Agaro oligosaccharide as well as preparation method and application thereof

Abstract

The invention provides agaro-oligosaccharide as well as a preparation method and application thereof. The agaro-oligosaccharide is obtained by carrying out enzymolysis on a strain and agar; the bacterial strain comprises at least one of flavobacterium galactivorans and phycophagella albicans. The main component of the agaro-oligosaccharide is neoagarotetraose, and the agaro-oligosaccharide has lower polymerization degree, so that the agaro-oligosaccharide can be more easily absorbed by a human body.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to an agar oligosaccharide and a method for preparing the same. Background Art

[0002] Agar is the main component of the cell wall of red algae, a polymer composed of agarose and agaropectin. Agar has strong gel properties and cannot be directly absorbed and utilized by the human body. However, agar oligosaccharides formed through hydrolysis have good water solubility, are easily absorbed by the human body, and at the same time, agar oligosaccharides have good physiological activities. Agar oligosaccharides are linear chain molecules composed of alternating (1→3)-β-D-galactose residues and (1→4)-3,6-anhydro-α-L-galactose residues. According to the difference in the non-reducing end of agar oligosaccharides, agar oligosaccharides can be divided into neoagarooligosaccharides with a non-reducing end of 3,6-anhydro-L-galactose and agarooligosaccharides with a non-reducing end of D-galactose.

[0003] Currently, the main methods for preparing agar oligosaccharides are acid hydrolysis and enzymatic hydrolysis. During the acid hydrolysis process, it is more inclined to hydrolyze the α-1,3 glycosidic bond to produce agarooligosaccharides with a non-reducing end of D-galactose. The enzymatic method is used to prepare even-numbered neoagarooligosaccharides and even-numbered agarooligosaccharides. Among them, the preparation of odd-numbered agar oligosaccharides by chemical method, i.e., acid hydrolysis, has the advantages of rapid reaction and large substrate concentration for treatment. However, the products are more complex after acid hydrolysis, the separation is more difficult, and it cannot be used to prepare neoagarooligosaccharides with a non-reducing end of 3,6-anhydro-L-galactose. Compared with acid hydrolysis, the enzymatic method has the advantages of mild reaction, single product, and easy separation. However, due to the gel properties of agar, the substrate concentration for the enzymatic method is low, resulting in low yield.

[0004] Therefore, it is of great significance to develop a high-efficiency preparation technology for agar oligosaccharides. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent.

[0006] Accordingly, in the first aspect of the present invention, the present invention provides an agar oligosaccharide. According to an embodiment of the present invention, the agar oligosaccharide is obtained by enzymatically hydrolyzing agar with a strain; the strain includes at least one of Zobellia galactanivorans and Agarivorans albus. Through a large number of experiments, the inventors found that Zobellia galactanivorans and Agarivorans albus can be used to produce agar oligosaccharides, and during the enzymatic hydrolysis fermentation process, both Zobellia galactanivorans and Agarivorans albus have relatively high enzyme activities, which can improve the production efficiency of agar oligosaccharides and enhance the raw material conversion rate. In addition, the main component of the agar oligosaccharide of the present invention is neoagarotetraose, which has a low degree of polymerization, and thus can be more easily absorbed by the human body.

[0007] In the second aspect of the present invention, the present invention provides a method for preparing the agar oligosaccharide described in the first aspect. According to an embodiment of the present invention, the method includes: enzymatically hydrolyzing a strain with a first agar substrate to obtain the agar oligosaccharide, and the strain includes at least one of Zobellia galactanivorans and Agarivorans albus. Thus, by using the method of the present invention, the production efficiency of agar oligosaccharides can be improved, the raw material conversion rate can be enhanced, and the production cost and process complexity can be reduced.

[0008] In the third aspect of the present invention, the present invention provides the use of a strain in the preparation of agar oligosaccharides, and the strain includes at least one of Zobellia galactanivorans and Agarivorans albus.

[0009] In the fourth aspect of the present invention, the agar oligosaccharide described in the first aspect, or the agar oligosaccharide prepared by the method described in the second aspect has at least one of the following uses: use in the preparation of a drug for anti-inflammatory, anti-tumor, treating cardiovascular diseases, regulating blood sugar, and regulating the intestinal flora; use in the preparation of a health food; use in the preparation of a cosmetic for anti-ultraviolet and antioxidant.

