CN113491331A - Algal polysaccharide composition and composite fruit juice mixed fermentation powder as well as preparation method and application thereof - Google Patents
Algal polysaccharide composition and composite fruit juice mixed fermentation powder as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN113491331A CN113491331A CN202110745091.1A CN202110745091A CN113491331A CN 113491331 A CN113491331 A CN 113491331A CN 202110745091 A CN202110745091 A CN 202110745091A CN 113491331 A CN113491331 A CN 113491331A
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- polysaccharide
- composition
- fruit juice
- treatment
- algal
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Abstract
The invention discloses a seaweed polysaccharide composition and compound fruit juice mixed fermentation powder as well as a preparation method and application thereof. The method comprises the following steps: adding the algal polysaccharide composition into the compound fruit juice, uniformly mixing, heating for heating to obtain a mixed solution, adjusting the pH of the mixed solution to be acidic, adding a lactobacillus rhamnosus microbial inoculum, performing fermentation treatment, sterilizing, concentrating, and drying to obtain the algal polysaccharide composition and compound fruit juice mixed fermentation powder. The algal polysaccharide composition-compound fruit juice mixed fermentation powder has the effect of remarkably improving diabetes and complications thereof, and can be used for producing health-care food, food and medicines for the diabetes and the complications thereof.
Description
Technical Field
The invention belongs to the field of health-care food, and particularly relates to seaweed polysaccharide composition and compound fruit juice mixed fermentation powder as well as a preparation method and application thereof.
Background
Diabetes mellitus is a chronic disease mainly caused by pancreatic insulin deficiency, peripheral tissue insulin resistance or both, is a common disease and frequently encountered disease which currently endangers human health, and is called as the third major health killer after malignant tumor and cardiovascular disease. The international diabetes association (IDF) predicts that the number of adult diabetic patients will exceed 7.002 billion worldwide in 2045 years. Diabetes is characterized by polydipsia, polyphagia, polyuria and weight loss, and also includes metabolic disorders such as sugar, fat, protein, electrolytes and the like.
Treatment of diabetes and its complications should be undertaken with the attention paid to early, effective and sustained control principles to delay the onset of the disease and reduce the probability of complications occurring and worsening. At present, the means for treating diabetes are changing day by day, and mainly relate to drug therapy, optimization of dietary structure, exercise regulation and the like, wherein the drug therapy is the most mainstream and effective means in the treatment of diabetes. However, the hypoglycemic drugs commonly used in clinic all have the characteristics of certain toxicity or side effect, high treatment cost, different adverse effects on the physical health of the diabetic patients and the like. Therefore, searching effective components for remarkably improving diabetes and complications thereof from low-value food-borne animal and plant or microbial resources, and researching and developing hypoglycemic drugs or auxiliary hypoglycemic functional factors with green, high safety and low price become a hot field of current research.
The marine algae is an important marine plant resource, is widely distributed in tropical and subtropical sea areas in China, and has huge development and application potentials. The protein, pigment, polyphenol and polysaccharide extracted from Sargassum also have physiological effects of promoting health and preventing and treating chronic diseases. Therefore, the discovery, preparation, characterization or identification of active substances of seaweed source, as well as functional evaluation and mechanism discussion have become one of the hot scientific problems of the current research. Algal polysaccharides are the most important bioactive molecules in algae, and due to their unique structural characteristics, they confer multiple biological activities, such as: antioxidant, antitumor, blood sugar lowering, intestinal flora regulating, immunity regulating, etc.
The fresh fruit juice contains various nutrient components such as vitamins, calcium, phosphorus and the like, and has the effects of enhancing the activity of cells and the activity of gastrointestinal tracts in a human body, eliminating fatigue and the like. In recent years, with the deep development of fruit and vegetable deep processing technology, the wide application of probiotics in fruit juice fermentation is also accepted by more people. The probiotic fermented fruit juice not only improves the utilization rate of fruits, enables the fruit juice to have better taste, but also improves the safety and the storage resistance of fruit and vegetable foods. In addition, the probiotic fermented fruit juice product not only has the nutrient components of the fruit raw materials, but also has the health-care function of the probiotics, reduces the content of glucose in the fruits, and has the effects of reducing blood sugar, improving lactose intolerance and the like. Therefore, the probiotics is utilized to ferment the fruits and vegetables to obtain the leavening with unique flavor and good biological activity, and the leavening has wide development and utilization prospects.
However, the fruit and vegetable raw materials have high water content and relatively low carbohydrate content, so that a carbon source such as glucose and the like is often required to be additionally added in the conventional fruit and vegetable fermentation process to ensure the normal reproduction and metabolism of probiotics. The algal polysaccharide is added as a carbon source of a culture medium, so that the algal polysaccharide cannot cause blood sugar rise after being ingested by a human body, and the algal polysaccharide can be used as the carbon source to promote the proliferation of probiotics and improve the fermentation of fruits and vegetables.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a seaweed polysaccharide composition and compound fruit juice mixed fermentation powder as well as a preparation method and application thereof.
