A kind of alginate lyase and its application in preparing pet food

Abstract

The present invention relates to an alginate lyase and an application thereof in the preparation of pet food. Specifically, the present invention improves the alginate degradation activity of the alginate lyase by evolving the alginate lyase, and utilizes the evolved alginate lyase to degrade undaria pinnatifida to efficiently prepare brown algae oligosaccharides. The brown algae oligosaccharides are used in the preparation of pet food, which can help pet hair growth, prevent hair loss, and improve hair gloss and toughness. The alginate lyase of the present invention has good application prospects in the field of pet food.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an alginate lyase and an application thereof in preparing pet food. Background Art

[0002] Algin is abundant in brown algae and widely exists in the cell walls and intercellular matrix of large algae such as undaria, kelp, and sargassum, accounting for about 40% of their dry weight. Algin is a general term for alginic acid (also known as alginic acid) and its derivatives. It is a linear high molecular polysaccharide formed by the β-1,4 glycosidic bond connection of L-guluronic acid (α-L-guluronic acid, G) and D-mannuronic acid (β-D-mannuronic acid, M) sugar units. According to the arrangement of G and M, it can be divided into polyguluronic acid segments (poly guluronic acid, polyG), polymannuronic acid segments (poly mannuronic acid, polyM), and heteropolysaccharide segments (polyMG) with alternating or random arrangement of MG.

[0003] Alginate degradation produces functional active oligosaccharides - brown algae oligosaccharides. As a multi-component mixture, brown algae oligosaccharides contain a variety of sugar units and have a variety of biological activities, including antiviral and immune regulation. For pets, brown algae oligosaccharides can be used as a nutritional additive in pet food. The rich nutrients it contains contribute to the healthy growth of pets, and its biological activity may have a positive impact on the health of pets' fur.

[0004] Brown algae oligosaccharides may achieve the effect of beautifying pet fur by promoting the metabolism and repair of pet skin cells, improving the gloss and texture of fur. In addition, the antioxidant and anti-inflammatory effects of brown algae oligosaccharides may also help reduce pet skin problems such as dandruff and dermatitis, further improving the health of pet fur.

[0005] Chinese patent application CN103689266A discloses a pet feed additive containing seaweed polysaccharides, which can increase the resistance of pets, promote hair growth, and reduce the incidence of skin diseases; Chinese patent application CN103315152A also discloses a pet feed containing seaweed powder, which helps prevent hair loss; in addition, Chinese patent application CN108740458A also discloses a cat food containing seaweed powder, which helps prevent hair loss.

[0006] Although the above patent applications all mention the use of seaweed polysaccharides or seaweed powder to prepare feed for preventing pet hair loss, they do not mention the preparation of seaweed powder or seaweed polysaccharides. This application improves the efficiency of preparing brown algae oligosaccharides using kelp by improving alginate lyase, and then uses the prepared brown algae oligosaccharides in pet food, which helps to increase the toughness and gloss of pet hair and reduce hair loss. Summary of the invention

[0007] In one aspect, the present invention provides an alginate lyase, the amino acid sequence of the alginate lyase is shown as SEQ ID No. 3.

[0008] The alginate lyase of the present invention is evolved from a parent alginate lyase. Compared with the parent alginate lyase, the activity of the alginate lyase of the present invention is increased by nearly 30%.

[0009] On the other hand, the present invention also provides a DNA encoding the above-mentioned alginate lyase, preferably, its DNA sequence is shown as SEQ ID No.4.

[0010] On the other hand, the present invention also provides a recombinant expression vector comprising the DNA, and a host cell comprising the recombinant expression vector.

[0011] In one embodiment, the host cell is selected from Pichia pastoris, Escherichia coli or Bacillus subtilis.

[0012] On the other hand, the present invention also provides a method for preparing the above-mentioned alginate lyase, the method comprising the step of fermenting the alginate lyase using the above-mentioned host cell; optionally, also comprising the step of purifying the alginate lyase.

[0013] On the other hand, the present invention also provides a method for degrading alginate, comprising the step of treating alginate with the alginate lyase.

[0014] In one embodiment, the alginate is derived from Undaria pinnatifida.