[0010] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. Description of the Drawings

[0011] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0012] Figure 1 is a morphological observation result diagram of four strains according to Embodiment 1 of the present invention;

[0013] Figure 2It is a Lugol's iodine staining result diagram of four kinds of bacteria according to Embodiment 1 of the present invention;

[0014] Figure 3 It is a growth curve diagram of four kinds of bacteria in a fermentation medium according to Embodiment 1 of the present invention;

[0015] Figure 4 It is a comparison diagram of the relative enzyme activities of four kinds of bacteria according to Embodiment 1 of the present invention;

[0016] Figure 5 It is a diagram showing the change of agar oligosaccharide concentration with time during the continuous fermentation of Ag bacteria and Zg bacteria according to Embodiment 2 of the present invention;

[0017] Figure 6 It is a diagram showing the change of the relative content of agar oligosaccharide with time during the continuous fermentation of Ag bacteria and Zg bacteria according to Embodiment 2 of the present invention;

[0018] Figure 7 It is a diagram showing the change of relative enzyme activity with time during the continuous fermentation of Ag bacteria and Zg bacteria according to Embodiment 2 of the present invention;

[0019] Figure 8 It is a mass spectrometry detection result diagram of agar oligosaccharide prepared from Zg bacteria according to Embodiment 2 of the present invention;

[0020] Figure 9 It is a mass spectrometry detection result diagram of agar oligosaccharide prepared from Ag bacteria according to Embodiment 2 of the present invention;

[0021] Figure 10 It is the change of relative enzyme activity with time during the single fermentation of Ag bacteria and Zg bacteria according to Embodiment 2 of the present invention;

[0022] Figure 11 It is a comparison diagram of the relative enzyme activities of Ag bacteria and Zg bacteria during single fermentation and continuous fermentation respectively according to Embodiment 2 of the present invention. Detailed implementation manners

[0023] The embodiments of the present invention are described in detail below, which are intended to explain the present invention and should not be construed as a limitation to the present invention.

[0024] Referring to "embodiment" herein means that the specific features, structures or characteristics described in connection with the embodiment may be included in at least one embodiment of the present invention. The phrase appears at various positions in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein may be combined with other embodiments.

[0025] For the sake of simplicity, only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form a range not explicitly recited; and any lower limit can be combined with other lower limits to form a range not explicitly recited, and similarly any upper limit can be combined with any other upper limit to form a range not explicitly recited. In addition, each individually disclosed point or single numerical value itself can serve as a lower limit or an upper limit and be combined with any other point or single numerical value or with other lower limits or upper limits to form a range not explicitly recited.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs; the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention; the terms "comprising" and "having" and any variations thereof in the specification and claims of the present invention and the above drawings are intended to cover non-exclusive inclusion.

[0027] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, the meaning of "a plurality of" is two or more.

[0028] In this article, the terms "containing", "comprising" or "including" are open expressions, that is, they include the content specified in the present invention, but do not exclude other aspects.

[0029] In this article, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur and the cases where the events or conditions do not occur.

[0030] Currently, the common idea of the enzymatic hydrolysis method is to produce enzymes through bacterial fermentation, and then separate the enzymes for use in the degradation of agar. However, it often ignores that in the process of microbial fermentation to produce agarase, an appropriate amount of agar substrate usually needs to be added to induce the bacteria to secrete a large amount of agarase. In this process of enzyme production by fermentation, the agar in the fermentation system will also be gradually degraded into agar oligosaccharides. Although under laboratory conditions, the consumption of this part of the agar substrate is relatively insignificant, in actual industrial production, if this part of the agar oligosaccharides can be utilized, it will help to improve the comprehensive utilization rate of agar raw materials and thus reduce the enterprise cost.

[0031] In view of the problems existing in the prior art, namely: ① There are certain differences in the enzyme production ability, enzyme activity, stability and degradation products of the potential candidate strains for agar oligosaccharide production reported in the prior art, which brings certain difficulties to the industrial standardization production of agarase; ② The separation and purification cost of agar degradation enzyme is high. Traditional production of agar oligosaccharides usually requires separation and purification of agarase, and then enzymatic hydrolysis preparation of agar oligosaccharides, and the process is very cumbersome; ③ Existing agarase fermentation production is usually batch fermentation production, and the activation and scale-up culture of strains need to be carried out again for each batch, and the production cycle is relatively long. In view of this, the present invention proposes a strain that can be used for industrial production of agar oligosaccharides. This strain has excellent enzyme production ability and enzyme activity, and can prepare agar oligosaccharides with a lower degree of polymerization. In addition, the present invention also improves the method for preparing agar oligosaccharides, enabling continuous production of agar oligosaccharides, reducing production costs and process cumbersome degree, and improving raw material conversion rate. The following will introduce agar oligosaccharides, the preparation method of agar oligosaccharides, and their uses in detail.