The purpose of the invention is realized by at least one of the following technical solutions.
The preparation method of the algal polysaccharide composition and compound fruit juice mixed fermentation powder provided by the invention comprises the following steps:
adding the algal polysaccharide composition into the compound fruit juice, uniformly mixing, heating for heating to obtain a mixed solution, adjusting the pH of the mixed solution to be acidic, adding a lactobacillus rhamnosus microbial inoculum, performing fermentation treatment, sterilizing, concentrating, and drying to obtain the mixed fermentation powder of the algal polysaccharide composition and the compound fruit juice.
Further, the algal polysaccharide composition has a particle size of 2-20 μm;
further, the algal polysaccharide composition is two or more of the following algal polysaccharides: fine micropowder of polysaccharide from Ascophyllum Nodosum, fine micropowder of polysaccharide from Cyrtymenia Sparsa, fine micropowder of polysaccharide from Fucus vesiculosus, fine micropowder of polysaccharide from Sargassum, fine micropowder of polysaccharide from Undaria pinnatifida, and fine micropowder of polysaccharide from Macrocystis.
Preferably, the algal polysaccharide composition is more than one of a composition of fine polysaccharide micropowder of Ascophyllum nodosum and fine polysaccharide micropowder of Cyrtymenia Sparsa, a composition of fine polysaccharide micropowder of Fucus vesiculosus and fine polysaccharide micropowder of Ascophyllum nodosum, a composition of fine polysaccharide micropowder of Sargassum and fine polysaccharide micropowder of Undaria pinnatifida, a composition of fine polysaccharide micropowder of Macrocystis, a composition of fine polysaccharide micropowder of Fucus vesiculosus and fine polysaccharide micropowder of Sargassum, a composition of fine polysaccharide micropowder of Ascophyllum nodosum and fine polysaccharide micropowder of Sargassum, and a composition of fine polysaccharide micropowder of Ascophyllum nodosum and fine polysaccharide micropowder of Sargassum.
More preferably, the seaweed polysaccharide composition is a composition with the mass ratio of the fine polysaccharide micro powder of the ascophyllum nodosum to the fine polysaccharide micro powder of the hizikia fusiforme of 1:1-4, a composition with the mass ratio of the fine polysaccharide micro powder of the fucoidan to the fine polysaccharide micro powder of the ascophyllum nodosum of 1:10-12, a composition with the mass ratio of the fine polysaccharide micro powder of the gulfweed to the fine polysaccharide micro powder of the undaria pinnatifida of 1:7-8, a composition with the mass ratio of the fine polysaccharide micro powder of the kelp to the fine polysaccharide micro powder of the undaria pinnatifida of 1:1-2, the mass ratio of the fucus vesiculosus polysaccharide fine micro powder to the gulfweed polysaccharide fine micro powder is 1:10-15, the mass ratio of the ascophyllum vesiculosus polysaccharide fine micro powder to the macroalgae polysaccharide fine micro powder is 1:8-10, and the mass ratio of the ascophyllum vesiculosus polysaccharide fine micro powder to the gulfweed polysaccharide fine micro powder is 1: 1-2.
Further, the preparation of the algal polysaccharide (one of fucus polysaccharide fine powder, sargassum fusiforme polysaccharide fine powder, undaria pinnatifida polysaccharide fine powder, gulfweed polysaccharide fine powder, ascophyllum polysaccharide fine powder and macroalgae polysaccharide fine powder) comprises the following steps:
(1) cleaning seaweed, air drying, soaking in ethanol solution, heating for reflux extraction, filtering to obtain filter residue, and drying to obtain ethanol-treated seaweed;
(2) soaking the seaweed treated by the ethanol in the step (1) in water, pulping to obtain paste, adding water into the paste to obtain mixed solution 1, refrigerating, melting, leaching, centrifuging, and taking supernatant to obtain extract 1;
(3) adjusting the pH value of the extracting solution 1 in the step (2) to be acidic, adding papain to obtain a mixed solution 2, heating for oscillation treatment, then inactivating enzyme, centrifuging to obtain a supernatant, and concentrating to obtain an extracting solution 2;
(4) and (3) passing the extracting solution 2 obtained in the step (3) through an ultrafiltration membrane, taking components with the molecular weight of more than 10kD to obtain an extracting solution 3, concentrating the extracting solution 3 to obtain a concentrated solution, adding absolute ethyl alcohol into the concentrated solution, standing, centrifuging to obtain a precipitate, redissolving the precipitate, carrying out reduced pressure concentration to remove the ethyl alcohol, and carrying out freeze drying to obtain the algal polysaccharide with the particle size of 2-20 microns.