[0015] On the other hand, the present invention also provides a method for preparing brown algal oligosaccharides, which comprises the step of treating Undaria pinnatifida with the above-mentioned alginate lyase.

[0016] In one embodiment, the pH of the treatment is 6-8, preferably 7; the temperature of the treatment is 30°C-40°C, preferably 35°C; and the time of the treatment is 4h-12h, preferably 6h.

[0017] In one embodiment, the method for preparing brown algae oligosaccharides comprises the following steps:

[0018] (1) mixing dried kelp with phosphate buffer; preferably, the dried kelp and phosphate buffer are soaked and mixed at a mass:volume ratio of 10% to 20%;

[0019] (2) adding alginate lyase to the mixed system of step (1) to perform enzymatic hydrolysis reaction; preferably, the final concentration activity of alginate lyase (alginate lyase activity) is 200 U / mL-1000 U / mL;

[0020] (3) After the enzymatic hydrolysis reaction is completed, the kelp hydrolyzate is collected, and the supernatant is collected by centrifugation. The supernatant is the liquid containing brown algae oligosaccharides;

[0021] Optionally, the method further comprises the following steps:

[0022] (4) Drying the supernatant of step (3) to obtain brown algae oligosaccharide powder.

[0023] In one embodiment, the step (4) comprises: concentrating the supernatant by rotary evaporation to obtain a concentrated solution, adding anhydrous ethanol to the concentrated solution and collecting the precipitate, and freeze-drying the collected precipitate to obtain brown algae oligosaccharide powder.

[0024] On the other hand, the present invention also provides the use of brown algae oligosaccharides prepared by the above method in the preparation of pet food additives.

[0025] On the other hand, the present invention also provides a method for preparing a pet food additive, the method comprising:

[0026] (1) treating Undaria pinnatifida with the alginate lyase to obtain brown algal oligosaccharides;

[0027] (2) The brown algae oligosaccharide obtained in step (1) is mixed with other ingredients to prepare a pet food additive, wherein the other ingredients include L-cysteine, deep-sea fish oil, vitamin B12 and zinc citrate.

[0028] On the other hand, the present invention also provides a pet food additive, which includes the brown algae oligosaccharide prepared by the above method, and optionally, the pet food additive also includes L-cysteine, deep-sea fish oil, vitamin B12 and zinc citrate.

[0029] In one embodiment, the content of the brown algae oligosaccharide in the pet food additive is 40%-50%, preferably 40%; the content of the deep-sea fish oil in the pet food additive is 40%-60%, preferably 45%; the content of L-cysteine ​​or vitamin B12 or zinc citrate in the pet food additive is 2%-10%, preferably 5%; the above contents are all weight percentages.

[0030] On the other hand, the present invention also provides the use of the brown algae oligosaccharide prepared by the above method or the above pet food additive in the preparation of pet food or pet feed that is beneficial to pet hair growth.

[0031] The above benefits for hair growth include preventing hair loss, improving hair gloss and toughness, etc.

[0032] In one embodiment, the pet is selected from cats and dogs, preferably, cats.

[0033] The invention improves the alginate degradation activity of alginate lyase by evolving it, and utilizes the evolved alginate lyase to degrade undaria pinnatifida to efficiently prepare brown algae oligosaccharides; and the brown algae oligosaccharides are used to prepare pet food, which can help pet hair growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 .Comparison of enzyme activities of different alginate lyases.

[0035] Figure 2 .TLC analysis of the products of Undaria pinnatifida hydrolyzed by alginate lyase A51R.

[0036] Figure 3 .The brown algae oligosaccharide product obtained by hydrolyzing kelp with alginate lyase A51R. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0038] In order to make the experimental contents and technical solutions of the present invention more clearly described, the present invention is described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0039] Example 1. Optimization and improvement of alginate lyase

[0040] In the previous research (Chinese patent application number CN202311345083.3), the applicant increased the expression level of alginate lyase by optimizing the alginate lyase gene sequence. The amino acid sequence of alginate lyase is shown in SEQ ID No. 1, and the optimized nucleic acid sequence is shown in SEQ ID No. 2.