[0032] Agar oligosaccharide

[0033] In the first aspect of the present invention, the present invention proposes an agar oligosaccharide. According to an embodiment of the present invention, the agar oligosaccharide is obtained by enzymatic hydrolysis of a strain and agar; the strain includes at least one of Zobellia galactanivorans and Agarivorans albus. Through a large number of experiments, the inventors found that Zobellia galactanivorans and Agarivorans albus can be used for the production of agar oligosaccharides, and during the enzymatic hydrolysis fermentation process, both Zobellia galactanivorans and Agarivorans albus have relatively high enzyme activities, which can improve the production efficiency of agar oligosaccharides and enhance the raw material conversion rate. In addition, the main component of the agar oligosaccharide of the present invention is neoagaro - tetraose, which has a lower degree of polymerization, and the average degree of polymerization is 5.51. Thus, it can be more easily absorbed by the human body.

[0034] In some embodiments of the present invention, the Zobellia galactanivorans was deposited at the China Center for Industrial Culture Collection on November 2, 2018, with the deposit number CICC 24669; the Agarivorans albus was deposited at the China Center for Industrial Culture Collection on October 30, 2013, with the deposit number CICC 10798.

[0035] In some embodiments of the present invention, the average degree of polymerization of the agar oligosaccharide is 5 - 6. For example, it can be 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, etc., or it can be a range composed of any of the above values.

[0036] Method for preparing agar oligosaccharide

[0037] In a second aspect of the present invention, the present invention provides a method for preparing the agar oligosaccharide described in the first aspect. According to an embodiment of the present invention, the method includes: subjecting the strain and agar to enzymatic hydrolysis treatment to obtain the agar oligosaccharide, and the strain includes at least one of Flavobacterium galactosidilyticum and Algicola albus. Thus, by using the method of the present invention, the production efficiency of agar oligosaccharide can be improved, the raw material conversion rate can be increased, and the production cost and process complexity can be reduced.

[0038] In some embodiments of the present invention, Flavobacterium galactosidilyticum was deposited at the China Center for Industrial Culture Collection on November 2, 2018, with the deposit number CICC 24669; Algicola albus was deposited at the China Center for Industrial Culture Collection on October 30, 2013, with the deposit number CICC 10798.

[0039] In some embodiments of the present invention, the method includes: subjecting the strain to fermentation treatment to obtain a fermentation broth; subjecting the fermentation broth and the first agar substrate to the enzymatic hydrolysis treatment to obtain the agar oligosaccharide.

[0040] In some embodiments of the present invention, the fermentation treatment is continuous fermentation, and the continuous fermentation is carried out in the following manner: 1) subjecting the strain to a first fermentation treatment in a fermentation medium to obtain a first fermentation broth; 2) extracting 50%-70% of the volume of the first fermentation broth for the enzymatic hydrolysis treatment, and at the same time adding an equal volume of the fermentation medium to the remaining first fermentation broth for a second fermentation treatment to obtain a second fermentation broth; repeating step 2) in a cycle. Thus, by adopting the continuous fermentation production process, the strain can be continuously fermented for more than three days and maintain high production vitality after activation, greatly reducing the production cost and process complexity.

[0041] It should be noted that the "equal volume of the fermentation medium" refers to the volume equal to the volume of the extracted first fermentation broth.

[0042] In some embodiments of the present invention, the fermentation medium contains a second agar substrate. Thus, this medium can induce the strain to produce a large amount of agarase during fermentation, thereby generating agar oligosaccharide.

[0043] In some embodiments of the present invention, based on the total mass of the fermentation medium, the mass ratio of the second agar substrate is 0.1%-0.3%. Thus, an appropriate amount of agar substrate can induce the strain to produce a large amount of agarase during fermentation and improve the enzyme activity.

[0044] In some embodiments of the present invention, the fermentation medium comprises: yeast powder 0.5 - 1.5 g / L, peptone 4.5 - 5.5 g / L, ferric citrate 0.05 - 0.15 g / L, sodium chloride 19 - 20 g / L, magnesium chloride 5.5 - 6.5 g / L, sodium sulfate 3 - 4 g / L, calcium chloride 1.5 - 2.5 g / L, potassium chloride 0.4 - 1 g / L, sodium carbonate 0.1 - 0.2 g / L, potassium bromide 0.05 - 0.1 g / L, strontium chloride 0.01 - 0.05 g / L, boric acid 0.01 - 0.05 g / L, sodium silicate 0.001 - 0.007 g / L, sodium fluoride 0.001 - 0.005 g / L, ammonium nitrate 0.001 - 0.002 g / L, disodium hydrogen phosphate 0.005 - 0.01 g / L, and a second agar substrate 1.5 - 2.5 g / L.