Further, in the step (1) of preparing algal polysaccharides, the seaweed is one of Ascophyllum nodosum, Cyrtymenia Sparsa, Fucus vesiculosus, Sargassum fusiforme, Undaria pinnatifida and Macrocystis japonica;
further, in the step (1) of preparing the algal polysaccharide, the volume percentage concentration of the ethanol solution is 90-100%;
further, in the step (1) of preparing the algal polysaccharide, the temperature of the reflux extraction treatment is 75-85 ℃, and the time of the reflux extraction treatment is 2-6 h;
further, in the step (2) of preparing the algal polysaccharide, the time of soaking the seaweed after ethanol treatment in water is 3-5 h;
further, in the step (2) of preparing the algal polysaccharide, the mass ratio of the seaweed after ethanol treatment to the water in the paste is 1: 3-8;
further, in the step (2) of preparing the algal polysaccharide, the mass ratio of the alga treated by the ethanol to the water in the mixed solution is 1: 15-30;
further, in the step (2) for preparing the algal polysaccharide, the temperature of the refrigeration treatment is-80 to-40 ℃, and the time of the refrigeration treatment is 2 to 5 hours;
further, in the step (2) of preparing the algal polysaccharide, the temperature of the melting treatment is 60-80 ℃;
further, in the step (2) of preparing the algal polysaccharide, the number of the cold storage treatment and the melting treatment is 2-3;
further, in the step (2) for preparing the algal polysaccharide, the temperature of the leaching treatment is 80-100 ℃, and the time of the leaching treatment is 2-8 h.
Further, in the step (3) of preparing the algal polysaccharide, the pH of the extracting solution 1 is adjusted to 5.5-6.0;
further, in the step (3) of preparing the algal polysaccharide, the enzyme activity of the papain in the mixed solution 2 is 50-200U/L; the volume of the papain is 0.5-2% of the volume of the mixed solution 2;
further, in the step (3) of preparing the algal polysaccharide, the temperature of the oscillation treatment is 55-65 ℃, and the time of the oscillation treatment is 50-80 min;
further, in the step (3) of preparing the algal polysaccharide, the enzyme deactivation treatment is boiling water bath for 5-10 min;
preferably, in the step (3) of preparing algal polysaccharide, after enzyme deactivation treatment, the mixed solution after enzyme deactivation treatment is cooled to 20-35 ℃ and then centrifuged.
Further, in the step (3) of preparing the algal polysaccharide, the solid content in the extracting solution 2 is 2-8 wt%;
further, in the step (4) of algal polysaccharide preparation, the ultrafiltration membrane (hollow ultrafiltration membrane) has a molecular weight cut-off of 10 kD;
preferably, in the step (4) of algal polysaccharide preparation, the extract 2 is repeatedly passed through an ultrafiltration membrane 4-8 times.
Further, in the step (4) of preparing the algal polysaccharide, the adding rate of the absolute ethyl alcohol is 3-8 mL/min;
further, in the step (4) of preparing the algal polysaccharide, the solid content in the concentrated solution is 5-12 wt%;
further, in the step (4) of preparing the algal polysaccharide, the volume ratio of the concentrated solution to the absolute ethyl alcohol is 1: 4;
preferably, in the step (4) of preparing the algal polysaccharide, anhydrous ethanol is added to the concentrated solution using a separatory funnel.
Further, in the step (4) for preparing the algal polysaccharide, the standing temperature is 4 ℃, and the standing time is 12-16 h.
Further, the preparation of the compound fruit juice comprises the following steps:
cleaning pawpaw, kiwi fruit and guava, cutting into pieces, mixing to obtain a mixture, adding water, pulping, heating in a shaking table for extraction treatment, centrifuging to remove precipitates, filtering to obtain filtrate to obtain the compound fruit juice;
further, in the preparation process of the compound fruit juice, the mass ratio of the pawpaw, the kiwi fruit and the guava is 5:2: 6;
further, in the preparation process of the compound fruit juice, the mass ratio of the mixture to water is 1: 10-15;
further, in the preparation process of the compound fruit juice, the temperature of the extraction treatment is 60-70 ℃, and the time of the extraction treatment is 6-8 h;
further, in the preparation process of the composite fruit juice, the rotation speed of the centrifugation is 8000-12000r/min, and the time of the centrifugation is 10-15 min.
Further, the mass ratio of the algal polysaccharide composition to the compound fruit juice is 1: 50-80;
further, the temperature of the heating treatment is 100-115 ℃, and the time of the heating treatment is 10-30 s;
further, the pH value of the mixed solution is adjusted to 3.0-4.0;
preferably, the pH of the mixed solution is adjusted to 4.0.
Further, the volume of the lactobacillus rhamnosus microbial inoculum is 0.5-2% of the volume of the mixed solution;
further, the temperature of the fermentation treatment is 37-40 ℃, and the time of the fermentation treatment is 12-24 h;
preferably, the fermentation treatment time is 24 h.