[0041] SEQ ID No.1 (algin lyase):

[0042] mksklvnivgsavllssfaahsaevnlvnpsfeqdfsgwtevdptavsgvaydgaksakfsgngarleqsvpvtsnteytlsayvladanigvevgsdtfsktasnsdwaqttitfnsgdateitifgeysgaegrvdlfkltsseiidppttslpvfdldpalppsgnfdlldwkldlpvddngnasgdaqevkegelssgfenseffytgddgglvfispvegattsantkytrsemremlrrgdtsisttgitknnwvfasapsddqnnsggvdgvleatlavnavtttgdssqvgrvivgqihandepirlyyrllpghtkgslyfahepnedassdpeqfinligssasnasepedgialnelffyrievqgnqlivtikrddhedvtetvdmttsgydvsgqymyfkagvynqnnsgdptdyvqatfyyltnshdgyefp

[0043] SEQ ID No.2:

[0044]

[0045] The applicant further optimized and improved the amino acid sequence of the alginate lyase. Through sequence analysis, it was found that the alginate lyase includes a CBM family domain and a domain that exerts alginate cleavage activity; the modification of the CBM family domain may improve the binding ability of the alginate lyase to the substrate, thereby improving its efficiency in degrading alginate; the modification of the domain of alginate cleavage activity may also improve its efficiency in degrading alginate. The key amino acid sites of the CBM domain were predicted by bioinformatics, and the 51st amino acid A of SEQ ID No.1 was selected; the key amino acid sites of the domain of alginate cleavage activity were predicted by bioinformatics, and the 310th amino acid N of SEQ ID No.1 was selected. Using conventional techniques in the art, the 51st amino acid A was mutated to D or R, and the 310th amino acid N was mutated to K or H, and the mutant alginate lyases A51D, A51R, N310K, and N310H were obtained.

[0046] Specifically, the variant of alginate lyase is generated by site-directed mutagenesis based on PCR. The specific method is to divide the DNA sequence of alginate lyase (SEQ ID No. 2) into two parts with the mutation site as the center, design two pairs of primers to amplify the two parts of the DNA sequence respectively, and introduce the sequence to be mutated on the primers at the same time, and finally load the two fragments into the pcDNA3.3-eGFP vector by Gibson cloning. Fragment amplification kit: TransStart FastPfu DNAPolymerase (containing 2.5mM dNTPs), the specific experimental process is detailed in the instructions. Gel recovery kit: GelDNAExtraction Mini Kit, please refer to the instruction manual for the specific experimental process. Kit used for vector construction: pEASY-BasicSeamless Cloning and Assembly Kit (CU201-03), please refer to the instruction manual for the specific experimental process.

[0047] The sequence of the alginate lyase obtained in this embodiment is shown in the following table:

[0048] Alginate lyase name Amino acid sequence information WT SEQ ID No.1 A51D SEQ ID No.1 position 51 A mutated to D A51R SEQ ID No.1, position 51, A mutated to R N310K SEQ ID No.1 No. 310 N mutated to K N310H SEQ ID No.1 No. 310 N mutated to H

[0049] In other embodiments, those skilled in the art may also obtain the protease sequence by other operation methods in the art, for example, obtaining the protease sequence by de novo synthesis.

[0050] Example 2. Expression and activity verification of alginate lyase

[0051] The alginate lyase WT, A51D, A51R, N310K, and N310H in Example 1 were expressed and purified in a manner similar to that of the Chinese patent application (application number: CN202311345083.3), and the DNA sequence was obtained by performing site-directed mutagenesis based on the mutated amino acid site based on the sequence shown in SEQ ID No. 2.

[0052] In this example, the Pichia pastoris expression strain Pichia pastoris X-33 was used, and the expression vector was pPICZαA.

[0053] Single colonies of fresh recombinant yeast strains expressing alginate lyase WT, A51D, A51R, N310K, and N310H correctly and control yeast strains without alginate lyase gene growing on YPD plates were picked and inoculated into BMGY medium respectively. They were cultured at 30°C and 200 rpm overnight, and then the cells were transferred into 50 mL BMMY medium to a concentration of OD 600 The pH value was about 1.0, and the culture was continued at 30°C and 200rpm. 1% methanol was added every 24h to induce expression for 120h. The supernatant obtained by centrifugation was the crude enzyme solution of alginate lyase WT, A51D, A51R, N310K or N310H. The results showed that the above alginate lyases could be successfully expressed. The crude enzyme solutions were diluted to 0.2mg / ml for later use.