[0045] In some embodiments of the present invention, the second agar substrate is agar powder.

[0046] In some embodiments of the present invention, the fermentation medium comprises: yeast powder 1.0 g / L, peptone 5.0 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.98 g / L, sodium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium carbonate 0.16 g / L, potassium bromide 0.08 g / L, strontium chloride 0.034 g / L, boric acid 0.022 g / L, sodium silicate 0.004 g / L, sodium fluoride 0.0024 g / L, ammonium nitrate 0.0016 g / L, disodium hydrogen phosphate 0.008 g / L, and agar powder 2 g / L.

[0047] In some embodiments of the present invention, the temperature of the first fermentation treatment is 25°C - 35°C. For example, it can be 25°C, 27°C, 29°C, 30°C, 32°C, 34°C, 35°C, etc., or it can be a range composed of any of the above values. In some embodiments of the present invention, the time of the first fermentation treatment is 70 h - 75 h. For example, it can be 70 h, 71 h, 72 h, 73 h, 74 h, 75 h, etc., or it can be a range composed of any of the above values. Thus, the initially inoculated strains can be fully fermented to produce agarase.

[0048] In some embodiments of the present invention, the temperature of the second fermentation treatment is 25°C - 35°C. For example, it can be 25°C, 27°C, 29°C, 30°C, 32°C, 34°C, 35°C, etc., or it can be a range composed of any of the above values. In some embodiments of the present invention, the time of the second fermentation treatment is 20 h - 25 h. For example, it can be 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, etc., or it can be a range composed of any of the above values. Thus, the proliferation and utilization of the strains can be effectively realized, and the cost can be saved.

[0049] In some embodiments of the present invention, the first agar substrate includes, but is not limited to, at least one of agar, agarose, Gelidium amansii extract, Gracilaria lemaneiformis extract, as long as it can undergo an enzymatic hydrolysis reaction with agarase to generate agar oligosaccharides.

[0050] In some embodiments of the present invention, the volume ratio of the first agar substrate in the enzymatic hydrolysis treatment system is 1%-3%. For example, it can be 1%, 1.5%, 2%, 2.5%, 3%, etc.

[0051] In some embodiments of the present invention, the enzymatic hydrolysis treatment is carried out under the condition that the pH value is 7.2-7.4. For example, it can be 7.2, 7.3 or 7.4. It should be noted that this pH value can be adjusted by adding an appropriate amount of phosphonate to the second agar substrate to maintain the stability of the enzyme activity.

[0052] In some embodiments of the present invention, the temperature of the enzymatic hydrolysis treatment is 35°C-45°C. For example, it can be 35°C, 37°C, 40°C, 42°C, 45°C, etc., or it can be a range composed of any of the above values. In some embodiments of the present invention, the time of the enzymatic hydrolysis treatment is 3h-12h. For example, it can be 3h, 5h, 7h, 10h, 12h, etc., or it can be a range composed of any of the above values. Thus, the agarase in the fermentation broth can fully undergo enzymatic hydrolysis treatment with the first agar substrate to generate agar oligosaccharides.

[0053] In some embodiments of the present invention, the enzymatic hydrolysis treatment is carried out under stirring conditions. Thus, the efficiency of enzymatic hydrolysis can be improved.

[0054] In some embodiments of the present invention, before the enzymatic hydrolysis treatment, it further includes: performing a first separation treatment on the fermentation broth obtained after the fermentation treatment. Thus, the undegraded agar substrate and bacteria in the fermentation broth can be removed.

[0055] In some embodiments of the present invention, the first separation treatment can be carried out by centrifugal separation or membrane filtration separation. According to the embodiments of the present invention, the rotation speed of the centrifugal separation is 3700r / min-4300r / min. For example, it can be 3700r / min, 3800r / min, 3900r / min, 4000r / min, 4100r / min, 4200r / min, 4300r / min, etc., or it can be a range composed of any of the above values. Thus, the undegraded agar substrate and bacteria in the fermentation broth can be removed.

[0056] In some embodiments of the present invention, after the enzymatic hydrolysis treatment, it further includes: performing a second separation treatment on the enzymatic hydrolysis treatment product to obtain the agar oligosaccharide. Thus, it is possible to effectively remove the bacteria, undegraded agar substrate, and protease substances in the enzymatic hydrolysis treatment product, and obtain a pure agar oligosaccharide solution.

