a microalgae beverage additive. The additive is prepared using active microalgae and can retain various nutrients to the greatest extent. At the same time, it is supplemented by ultrasonic treatment, which is conducive to the exudation of intracellular substances. It also combines the addition of CO2 to improve the taste of the beverage and provide conditions for the survival of active microalgae.

[0024] (2) In the process of preparing the microalgae beverage additive, this invention also adds psyllium husk powder, fructooligosaccharides, and inulin. Under normal circumstances, beneficial bacteria such as lactobacilli and bifidobacteria in the intestinal microbiota play an important role in protecting the body's health. Feeding dairy cows an appropriate amount of fructooligosaccharides has a significant effect on the abundance of intestinal microbiota, significantly increasing the relative abundance of Bacteroides and significantly decreasing the relative abundance of Clostridium, thereby significantly reducing the occurrence of diarrhea. Fructooligosaccharides can also remove some of the fishy smell of microalgae, making microalgae more acceptable to dairy cows. Inulin can increase the number and activity of bifidobacteria and lactobacilli in the colon, thereby protecting intestinal health. Psyllium husk powder contains 70%-80% water-soluble dietary fiber, which is 8 times that of the same weight of oat bran. As a water-soluble dietary fiber, psyllium husk powder has good physiological effects, which can promote the excretion of animal urine, reduce uric acid levels, promote gastric juice secretion and intestinal digestion, improve the immunity of cattle, and also promote intestinal peristalsis and reduce blood lipids. The addition of plantain husk powder, fructooligosaccharides, and inulin allows for a more comprehensive retention of nutrients in the liquid active microalgae.

[0025] (3) This invention takes into account dosage control. First, the ultrasonic power is controlled at 100W and the ultrasonic time is 5min. Under these conditions, the lethality of microalgal cells and the leakage of intracellular substances can be better controlled. Second, the addition of 2% CO2 maximizes the activity of microalgae and provides conditions for the survival of active microalgae. Finally, the addition of 1% inulin, 0.5% fructooligosaccharides and 5% plantain husk powder achieves the best effect under specific dosage control, resulting in a microalgae beverage additive with good stability, palatability, no obvious clumping or algal odor. It can significantly improve the diversity and structure of the intestinal microbiota of dairy cows, increase the abundance of beneficial microorganisms in the dairy cow intestine, and reduce diarrhea. Combined with feeding trials, it is known that it significantly increases the DHA content in milk, increases the content of unsaturated fatty acids in milk, and improves the quality of milk.

[0026] (4) The method of the present invention is simple, requires little equipment investment, and is more environmentally friendly; the microalgae beverage additive prepared can be added with daily drinking water, making it more convenient to use and having broad market application prospects. Attached Figure Description

[0027] Figure 1 This is the growth curve of microalgae.

[0028] Figure 2 The cell wall damage rate of *Microcystis globulus* is shown.

[0029] Figure 3 The cell wall damage rate of Chlorella.

[0030] Figure 4 The lethality rate of *Microcystis globulus* cells.

[0031] Figure 5 The lethality rate of Chlorella cells.

[0032] Figure 6 This represents the change in the quality of milk from lactating cows.

[0033] Figure 7 For calf growth performance. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] Culture medium:

[0039] (1) Microcystis culture medium: composed of seawater and f / 2 culture nutrient components; the seawater was brought to a final volume of 1L, wherein the f / 2 culture nutrient components were NaNO3 75mg / L, NaH2PO4·H2O 5mg / L, Na2SiO3·9H2O 30mg / L, FeCl3·6H2O 3.15mg / L, and Na2EDTA·2H2O 4.36mg / L.

[0040] (2) Chlorella culture medium: The composition is CO(NH2)2 300mg / L, KH2PO4 60mg / L, MgSO4·7H2O 60mg / L, FeSO4·7H2O 1mg / L, Na2SiO3·9H2O 4mg / L, CaCl2·2H2O2 2mg / L, MgCl2·6H2O 1mg / L, and diluted to 1L with distilled water.

[0041] After preparing the culture media according to the components of (1) and (2), sterilize them at 121℃ for 20 minutes and set them aside.