Further, the sterilization treatment is boiling water bath for 15-20 min;
further, the concentration is carried out until the content of the solid matters is 15-20 wt%.
The invention provides a seaweed polysaccharide composition prepared by the preparation method and composite fruit juice mixed fermentation powder.
The algal polysaccharide composition and the compound fruit juice mixed fermentation powder provided by the invention can be applied to the preparation of health-care food, food and medicines for treating diabetes and complications thereof.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) compared with the conventional method for preparing the polysaccharide by hot water extraction, the preparation method provided by the invention has the advantages that the steps of soaking, pulping and repeated freezing and thawing are added before the hot water extraction, and the adding speed of ethanol is controlled by using a separating funnel in the alcohol precipitation process, so that the yield of the polysaccharide is improved.
(2) According to the invention, the algal polysaccharides from different sources are compounded to obtain a plurality of algal polysaccharide compositions with more remarkable in-vitro alpha-glucosidase inhibitory activity than that of single algal polysaccharide, the optimal combination proportion range is limited through process optimization, and the harm of the algal polysaccharide composition and the compound fruit juice mixed fermentation powder to patients caused by blood sugar fluctuation caused by food intake can be reduced.
(3) The invention utilizes the mixture of the seaweed polysaccharide composition and the compound fruit juice fermented by the probiotics to cover or eliminate the fishy smell of the pure seaweed polysaccharide (or the seaweed polysaccharide composition) to a certain extent, and the flavor and the color are more easily accepted by various people.
(4) In the aspect of blood sugar reducing efficacy, the algal polysaccharide composition and the compound fruit juice mixed fermentation powder provided by the invention are obviously superior to those of the control example related to the invention.
Drawings
Fig. 1 is a graph of fasting plasma glucose results at week 8 for each group of experimental mice in the example, and different lower case letters in fig. 1 represent significant differences (P <0.05) between data.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
Analysis method of alpha-glucosidase inhibition rate
Respectively taking 30 mu L of test sample solution with different concentrations, adding 30 mu L of alpha-glucosidase diluent with the enzyme activity unit of 0.2U/mL, preserving the temperature for 10 minutes at 37 ℃, then adding 30 mu L of p-nitrophenyl-alpha-D-glucopyranoside solution for reacting for 15 minutes, and then adding 100 mu L of Na with the concentration of 0.1mol/L2CO3The reaction was stopped with solution and the absorbance was measured at 405nm with acarbose (ACAR) as a positive control.The alpha-glucosidase inhibition rate of the test sample was calculated according to equation 1.
Example 1: preparation of algal polysaccharides and interaction thereof on alpha-glucosidase inhibition
(1) Preparing the seaweed polysaccharide fine powder:
cleaning Sargassum (one of Ascophyllum Nodosum, Cyrtymenia Sparsa, Fucus vesiculosus, Sargassum, Undaria Pinnatifida, and Macrocystis), air drying, soaking in 100% ethanol (anhydrous ethanol), heating with 100% ethanol (anhydrous ethanol), reflux-extracting for 2 hr at 80 deg.C, filtering to obtain filter residue, and drying to obtain ethanol-treated Sargassum;
soaking the seaweed subjected to ethanol treatment in water (the mass of the water is 6 times that of the seaweed subjected to ethanol treatment), soaking for 4 hours, pulping to paste to obtain paste, and then adding water into the paste to obtain a mixed solution 1, wherein the mass ratio of the seaweed subjected to ethanol treatment to the water in the mixed solution 1 is 1: 20; placing in-80 deg.C for freezing for 3h, thawing at 60 deg.C, repeatedly freezing and thawing for 3 times; hot water extraction is carried out for 4 hours, and the temperature of the hot water extraction is 90 ℃; filtering to obtain filtrate, centrifuging, and collecting supernatant to obtain extractive solution S1;
adjusting pH of the extractive solution S1 to 6.0, adding 1 wt% papain, shaking in 55 deg.C water bath shaker for 60min, inactivating enzyme in boiling water bath for 10min, cooling to 25 deg.C, centrifuging to obtain supernatant, and concentrating solid content to 6 wt% to obtain extractive solution S2.
Separating the extractive solution S2 with hollow ultrafiltration membrane with cut-off molecular weight of 10kD, repeating the operation for 6 times, and collecting components with molecular weight greater than 10kD to obtain extractive solution S3. Concentrating the extracting solution S3 under reduced pressure until the solid content is 10 wt% to obtain a concentrated solution, adding anhydrous ethanol with 4 times volume of the concentrated solution by using a separating funnel (the dropping flow rate of the separating funnel is controlled to be 4mL/min), standing for 16h at 4 ℃, centrifuging to obtain a precipitate, adding water for redissolution, concentrating under reduced pressure to remove ethanol, and freeze-drying to prepare the algal polysaccharide fine powder with the particle size of 2 mu m, which is respectively recorded as: fucus vesiculosus fine powder (FVP), Cyrtymenia Sparsa Fine Powder (SFP), Undaria pinnatifida fine powder (USP), Sargassum fine powder (SCP), Ascophyllum fine powder (ANP), and Macrocystis fine powder (MPP).