[0057] In some embodiments of the present invention, the second separation treatment can be centrifugal separation or membrane filtration separation. According to the embodiments of the present invention, the rotation speed of the centrifugal separation is 7800 r / min - 8200 r / min. For example, it can be 7800 r / min, 7900 r / min, 8000 r / min, 8100 r / min, 8200 r / min, etc., or it can be a range composed of any of the above values. Thus, it is possible to effectively remove the bacteria, undegraded agar substrate, and protease substances in the enzymatic hydrolysis treatment product, and obtain a pure agar oligosaccharide solution.

[0058] Use

[0059] In the third aspect of the present invention, the present invention proposes the use of the strain in the preparation of agar oligosaccharide, and the strain includes at least one of Flavobacterium galactosidilyticum and Algicola candidus.

[0060] In some embodiments of the present invention, Flavobacterium galactosidilyticum was deposited at the China Center for Industrial Culture Collection on November 2, 2018, with the deposit number CICC 24669; Algicola candidus was deposited at the China Center for Industrial Culture Collection on October 30, 2013, with the deposit number CICC 10798.

[0061] In the fourth aspect of the present invention, the agar oligosaccharide described in the first aspect of the present invention, or the agar oligosaccharide prepared by the method described in the second aspect has at least one of the following uses: use in the preparation of a drug for anti - inflammation, anti - tumor, treating cardiovascular diseases, regulating blood sugar, and regulating the intestinal flora; use in the preparation of a health food; use in the preparation of a cosmetic for anti - ultraviolet and antioxidant.

[0062] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0063] Example 1: Screening of bacteria with high - yield agarase

[0064] During the experiment, the inventors unexpectedly discovered that Agarivorans albus (referred to as Ag for short), Zobelliagalactanivoran (referred to as Zg for short), Microbulbifer aggregans (referred to as Mi for short), and Vibrio sp. (referred to as Vi for short) can encode agarase. Therefore, in order to study the actual agarase production ability of these four strains, relevant experiments were conducted. Among them, the four strains were purchased from the China Center of Industrial Culture Collection (CICC). The preservation number of Ag is CICC 10798, that of Zg is CICC 24669, that of Mi is CICC 24666, and that of Vi is CICC 24670.

[0065] 1. Activation and preservation of strains

[0066] The above-mentioned strains were activated in 2216E liquid medium for 24 h and stored in large quantities using 60% glycerol. The stored strains were placed at -80 °C.

[0067] Among them, the composition of 2216E liquid medium is: yeast powder 1.0 g / L, peptone 5.0 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.98 g / L, sodium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium carbonate 0.16 g / L, potassium bromide 0.08 g / L, strontium chloride 0.034 g / L, boric acid 0.022 g / L, sodium silicate 0.004 g / L, sodium fluoride 0.0024 g / L, ammonium nitrate 0.0016 g / L, disodium hydrogen phosphate 0.008 g / L.

[0068] 2. Observation of transparent zone by Lugol's iodine solution staining

[0069] Each strain was cultured on 2216E agar solid medium (2216E agar solid medium was prepared by adding 15 g / L (1.5%) agar powder to 2216E liquid medium). The results are as Figure 1 shown. The morphology of each strain was good, indicating that they can all grow stably on 2216E agar solid medium.

[0070] After each strain formed colonies of a certain size, Lugol's iodine solution was used to stain the colonies. After staining, let it stand for a period of time, and observe the size and light transmittance of the transparent zone around the colonies, so as to preliminarily judge the agar degradation ability of different strains. The experimental results are as Figure 2 shown. It can be seen that all four strains can produce transparent zones, among which the transparent zone of Zg is the largest and the light transmittance is the strongest.

[0071] 3. Growth curve plotting

[0072] On the basis of 2216E liquid medium, 2 g / L (0.2%) of sterilized agar powder was added to prepare a liquid fermentation medium. Subsequently, the four strains were cultured separately in the fermentation medium, and samples were taken at regular intervals to measure the absorbance value of the bacterial solution at 600 nm, and the growth curves of the strains were plotted. The experimental results are as Figure 3 shown. All four strains could grow rapidly in the fermentation medium, and among them, Zg had the fastest growth rate.