[0042] The specific selection process for the technical solutions is as follows:

[0043] 1. Determination of microalgae culture time

[0044] Microalgae selected were *Chlorella vulgaris* and *Microcystis microsporum*, inoculated at a rate of 0.2 g / L each in the logarithmic growth phase. Microalgae growth curves were monitored to determine the optimal growth time. The microalgae were cultured in a constant temperature and light incubator at a set temperature of 22.4℃ and a light intensity of 3000 lux, with daily shaking to obtain microalgae growth curves, as shown below. Figure 1 As shown. According to production needs, the growth cycle should not be too long, and the microalgal biomass should not be too low. The microalgal biomass increases rapidly from 0 to 12 days after inoculation, reaches its peak at 12 days, and then (until 14 days) the biomass is basically stable. Therefore, the optimal culture time is determined to be 12 days.

[0045] 2. Ultrasonic treatment

[0046] After 12 days of microalgae culture, the microalgae culture medium was harvested. Different ultrasonic powers were used to treat the microalgae. By varying the ultrasonic power and time, the cell wall damage rate, cell mortality rate, lipid extraction rate after ultrasound, and small molecule active substance exudation rate after ultrasound were measured to determine the optimal power and time. Specific results are shown in [link to results]. Figure 2-5 ;

[0047] First, through Figure 2-3 It is evident that ultrasonic power is crucial for both *Microcystis aeruginosa* and *Chlorella vulgaris*. At 100W, the cell wall damage rate of microalgae is highest; excessively high or low power will affect the effectiveness. Secondly, through... Figure 4-5 It is known that the mortality rate of microalgal cells increases with increasing ultrasonic power; the mortality rate of microalgal cells is relatively low when the ultrasonic power is between 50-100W. However, at the same time, the higher the cell wall damage rate, the more conducive it is to the leakage of intracellular substances, which is an important reference indicator. Therefore, considering all factors, an ultrasonic power of 100W was selected.

[0048] Finally, the effect of ultrasound time was investigated when the ultrasound power was controlled at 100W. The results showed that the cell mortality rates of microalgae were 0%, 10.3%, and 27.5% when the ultrasound time was 0, 5 min, and 10 min, respectively. The ultrasound time should not be too long, so the ultrasound time was finally selected as 5 min. The ultrasound treatment method was: 5 seconds of ultrasound followed by 5 seconds of interval.

[0049] 3. Inflate with CO2

[0050] (1) 600 mL of the microalgae solution after sonication (100 W, 5 min) was taken and divided into 3 equal portions of 200 mL each. The portions were then filled with CO2 at 2, 3, and 4 times the volume of the algae solution, respectively, and the activity of the microalgae cells was compared. The results showed that the microalgae activities corresponding to the CO2 volume from low to high were 0.32, 0.25, and 0.22, respectively. This indicates that excessive CO2 will affect the activity of the microalgae. Therefore, the CO2 volume was selected to be 2 times the volume of the algae solution.

[0051] (2) Measure 600 mL of the sonicated microalgae solution (sonication 100 W, 5 min), and divide it into three equal portions of 200 mL each. The first portion was filled with CO2 at twice the volume of the algae solution, with a CO2 concentration of 1%. The second portion was filled with CO2 at twice the volume of the algae solution, with a CO2 concentration of 3%. The third portion was filled with CO2 at twice the volume of the algae solution, with a CO2 concentration of 5%. The activity of the microalgae cells was then compared. The measured CO2 concentrations from low to high corresponded to microalgae activities of 0.27, 0.29, and 0.26, respectively. Therefore, a CO2 concentration of 3% was chosen.

[0052] Example 1:

[0053] (1) Microalgae culture

[0054] Under aseptic conditions in a clean bench, Chlorella vulgaris and Micrococcus microcarpa in the logarithmic growth stage were inoculated into their respective culture media at an inoculum size of 0.2 g / L. They were then cultured for 12 days under conditions of 3000 Lux light and 22.4 °C, with the flasks shaken three times a day. After the culture was completed, the microalgae culture medium was obtained.