(2) Interaction analysis of algal polysaccharides on α -glucosidase inhibition:
for six algal polysaccharide samples, the half Inhibition Concentration (IC) of each test sample on the inhibition of alpha-glucosidase is calculated according to the curve equation of the test sample concentration to the inhibition rate50) Values and select 0.125 times IC respectively500.25 times IC500.5 times IC500.75 times IC50，IC501.25 times IC50And 1.5 times IC50The concentration of the polysaccharide is mixed in pairs according to the ratio of 1:1 respectively to obtain a series of concentration gradients of the mixed polysaccharides, a relation curve of the concentration of the polysaccharide composition to the inhibition rate is drawn according to the method of the alpha-glucosidase inhibition activity, and the IC of each polysaccharide in the mixed polysaccharides is calculated50And calculating a joint index CI value and a decrement index DRI value through software Calcusyn. And the combination ratio of the polysaccharide composition is defined by process optimization.
IC50The value refers to half dosage concentration, IC, of the antagonist in inhibiting enzyme activity and cell receptor50The smaller the value, the greater the antagonist's antagonistic ability. As is clear from Table 1, the α -glucosidase inhibitory activity IC of the respective polysaccharides alone is shown after the polysaccharides are used in combination50Is obviously reduced. Specifically, IC of FVP combined with SFP, ANP, MPP, SCP, USP50The reduction is 42.86%, 52.38%, 38.10%, 80.95% and 42.86% respectively; IC of SFP combined with FVP, ANP, MPP, SCP50The reduction is 49.55%, 65.10%, 55.15% and 56.24% respectively; IC of ANP combined with FVP, SFP, MPP, SCP, USP50The reduction is 62.77%, 66.67%, 63.48%, 65.60% and 20.21% respectively; IC of MPP combined with FVP, SFP, ANP, SCP, USP50Respectively reduced by 46.66%, 53.33%, 59.92%, 41.03% and 71.90%; IC of SCP combined with FVP, SFP, ANP, MPP, USP50Respectively fall down 8648%, 65.56%, 71.43%, 55.36% and 78.06%; IC of USP combined with FVP, ANP, MPP, SCP50The decrease was 42.97%, 31.85%, 78.14% and 77.51%, respectively. The results show that the algal polysaccharide composition has alpha-glucosidase inhibitory activity and can effectively prevent the blood glucose fluctuation caused by eating.
TABLE 1 algal polysaccharides and compositions for their alpha-glucosidase inhibitory activity
The reduction index (DRI) is the multiple of the reduction in the dose allowed compared to the dose of antagonist alone, at a certain antagonistic effect, such as inhibition of enzyme activity, etc. Generally, a larger DRI value indicates a better synergistic effect. As shown in Table 2, the DRI values of the individual polysaccharides increased with the increase in inhibition rate after the combination of FVP-SFP, FVP-ANP, SFP-USP, ANP-MPP, ANP-USP, MPP-SCP and SCP-USP. Wherein, when the inhibition ratio of the SCP-USP combination is 95%, the DRI values of SCP and USP are 7.812 and 12.573 respectively.
TABLE 2 DRI values for alpha-glucosidase inhibition of 50%, 75%, 90% and 95%
Synergy index (CI) is a powerful means to assess whether there is a synergistic effect between active and active. Since the analog calculation of CI value has no strict requirement for setting the dose of the substance, it is widely used in animal experiments, cell experiments, and enzyme inhibition experiments. Depending on whether there is a synergistic interaction between substances for a chemical or biological process, the CI value is divided into three segments, namely: a CI value of <0.9 indicates synergy, and the smaller the value, the stronger the synergy; 0.9< CI value <1.1 indicates that the effects have additive effects; CI >1.1 indicates that the effect has antagonistic effect, with the greater the number, the more pronounced the antagonistic effect. As shown in Table 3, the seven compositions of ANP-SFP, FVP-ANP, SCP-USP, MPP-USP, FVP-SCP, ANP-MPP and ANP-SCP have significant synergistic effect in the concentration range related to the experiment, and the greater the concentration is, the more significant the synergistic effect is.