[0054] The enzyme activity was determined by referring to the method of Chinese patent application (application number: CN202311345083.3): substrates (1% alginate (MV-Algin, LV-Algin), polyG and polyM) were prepared with pH 7.0, 50 mM phosphate buffer, 900 μL of the above substrates were added to 1.5 mL centrifuge tubes, and then 100 μL of alginate lyase WT, A51D, A51R, N310K or N310H crude enzyme solution of appropriate dilution was added, mixed quickly and evenly, reacted at 35 ° C for 10 min, and the reaction was terminated by boiling in a boiling water bath for 10 min. The inactivated enzyme-substrate-buffer reaction system was used as a blank control to measure OD 235 One activity unit (U) is defined as the OD per minute under the optimal reaction conditions (pH 7.0, temperature 35°C). 235 The enzyme amount required to increase the absorbance by 0.1. The results of enzyme activity test of crude enzyme solution of alginate lyase WT, A51D, A51R, N310K or N310H are as follows Figure 1 As shown. Figure 1As shown, compared with WT, the activities of N310K and N310H were significantly reduced, indicating that the mutation of amino acid position 310 would seriously affect the activity of alginate lyase. The activity of A51D was comparable to that of WT, with almost no change, but the activity of A51R was significantly improved compared with WT. Next, A51R will be used for more analysis and research.

[0055] The amino acid sequence of A51R is shown in SEQ ID No.3, and the DNA sequence thereof is shown in SEQ ID No.4.

[0056] SEQ ID No.3:

[0057] mksklvnivgsavllssfaahsaevnlvnpsfeqdfsgwtevdptavsgvrydgaksakfsgngarleqsvpvtsnteytlsayvladanigvevgsdtfsktasnsdwaq ttitfnsgdateitifgeysgaegrvdlfkltssseiidppttslpvfdldpalppsgnfdlldwkldlpvddngnasgdaqevkegelssgfenseffytgddgglvfispv egattsantkytrsemremlrrgdtsisttgitknnwvfasapsddqnnsggvdgvleatlavnavtttgdssqvgrvivgqihanndepirlyyrllpghtkgslyfahep nedassdpeqfinligssasnasepedgialnelffyrievqgnqlivtikrddhedvtetvdmttsgydvsgqymyfkagvynqnnsgdptdyvqatfyyltnshdgyefp

[0058] SEQ ID No.4

[0059]

[0060] Example 3. Degradation of Undaria pinnatifida by alginate lyase A51R

[0061] In this example, dried kelp was purchased from Dalian Yongtai Food Co., Ltd.; standard saturated α-L-guluronic acid oligosaccharide was purchased from Qingdao Bozhihuili Biotechnology Co., Ltd.; the chromatography plate TLC Silica gel60F254 used for thin layer chromatography was purchased from Sigma-aldrich; other reagents can be purchased from ordinary biochemical reagent companies.

[0062] The dried kelp was soaked and mixed with pH 7.0, 50 mM phosphate buffer at a mass: volume ratio of 14%, and alginate lyase A51R (final alginate lyase activity of 400 U / mL) was added at a volume ratio of 12% of the crude alginate lyase solution and the pH 7.0, 50 mM phosphate buffer. The stirring speed was adjusted to 180-200 rpm / min and the temperature was controlled to 35° C. The enzymatic hydrolysis reaction of the kelp was carried out, and the inactivated alginate lyase-substrate (kelp)-buffer reaction system was used as a blank control.

[0063] The kelp degradation reaction system after 6 hours of enzymatic reaction was filtered with a 60-mesh stainless steel sample sieve, and the kelp hydrolyzate and undegraded kelp residue were collected. The undegraded kelp residue was placed in a 105°C oven for drying. The reaction system with inactivated alginate lyase was used as a control. The calculation formula for the degradation rate of kelp hydrolyzed by alginate lyase is as follows:

[0064] M=(1-W T / W C )*100%

[0065] in:

[0066] M: Undaria pinnatifida degradation rate (%);

[0067] W T : dry weight of undegraded kelp after alginate lyase treatment (g);

[0068] W C : Dry weight of remaining kelp in control group (g).