[0055] (2) Ultrasonic treatment: After the microalgae culture is completed, the microalgae culture medium is placed in an ultrasonic cell disruptor and ultrasonically treated at 25°C. The ultrasonic power is 100W and the total time is 5min. The treatment method is: ultrasonic for 5s, intermittent for 5s, and repeated to obtain the ultrasonically treated microalgae culture medium. Ultrasonic treatment damages the cell wall of microalgae and causes the internal substances of the cells to seep out, while the microalgae still maintain their activity.

[0056] (3) Introducing CO2: Introduce CO2 at a concentration of 3% and twice the volume of the microalgae culture medium into the ultrasonically treated microalgae culture medium to obtain a CO2 mixed culture medium;

[0057] (4) Preparation of microalgae beverage additives:

[0058] (a) In a CO2 mixed culture medium, add 0.5% of the mass of fructooligosaccharide and 5% of plantain husk powder to the CO2 mixed culture medium, and change the amount of inulin added to add 0.5%, 1%, 1.5% and 2.0% of inulin respectively; mix evenly to obtain microalgae beverage additive.

[0059] Observations on the obtained microalgae beverage additives revealed that when the addition amount was 0.5%, the microalgae beverage additive was not stable enough, and the microalgae would precipitate and agglomerate; when the addition amount was 1%, the microalgae beverage additive did not agglomerate and maintained good stability; when the addition amount was 1.5%, the stability was good and there was no agglomeration; when the addition amount was 2.0%, the microalgae beverage additive became viscous, which was not conducive to application.

[0060] (b) In the CO2 mixed culture medium, 1% of inulin and 5% of plantain husk powder by mass of the CO2 mixed culture medium were added, and the amount of fructooligosaccharides added was changed to 0.25%, 0.35%, 0.5% and 0.8% respectively; the mixture was mixed evenly to obtain the microalgae beverage additive.

[0061] When the addition amount is 0.25% and 0.35%, the algae beverage additive has a slightly strong algae smell; when the addition amount is 0.5%, the microalgae beverage additive has no obvious algae smell, and the beverage has a slightly sweet taste; when the addition amount is 0.8%, the sweetness of the microalgae beverage additive is obviously too strong; taking all factors into consideration, the addition amount of fructooligosaccharide is selected as 0.5%.

[0062] (c) In the CO2 mixed culture medium, 1% of inulin and 0.5% of fructooligosaccharides by mass of the CO2 mixed culture medium were added. The amount of plantain husk powder added was changed to 2%, 3.5%, 5% and 8% of plantain husk powder respectively, and mixed evenly to obtain microalgae beverage additive.

[0063] When the addition amount is 2%, the microalgae beverage additive is not stable enough, and the microalgae precipitate and agglomerate; when the addition amount is 3.5%, the microalgae beverage additive is basically stable, with slight layering; when the addition amount is 5%, the microalgae beverage additive is in a stable suspension state and does not exhibit layering or agglomeration; when the addition amount is 8%, the microalgae beverage additive becomes thick; because plantain husk powder has water-absorbing properties, adding too much will cause the microalgae beverage additive to lose moisture and become too thick, so a 5% addition amount of plantain husk powder is chosen.

[0064] In summary, the microalgae beverage additive obtained by adding 1% inulin, 0.5% fructooligosaccharides and 5% psyllium husk powder was the final product of Example 1. At this ratio, the microalgae beverage additive maintained good stability, without obvious clumping or algae-like odor, and had good palatability.

[0065] Application Example 1:

[0066] The test site was a dairy farm in Matou Town, Huai'an City, Jiangsu Province, where the microalgae beverage additive prepared in Example 1 was used for feeding trials.

[0067] Twenty healthy adult dairy cows were divided into four groups of five cows each. Groups B, C, and D had 1%, 3%, and 5% of microalgae beverage additive added to their daily drinking water, respectively. Group A served as the control group, and no microalgae beverage additive was added to their drinking water. All four groups of cows had free access to feed and water 24 hours a day. However, for groups B, C, and D, the microalgae beverage additive was added to their drinking water at 8:00 AM and 2:00 PM, respectively.