TABLE 3 CI values for 50%, 75%, 90% and 95% alpha-glucosidase inhibition
As shown in Table 3, the seven compositions of ANP-SFP, FVP-ANP, SCP-USP, MPP-USP, FVP-SCP, ANP-MPP and ANP-SCP have significant synergistic effect in the concentration range related to the experiment, and the greater the concentration is, the more significant the synergistic effect is. By optimizing the combination proportion, the coordination and synergism of the seven compositions are the strongest under the condition that the mixture ratio of the seven compositions is the ratio of the IC50 values of each single polysaccharide in the compositions. Therefore, the seven groups of algal polysaccharide compositions of the invention have the following mixture ratio: mixing ANP and SFP at a mass ratio of 1:1-4 to obtain a seaweed polysaccharide composition, which is marked as ANP-SFP; mixing the FVP and the ANP according to the mass ratio of 1:10-12 to obtain an algal polysaccharide composition which is marked as FVP-ANP; mixing SCP and USP according to the mass ratio of 1:7-8 to obtain a seaweed polysaccharide composition, and marking as SCP-USP; mixing MPP and USP according to the mass ratio of 1:1-2 to obtain a seaweed polysaccharide composition, and marking as MPP-USP; mixing the FVP and the SCP according to the mass ratio of 1:10-15 to obtain a seaweed polysaccharide composition which is marked as FVP-SCP; mixing ANP and MPP at a mass ratio of 1:8-10 to obtain algal polysaccharide composition, which is marked as ANP-MPP; and mixing the ANP and the SCP according to the mass ratio of 1:1-2 to obtain the algal polysaccharide composition which is marked as ANP-SCP. The algal polysaccharide composition is one or a combination of more of the above.
Example 2: preparation of fruit juice and compound fruit juice and analysis of alpha-glucosidase inhibition activity thereof
Cleaning fructus Chaenomelis, fructus Actinidiae chinensis, and fructus Psidii Guajavae Immaturus respectively, cutting into fruit pieces, adding water respectively at a weight ratio of 1:10, pulping, heating to 70 deg.C in a shaking table, extracting for 6h, centrifuging at 8000r/min for 15min, collecting supernatant, and filtering to obtain corresponding fruit juice (fructus Chaenomelis juice, fructus Actinidiae chinensis juice, and fructus Psidii Guajavae Immaturus juice);
washing pawpaw, kiwi fruit and guava respectively, cutting into pawpaw fragments, kiwi fruit fragments and guava fragments respectively, and cutting into the pawpaw fragments: kiwifruit pieces: mixing fructus Psidii Guajavae Immaturus pieces at a mass ratio of 5:2:6 to obtain a mixture, adding 10 times of water, pulping, and extracting at 70 deg.C for 6 h. Centrifuging at 8000r/min for 15min, collecting supernatant, and filtering to obtain filtrate to obtain compound fruit juice.
Papaya juice, kiwi fruit juice, guava juice and compound juice are respectively diluted by 10 times, and alpha-glucosidase inhibition rate determination tests are carried out, wherein the results are as follows:
TABLE 4 inhibitory Activity of fresh fruit and vegetable juices on alpha-glucosidase
As can be seen from table 4, under the test conditions, guava juice, blueberry juice, kiwi fruit juice and compound juice all showed strong α -glucosidase inhibitory activity.
Example 3: preparation of ANP-SFP composition and composite fruit juice mixed fermentation powder
In the specific implementation, the method can be realized by the following steps:
respectively cleaning and air drying Ascophyllum nodosum and Cyrtymenia Sparsa, respectively soaking in 100% alcohol (anhydrous ethanol), respectively heating for reflux extraction at 80 deg.C for 2 hr, respectively filtering to obtain filter residue, and drying to obtain ethanol-treated Sargassum.
Soaking the seaweed subjected to ethanol treatment in water (the mass of the water is 6 times that of the seaweed subjected to ethanol treatment), soaking for 4 hours, pulping to paste to obtain paste, and then adding water into the paste to obtain a mixed solution 1, wherein the mass ratio of the seaweed subjected to ethanol treatment to the water in the mixed solution 1 is 1: 20; placing in-80 deg.C for freezing for 3h, thawing at 60 deg.C, repeatedly freezing and thawing for 3 times; leaching for 4 hours by hot water; the temperature of hot water extraction is 90 ℃; filtering to obtain filtrate, centrifuging, and collecting clear liquid to obtain extract S1;
adjusting pH of the extractive solution S1 to 6.0, adding 1 wt% papain, shaking in 55 deg.C water bath shaker for 60min, inactivating enzyme in boiling water bath for 10min, cooling to 25 deg.C, centrifuging to obtain supernatant, and concentrating solid content to 6 wt% to obtain extractive solution S2.
Separating the extractive solution S2 with hollow ultrafiltration membrane with cut-off molecular weight of 10kD, repeating the operation for 6 times, and collecting components with molecular weight greater than 10kD to obtain extractive solution S3. Concentrating the extractive solution S3 under reduced pressure to solid content of 10 wt% to obtain concentrated solution, adding 4 times volume of anhydrous ethanol (dropping flow rate of 4mL/min in separating funnel) into the concentrated solution, standing at 4 deg.C for 16h, centrifuging to obtain precipitate, adding water for redissolving, concentrating under reduced pressure to remove ethanol, freeze drying, and making into fine powder of algal polysaccharide with particle size of 2 μm, which is respectively marked as Ascophyllum Nodosum Polysaccharide (ANP) and Cyrtymenia Sparsa polysaccharide (SFP). Mixing ANP and SFP at a mass ratio of 1:2 to obtain algal polysaccharide composition, which is marked as ANP-SFP.