[0073] 4. Results of fermentation culture and enzyme activity determination

[0074] After the above four cryopreserved strains were activated and cultured for 24 hours, activated bacterial solutions were obtained. 150 ml of sterilized fermentation medium was added to a 250 ml conical flask, and 3 ml (2%) of the activated bacterial solution was inoculated. After sealing with a breathable membrane, it was placed in a shaker for shaking culture. Samples were taken every 24 hours during the fermentation process. After the samples were centrifuged at 4000 r / min, the supernatant was taken as the crude enzyme solution and the content of agar oligosaccharides in it was measured.

[0075] In PBS solution (pH 7.2 - 7.5, 0.01 M), 2% agar powder was added to prepare an agar substrate solution, which was placed in a 40°C constant temperature water bath for heating and insulation. After the substrate temperature reached 40°C, 5 ml of the agar substrate solution was added to a clean glass test tube, and then 3 ml of the crude enzyme solution was added. After standing and reacting at 40°C for 1 hour, the reaction was terminated by centrifuging the enzyme and the substrate at 4°C. The supernatant was taken to measure the content of agar oligosaccharides in it and calculate the enzyme activity.

[0076] Among them, the enzyme activity of the crude enzyme solution was equal to the content of agar oligosaccharides in the supernatant after the reaction minus the content of agar oligosaccharides in the crude enzyme solution. The enzyme activity unit U was defined as the amount of enzyme required for 1 mL of the crude enzyme solution to degrade and produce 1 μmol of reducing sugar per minute. In order to facilitate the comparison of the enzyme activity differences between different groups, the enzyme activity unit was further converted into relative enzyme activity, that is, the group with the highest enzyme activity was taken as 100%, and the other groups were converted according to the relative size.

[0077] The experimental results are as Figure 4 shown. Zg had the highest relative enzyme activity, followed by Ag, and Mi and Vi were relatively weak.

[0078] Therefore, in summary, the strain with the largest clear zone, the largest area under the growth curve at the same culture time, and the highest relative enzyme activity was selected as the dominant strain. In the present invention, Flavobacterium zobellii that can utilize galactan was determined as the dominant strain, and Algicola oricola was determined as the sub-dominant strain. Next, the effects of these two strains in the actual fermentation and enzymatic hydrolysis processes for preparing agar oligosaccharides were studied.

[0079] Example 2: Optimization of Fermentation Conditions

[0080] During the experiment, the inventors unexpectedly found that the fermentation method would affect the relative enzyme activities of Flavobacterium galactoglucuronovorans and Algicola oricola during fermentation. In order to make Flavobacterium galactoglucuronovorans and Algicola oricola have relatively high enzyme activities and improve the production efficiency of agar oligosaccharides, the fermentation method was studied.

[0081] 1. Continuous Fermentation Mode

[0082] (1) Continuous fermentation: After the cryopreserved Flavobacterium galactoglucuronovorans and Algicola oricola were separately activated and cultured for 24 hours, the activated bacterial solutions were obtained. 150 ml of sterilized fermentation medium was added to a 250 ml conical flask, and 3 ml (2%) of the activated bacterial solution was inoculated. After sealing with a breathable membrane, continuous fermentation was carried out in a shaker at 29°C. After the initial inoculation and fermentation for 72 hours, about 2 / 3 of the fermentation broth in the system was extracted for preparing crude enzyme solution, and then an equal amount of sterilized fermentation medium was added for the next round of fermentation. Thereafter, the above cycle could be carried out every 24 hours, so as to realize the proliferation and utilization of the strains and save production costs.

[0083] (2) Enzymolysis process: The extracted fermentation broth was centrifuged at 4000 r / min to remove undegraded substrates and cell bodies to prepare crude enzyme solution. Subsequently, agar powder was added to the crude enzyme solution, and the concentration of agar substrate in the enzymolysis system was controlled at about 2%. An appropriate amount of phosphate was also added to the substrate as a buffer to maintain the pH of the enzymolysis environment at about 7.2 - 7.4 to maintain the stability of enzyme activity. In addition, the optimal enzymolysis temperature was controlled at 40°C, and the enzymolysis efficiency was improved by uniform stirring. The enzymolysis time was preferably controlled at 3 - 6 h.

[0084] (3) Separation of agar oligosaccharides: After the enzymolysis was completed, the enzymolysis system was centrifuged at 8000 r / min to effectively remove cell bodies, undegraded agar substrates and protease substances, and a pure agar oligosaccharide solution was obtained.