[0069] The kelp hydrolyzate obtained by filtration was centrifuged at 12000rpm for 5min, and the supernatant was collected. The uronic acid content in the kelp hydrolyzate was determined by the m-hydroxybiphenyl method. 25μL of kelp hydrolyzate diluted to an appropriate dilution was added to a 1.5mL centrifuge tube in an ice bath, and then 125μL of the cooled borate solution was added dropwise. After vortexing and mixing, it was boiled in a water bath for 15min, taken out and centrifuged at 12000rpm for 1min to collect all the liquid at the bottom of the tube, 2μL of m-hydroxybiphenyl solution was added to the ice bath, mixed and placed at room temperature for 30min, and the absorbance was detected at 525nm using an enzyme marker. According to the above operation steps, the absorbance values ​​of 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg / mL glucuronic acid standard solutions at 525 nm were measured. The concentration of the glucuronic acid standard solution was used as the abscissa and the absorbance value was used as the ordinate to establish a standard curve. The linear regression equation was obtained: y = 1.0223x + 0.0048, R 2 =0.999. According to the established standard curve regression equation and the detected absorbance value, the uronic acid content in the hydrolyzate obtained by degrading the kelp with alginate lyase was calculated.

[0070] The results showed that alginate lyase A51R has a high efficiency in degrading natural kelp. A single enzyme can degrade untreated kelp into brown algae oligosaccharides within 6 hours. With the increase of hydrolysis time, the degradation rate of kelp continued to increase. The final degradation rate can reach 62.15%, and the uronic acid production can reach 63.21 mg / ml, which is the best effect of a single enzyme in degrading natural kelp in the shortest time reported so far.

[0071] The soluble product obtained by hydrolyzing kelp with alginate lyase A51R was analyzed by thin layer chromatography (TLC) to clarify the composition of the brown algae oligosaccharide product. Commercially available guluronic acid oligosaccharides (G1-G7) were used as standard controls, and the inactivated alginate lyase-substrate-buffer reaction system was used as a blank control. The specific method is as follows:

[0072] (1) Spotting: Take 1 μL of the hydrolyzate with appropriate dilution and drop it onto the thin layer chromatography plate. Keep the spot diameter at about 1 mm and the spot distance at 1 cm. After spotting, place it in a 100°C oven to dry for 1-2 minutes.

[0073] (2) Development: Add a developing agent: a mixture of n-butanol, acetic acid, and water (2:1:2, v / v / v) to the chromatography cylinder, seal it and place it for 15 minutes for pre-equilibration, use tweezers to pick up the spotted chromatography plate and place it obliquely in the chromatography cylinder, seal it and develop for 2-3 hours. When the development reaches 1-2 cm from the front line, take out the thin layer plate and place it in a 65°C oven to dry the developing agent.

[0074] (3) Color development: Use sulfuric acid-ethanol solution as the color developer (concentrated sulfuric acid: ethanol = 1:19, v / v). Slowly push the dried chromatography plate downward to allow the color developer to evenly and fully stain the chromatography plate. Then place the chromatography plate in a 105°C oven for 10 min and observe after color development.

[0075] The analysis results of the brown algae oligosaccharide products obtained by hydrolyzing kelp with alginate lyase A51R are as follows Figure 2 As shown in the figure, the main oligosaccharide components of kelp hydrolyzed by alginate lyase are unsaturated fucoidan and trisaccharide, and a small amount of saturated fucoidan components. The low-polymerization degree fucoidan oligosaccharide components in the hydrolyzate of kelp have better biological activity and more application prospects.

[0076] The Undaria pinnatifida hydrolyzate obtained above was concentrated to 1 / 5 of the original volume by rotary evaporation at 55-60° C. and 50-60 rpm / min to obtain the Undaria pinnatifida concentrated hydrolyzate.

[0077] Add 3 times the volume of anhydrous ethanol to the above concentrated hydrolyzate, stir evenly and let it stand at 4°C for 12 hours, then centrifuge at 4000 rpm for 30 minutes, remove the supernatant, and collect and combine the precipitates.