Washing pawpaw, kiwi fruit and guava respectively, cutting into pieces of pawpaw, pieces of kiwi fruit and pieces of guava respectively, and cutting into pieces of pawpaw: kiwifruit pieces: mixing fructus Psidii Guajavae Immaturus pieces at a mass ratio of 5:2:6 to obtain a mixture, adding 10 times of water, pulping, and extracting at 70 deg.C for 6 h. Centrifuging at 8000r/min for 15min, collecting supernatant, and filtering to obtain filtrate to obtain compound fruit juice.
Adding ANP-SFP into the composite fruit juice 60 times of the mass of the ANP-SFP, uniformly mixing, treating for 10s at 115 ℃ to obtain a mixed solution, and adjusting the pH of the mixed solution to 4.0. The lactobacillus rhamnosus is firstly inoculated in fresh cow milk and activated for 12 hours at the temperature of 37 ℃, and the inoculation amount is 0.1 g/L. Adding the activated lactobacillus rhamnosus microbial inoculum into the mixed solution of the algal polysaccharide and the compound fruit juice according to the volume ratio of 1%, and fermenting for 24h at 37 ℃. Sterilizing in boiling water bath for 15min, concentrating to solid content of 15 wt%, and drying to obtain mixed fermented powder of ANP-SFP composition and composite fruit juice.
Example 4: preparation of mixed fermentation powder of SCP-USP composition and compound fruit juice
In the specific implementation, the method can be realized by the following steps:
respectively cleaning Sargassum and thallus laminariae, air drying, respectively soaking in (anhydrous ethanol), respectively heating for reflux extraction for 2 hr at 80 deg.C, respectively filtering to obtain filter residue, and drying to obtain ethanol-treated Sargassum;
soaking the seaweed subjected to ethanol treatment in water (the mass of the water is 6 times that of the seaweed subjected to ethanol treatment), soaking for 4 hours, pulping to paste to obtain paste, and then adding water into the paste to obtain a mixed solution 1, wherein the mass ratio of the seaweed subjected to ethanol treatment to the water in the mixed solution 1 is 1: 20; placing in-80 deg.C for freezing for 3h, thawing at 60 deg.C, repeatedly freezing and thawing for 3 times; leaching for 4 hours by hot water; the temperature of hot water extraction is 90 ℃; filtering to obtain filtrate, centrifuging, and collecting clear liquid to obtain extractive solution S1.
Adjusting the pH value of the extracting solution S1 to 6.0, adding 1 wt% of papain, enabling the enzyme activity to be 200U/L, oscillating for 60min in a water bath shaker at 55 ℃, then boiling for 10min to inactivate the enzyme, cooling to 25 ℃, centrifuging to obtain a supernatant, and concentrating the solid content to 6 wt% to obtain an extracting solution S2;
separating the extractive solution S2 with hollow ultrafiltration membrane with cut-off molecular weight of 10kD, repeating the operation for 6 times, and collecting components with molecular weight greater than 10kD to obtain extractive solution S3. Concentrating the extractive solution S3 under reduced pressure to solid content of 10 wt% to obtain concentrated solution, adding 4 times volume of anhydrous ethanol (dropping flow rate of 4mL/min in separating funnel) into the concentrated solution, standing at 4 deg.C for 16h, centrifuging to obtain precipitate, adding water for redissolving, concentrating under reduced pressure to remove ethanol, freeze drying, and making into Sargassum polysaccharide fine powder with particle size of 2 μm, which is respectively marked as Sargassum polysaccharide (SCP) and Undaria Pinnatifida polysaccharide (USP). And mixing the SCP and the USP according to the mass ratio of 1:7 to obtain the algal polysaccharide composition which is marked as SCP-USP.
Washing pawpaw, kiwi fruit and guava respectively, cutting into pieces of pawpaw, pieces of kiwi fruit and pieces of guava respectively, and cutting into pieces of pawpaw: kiwifruit pieces: the mass ratio of the broken guava blocks is as follows: 2:6 mixing to obtain a mixture, adding water with the mass being 10 times that of the mixture, pulping, and extracting for 6 hours at 70 ℃ in a shaking table. Centrifuging at 8000r/min for 15min, collecting supernatant, and filtering to obtain filtrate to obtain compound fruit juice.