[0085] The method described in Example 1 was used to detect the relative enzyme activity of the crude enzyme solution. The total sugar content before enzymolysis was determined by the phenol-sulfuric acid method, and the concentration of agar oligosaccharides after enzymolysis was determined by the DNS method. The average degree of polymerization (Dp) of the agar oligosaccharide sample was equal to the ratio of the total sugar content to the reducing sugar content in the sample. The conversion rate of the agar raw material was further calculated.

[0086] The concentration changes of agar oligosaccharides produced by Zg bacteria and Ag bacteria with the highest relative agarase activity under the continuous fermentation mode at different fermentation times are as Figure 5As shown, it can be seen that the strains in the continuous fermentation mode can still maintain a certain degree of growth after 72 h. Among them, the concentration of agar oligosaccharides of Zg bacteria reaches the highest at 96 h, while the Ag bacteria are still growing with the change of time, indicating that the continuous fermentation mode is beneficial to the production of agar oligosaccharides and improves the production efficiency.

[0087] The relative contents of agar oligosaccharides obtained from the crude enzyme solutions prepared by fermenting Zg bacteria and Ag bacteria at different enzymatic hydrolysis times are as Figure 6 shown. As can be seen from the figure, with the extension of the enzymatic hydrolysis time, the content of agar oligosaccharides in the system continuously increases, but the enzymatic hydrolysis rate gradually decreases. For Zg bacteria, compared with the enzymatic hydrolysis for 12 h, the enzymatic hydrolysis amount can reach more than 80% at 6 h of enzymatic hydrolysis, and more than 60% at 3 h of enzymatic hydrolysis; for Ag bacteria, the enzymatic hydrolysis amount can also reach more than 60% at 6 h. Therefore, in order to save time costs, it is recommended that the enzymatic hydrolysis time be about 6 h.

[0088] The results of the change of relative enzyme activity with time during continuous fermentation are as Figure 7 shown. The enzyme activity during continuous fermentation is still increased at 96 h compared with that at 72 h. At 120 h of fermentation, the enzyme activities of Ag and Zg begin to decline at 120 h of fermentation, but the difference from the enzyme activity at 96 h is not significant, indicating that continuous fermentation has a small impact on the enzyme activities of Ag and Zg. Therefore, the continuous fermentation mode is beneficial to improving the enzyme activity, thus contributing to improving the production efficiency of agar oligosaccharides.

[0089] The components of agar oligosaccharides prepared by continuous fermentation of Zg bacteria and Ag bacteria were determined, and the experimental results are respectively as Figure 8 and Figure 9 shown. After mass spectrometry detection, the main component of agar oligosaccharides prepared by fermenting Zg bacteria is neoagarotetraose, and it has a low degree of polymerization, with an average degree of polymerization of 5.51. The main component of agar oligosaccharides prepared by fermenting Ag bacteria is neoagarotetraose, and it has a low degree of polymerization, with an average degree of polymerization of 5.54.

[0090] It is calculated that continuous fermentation with Zg bacteria can make the conversion rate of agar raw materials reach more than 85%. Continuous fermentation with Ag bacteria can make the conversion rate of agar raw materials reach more than 81%.

[0091] 2. Single fermentation mode

[0092] After the cryopreserved Flavobacterium galactanolyticum and Algicola candidus were activated and cultured for 24 hours, an activated bacterial solution was obtained. 150 ml of sterilized fermentation medium was added to a 250 ml conical flask, and 3 ml (2%) of the activated bacterial solution was inoculated. After sealing with a breathable membrane, it was placed in a shaker for shaking culture. During the fermentation process, samples were taken every 24 hours. After centrifugal separation of the samples, the supernatant was taken to measure the content of agar oligosaccharides and prepare a crude enzyme solution.

[0093] Prepare a PBS solution (pH 7.2 - 7.5, 0.01 M) with an agar substrate content of 2%, place it in a constant temperature water bath at 40°C for heating and insulation. After the substrate temperature reaches 40°C, add 5 ml of the agar substrate solution to a glass test tube, and then add 3 ml of the crude enzyme solution. After standing and reacting at 40°C for 1 hour, centrifuge at 4°C to separate the enzyme and the substrate to terminate the reaction. Take the supernatant to measure the content of agar oligosaccharides and calculate the enzyme activity.

[0094] In the single fermentation process, the results of the change of relative enzyme activity over time are as Figure 10 shown. Zg and Ag have the highest relative enzyme activity at 72 h of fermentation, and then the enzyme activity begins to decline.