[0078] The collected precipitate is freeze-dried for 48 hours to obtain brown algae oligosaccharide. The obtained product is as follows Figure 3 shown.

[0079] Example 4. Preparation of pet food using brown algae oligosaccharides obtained in Example 3

[0080] In this embodiment, the brown algae oligosaccharide obtained in Example 3 was used to prepare a pet food additive; the brown algae oligosaccharide prepared by hydrolyzing kelp using alginate lyase A51R in Example 3 was used as the experimental group. At the same time, the brown algae oligosaccharide prepared by hydrolyzing kelp using inactivated alginate lyase A51R (high temperature inactivated) was used as the control group in the same manner as in Example 3.

[0081] The pet food additive in this embodiment includes the following ingredients (the amount of each substance is provided on a weight percentage basis):

[0082] Ingredients Content (wt%) Brown algae oligosaccharide-experimental group or brown algae oligosaccharide-control group 40 Deep sea fish oil 45 L-Cysteine 5 <![CDATA[Vitamin B 12 > 5 Zinc Citrate 5

[0083] L-cysteine, deep-sea fish oil, vitamin B in the above food additives 12 Both zinc citrate and zinc citrate are commercially available.

[0084] The above pet food additive (experimental group or control group) was added to the daily cat food at a ratio of 2% (wt%), that is, 20 grams of the above pet food additive was contained in every 1 kg of cat food.

[0085] The above cat food with food additives was used for feeding test of adult cats, and the hair growth was recorded. Each cat food was fed to 15 adult cats, and the hair growth of adult cats was recorded. The results were tracked for 480 hours, and the hair quality of each adult cat was scored according to the following scoring criteria.

[0086] Scoring criteria: 1-3 points: Partial hair loss occurs, and 1-3 points are scored according to the degree of hair loss. The more severe the hair loss, the lower the score; 4-7 points: Hair loss is significantly improved, and 4-7 points are scored according to the hair quality. The better the hair quality, the higher the score; 8-10 points: Hair is smooth, grows vigorously, has no hair loss, is tough and shiny, and the better the overall hair quality, the higher the score.

[0087] The results are shown in the table below. The hair quality of the experimental group cats is significantly better than that of the control group cats. This shows that the use of alginate oligosaccharides prepared by hydrolyzing kelp with alginate lyase A51R for pet food can help alleviate pet hair loss and improve the hair quality of pets.

[0088] Group Average score Experimental Group 8.3±0.99 Control group 6.2±0.98

[0089] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. An alginate lyase, the amino acid sequence of which is shown in SEQ ID No.

3.

2. A DNA encoding the alginate lyase according to claim 1.

3. A recombinant expression vector comprising the DNA of claim 2.

4. A host cell comprising the recombinant expression vector according to claim 3.

5. A method for degrading alginate, comprising the step of treating alginate with the alginate lyase according to claim 1.

6. A method for preparing brown algal oligosaccharides, comprising the step of treating Undaria pinnatifida with the alginate lyase according to claim 1.

7. The method according to claim 6, characterized in that The pH of the treatment is 6-8, the temperature of the treatment is 30° C.-40° C., and the time of the treatment is 4 h-12 h.

8. The method according to claim 6, characterized in that The method comprises the following steps: (1) Mix dried kelp and phosphate buffer; (2) adding alginate lyase to the mixed system in step (1) to perform enzymatic hydrolysis reaction; (3) After the enzymatic hydrolysis reaction is completed, the kelp hydrolyzate is collected, and the supernatant is collected by centrifugation. The supernatant is the liquid containing brown algae oligosaccharides; Optionally, the method further comprises the following steps: (4) Drying the supernatant of step (3) to obtain brown algal oligosaccharide powder.

9. A method for preparing a pet food additive, the method comprising: (i) preparing brown algal oligosaccharide using the method according to any one of claims 6 to 8; (ii) preparing a pet food additive by mixing the brown algae oligosaccharide obtained in step (i) with other ingredients, wherein the other ingredients include L-cysteine, deep-sea fish oil, vitamin B12 and zinc citrate.

Images