[0068] After 60 days of feeding, the milk quality of the cows was measured. The cows were milked three times a day, and the total milk quality was calculated. The changes in milk quality are shown in the table below. Figure 6 ;

[0069] from Figure 6 The results show that when the microalgae beverage additive was added at 1%, 3%, and 5%, the milk yield per cow increased, while the milk fat percentage and lactose percentage decreased compared to group A. At the same time, compared to group A, the DHA (docosahexaenoic acid) content in groups B, C, and D all increased by more than 10%. In addition, the results show that the effects of 3% and 5% addition were similar and had no significant impact. Therefore, 3% was selected as the optimal addition amount.

[0070] Application Example 2:

[0071] The test site was a dairy farm in Matou Town, Huai'an City, Jiangsu Province; the microalgae beverage additive prepared in Example 1 was used for feeding trials.

[0072] Twenty healthy calves were divided into four groups of five each: Groups B, C, and D had 1%, 3%, and 5% of microalgae beverage additive added to their daily drinking water, respectively; Group A served as the control group, and no microalgae beverage additive was added to their drinking water. All four groups of cows had free access to feed and water 24 hours a day, except for Groups B, C, and D, where the microalgae beverage additive was added to their drinking water at 8:00 AM and 2:00 PM, respectively.

[0073] The changes in calf growth performance were measured after 40 days of feeding. Figure 7 .

[0074] like Figure 7 As shown, when the microalgae beverage additive was added at 3% and 5%, the average daily weight gain (ADG) and body mass index (BMI) of calves increased, while the dry matter intake (DMI) decreased. For calves, the effects of 3% and 5% addition were similar and not substantially significant; therefore, 3% was chosen as the optimal addition level.

[0075] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a microalgae feed additive, characterized in that, The preparation steps are as follows: (1) Microalgae culture: First, microalgae in the logarithmic growth phase are inoculated in the microalgae culture medium and cultured. After the culture is completed, microalgae culture solution is obtained. The culture conditions are: light conditions of 3000-5000 Lux, temperature of 16-30℃, and culture time of 7-14 days. (2) Ultrasonic treatment: The microalgae culture medium obtained in step (1) is ultrasonically treated at 100W for 5 minutes at 20-25℃. The ultrasonic treatment method is 5 seconds of ultrasonic treatment followed by 5 seconds of intermittent treatment. This process is repeated to obtain the ultrasonically treated microalgae culture medium. (3) Introducing CO2: Introduce CO2 at a concentration of 3% and twice the volume of the microalgae culture medium into the ultrasonically treated microalgae culture medium to obtain a CO2 mixed culture medium; (4) Adding additives: Based on the mass of the CO2 mixed culture medium, add 1% of inulin, 0.5% of fructooligosaccharides and 5% of plantain seed husk powder to the CO2 mixed culture medium, mix evenly, and the microalgae feed additive is obtained.

2. The method for preparing a microalgae feed additive according to claim 1, characterized in that, The microalgae mentioned in step (1) include Microcystis and Chlorella; The microalgal culture medium corresponding to *Microcystis aeruginosa* consists of seawater and f / 2 culture nutrient components; the f / 2 culture nutrient components are: NaNO3 75 mg / L, NaH2PO4·H2O 5 mg / L, Na2SiO3·9H2O 30 mg / L, FeCl3·6H2O 3.15 mg / L, and Na2EDTA·2H2O 4.36 mg / L. The microalgal culture medium for Chlorella consists of the following components: CO(NH2)2 300 mg / L, KH2PO4 60 mg / L, MgSO4·7H2O 60 mg / L, FeSO4·7H2O 1 mg / L, Na2SiO3·9H2O 4 mg / L, CaCl2·2H2O 2 mg / L, MgCl2·6H2O 1 mg / L, and distilled water to a final volume of 1 L.

3. The method for preparing a microalgae feed additive according to claim 1, characterized in that, The inoculation amount of microalgae in step (1) is 0.2 g / L.

4. The method for preparing a microalgae feed additive according to claim 1, characterized in that, The culture temperature in step (1) is 22.4℃ and the culture time is 12 days.

5. The microalgae feed additive prepared according to any one of claims 1 to 4.

6. The use of the microalgae feed additive according to claim 5 for improving the quality of dairy cow milk.

7. The use according to claim 6, characterized in that, Add microalgae feed additives to the drinking water of dairy cows at a rate of 1% to 5% by volume of drinking water.

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