[0095] 3. Comparison between single fermentation and continuous fermentation

[0096] The comparison charts of the change of relative enzyme activity of the crude enzyme solution over time during the single fermentation and continuous fermentation of Zg bacteria and Ag bacteria are as Figure 11 shown. During the continuous fermentation process from 72 h to 96 h, the enzyme activity still increases, and slightly decreases at 120 h; while in the single fermentation, the enzyme activity drops significantly at 96 h compared to 72 h. Therefore, the continuous fermentation mode is more conducive to improving the enzyme activity than the single fermentation mode, thereby helping to improve the production capacity of agar oligosaccharides.

[0097] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0098] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An agar oligosaccharide, characterized in that: The agar oligosaccharide is obtained by enzymatically hydrolyzing a strain with agar; the strain includes at least one of the galactan-eating Flavobacterium zabelii and the white algae-eating bacteria.

2. Agar oligosaccharide according to claim 1, characterized in that The average polymerization degree of the agar oligosaccharide is 5-6.

3. A method for preparing the agar oligosaccharide according to claim 1 or 2, characterized in that: include: The strain and the first agar substrate are subjected to enzymatic hydrolysis to obtain the agar oligosaccharide, and the strain includes at least one of the galactan-eating Flavobacterium zabelii and the white algae-eating bacteria.

4. The method according to claim 3, characterized in that The method comprises: The strain is subjected to fermentation treatment to obtain a fermentation liquid; The fermentation liquid and the first agar substrate are subjected to the enzymatic hydrolysis treatment to obtain the agar oligosaccharide.

5. The method according to claim 4, characterized in that The fermentation process is a continuous fermentation, and the continuous fermentation is carried out in the following manner: 1) subjecting the strain to a first fermentation treatment in a fermentation medium to obtain a first fermentation liquid; 2) extracting 50%-70% of the volume of the first fermentation liquid for the enzymatic hydrolysis treatment, and adding an equal amount of the fermentation medium to the remaining first fermentation liquid to perform a second fermentation treatment to obtain a second fermentation liquid; Repeat step 2) in a loop.

6. The method according to claim 5, characterized in that The fermentation medium contains a second agar substrate; Optionally, based on the total mass of the fermentation medium, the mass proportion of the second agar substrate is 0.1%-0.3%; Optionally, the fermentation medium comprises: yeast powder 0.5-1.5 g / L, peptone 4.5-5.5 g / L, ferric citrate 0.05-0.15 g / L, sodium chloride 19-20 g / L, magnesium chloride 5.5-6.5 g / L, sodium sulfate 3-4 g / L, calcium chloride 1.5-2.5 g / L, potassium chloride 0.4-1 g / L, sodium carbonate 0.1-0.2 g / L, Potassium bromide 0.05-0.1g / L, strontium chloride 0.01-0.05g / L, boric acid 0.01-0.05g / L, sodium silicate 0.001-0.007g / L, sodium fluoride 0.001-0.005g / L, ammonium nitrate 0.001-0.002g / L, disodium hydrogen phosphate 0.005-0.01g / L, second agar substrate 1.5-2.5g / L; Optionally, the temperature of the first fermentation treatment is 25°C-35°C; Optionally, the first fermentation treatment time is 70h-75h; Optionally, the temperature of the second fermentation treatment is 25°C-35°C; Optionally, the second fermentation treatment lasts for 20 h to 25 h.

7. The method according to claim 4, characterized in that The volume proportion of the first agar substrate in the enzymatic treatment system is 1%-3%; Optionally, the enzymatic hydrolysis treatment is carried out at a pH value of 7.2-7.4; Optionally, the temperature of the enzymatic treatment is 35°C-45°C; Optionally, the enzymatic treatment time is 3h-12h; Optionally, the enzymatic treatment is carried out under stirring conditions.

8. The method according to claim 4, characterized in that Before the enzymatic hydrolysis treatment, the method further comprises: subjecting the fermentation liquid obtained after the fermentation treatment to a first separation treatment; Optionally, after the enzymatic hydrolysis treatment, the method further comprises: subjecting the enzymatic hydrolysis product to a second separation treatment to obtain the agar oligosaccharide.

9. Use of a strain in preparing agar-oligosaccharide, wherein the strain comprises at least one of the galactan-eating Flavobacterium zabelii and the algae-eating Bacillus.

10. The agar oligosaccharide according to claim 1 or 2, or the agar oligosaccharide prepared by the method according to any one of claims 3 to 8 has at least one of the following uses: Use in the preparation of drugs for anti-inflammation, anti-tumor, anti-cardiovascular disease, anti-diabetes, blood sugar regulation, and intestinal flora regulation; Use in the preparation of health foods; Use in the preparation of anti-ultraviolet and anti-oxidation cosmetics.

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