Adding SCP-USP into composite fruit juice 60 times of the weight of SCP-USP, mixing uniformly, treating for 10s at 115 ℃ to obtain a mixed solution, and adjusting the pH of the mixed solution to 4.0. The lactobacillus rhamnosus is firstly inoculated in fresh cow milk and activated for 12 hours at the temperature of 37 ℃, and the inoculation amount is 0.1 g/L. Adding the activated lactobacillus rhamnosus microbial inoculum into the mixed solution of the algal polysaccharide and the compound fruit juice according to the volume ratio of 1%, and fermenting for 24h at 37 ℃. Sterilizing in boiling water bath for 15min, concentrating to solid content of 15 wt%, and drying to obtain mixed fermentation powder of SCP-USP composition and compound fruit juice.
Example 5: preparation of ANP-MPP composition-composite fruit juice ferment mixed powder
In the specific implementation, the method can be realized by the following steps:
respectively cleaning and airing the Ascophyllum nodosum and the giant kelp, respectively soaking in absolute ethyl alcohol, respectively heating to perform reflux extraction treatment for 2 hours at 80 ℃, respectively filtering to obtain filter residues, and drying to obtain the alga after ethanol treatment.
Adding the seaweed treated by the ethanol into water (the mass of the water is 6 times of that of the seaweed treated by the ethanol), soaking for 4 hours, pulping to paste to obtain paste, then adding water into the paste to obtain mixed liquid 1, wherein the mass ratio of the seaweed treated by the ethanol to the water in the mixed liquid 1 is 1: 20; placing in-80 deg.C for freezing for 3h, thawing at 60 deg.C, repeatedly freezing and thawing for 3 times; hot water leaching is carried out for 4 hours, and the temperature of the hot water leaching is 90 ℃; filtering to obtain filtrate, centrifuging, and collecting clear liquid to obtain extractive solution S1.
Adjusting pH of the extractive solution S1 to 6.0, adding 1 wt% papain, shaking in 55 deg.C water bath shaker for 60min, inactivating enzyme in boiling water bath for 10min, cooling to 25 deg.C, centrifuging to obtain supernatant, and concentrating solid content to 6 wt% to obtain extractive solution S2.
Separating the extractive solution S2 with hollow ultrafiltration membrane with cut-off molecular weight of 10kD, repeating the operation for 6 times, and collecting components with molecular weight greater than 10kD to obtain extractive solution S3. Concentrating the extractive solution S3 under reduced pressure to solid content of 10 wt% to obtain concentrated solution, adding 4 times volume of anhydrous ethanol (dropping flow rate of 4mL/min in separating funnel) into the concentrated solution, standing at 4 deg.C for 16h, centrifuging to obtain precipitate, adding water for redissolving, concentrating under reduced pressure to remove ethanol, freeze drying, and making into fine powder of algal polysaccharide with particle size of 2 μm, which is respectively marked as Ascophyllum polysaccharide (ANP) and Macrocystin (MPP). Mixing ANP and MPP at a mass ratio of 1:10 to obtain algal polysaccharide composition, which is marked as ANP-MPP.
Washing pawpaw, kiwi fruit and guava respectively, cutting into pieces of pawpaw, pieces of kiwi fruit and pieces of guava respectively, and cutting into pieces of pawpaw: kiwifruit pieces: mixing fructus Psidii Guajavae Immaturus pieces at a mass ratio of 5:2:6 to obtain a mixture, adding 10 times of water, pulping, and extracting at 70 deg.C for 6 h. Centrifuging at 8000r/min for 15min, collecting supernatant, and filtering to obtain filtrate to obtain compound fruit juice.
Adding ANP-MPP into the composite fruit juice 60 times of the mass of the ANP-MPP, uniformly mixing, treating for 10s at 115 ℃ to obtain a mixed solution, and adjusting the pH of the mixed solution to 4.0. The lactobacillus rhamnosus is firstly inoculated in fresh cow milk and activated for 12 hours at the temperature of 37 ℃, and the inoculation amount is 0.1 g/L. Adding the activated lactobacillus rhamnosus microbial inoculum into the mixed solution of the algal polysaccharide and the compound fruit juice according to the volume ratio of 1%, and fermenting for 24h at 37 ℃. Sterilizing in boiling water bath for 15min, concentrating until the solid content is 15 wt%, and drying to obtain mixed fermented powder of ANP-MPP composition and compound fruit juice.
Example 6: animal experiment for reducing blood sugar by algal polysaccharide and composition thereof and compound fruit juice fermentation product
The blood sugar reducing animal experiment of the test sample and the result thereof are as follows:
the 96 SPF-grade db/db diabetic mice and 8 littermates of the SPF-grade db/m mice involved in the experiment were purchased from Changzhou Kavens laboratory animals Co., Ltd, and bred to Guangzhou Laidel Union Biotechnology Ltd, Guangzhou science City. Animal feed (SFP grade) was supplied by Hi Biotech, Guangzhou Laiden, and stored at 4 ℃. The temperature of the breeding room is maintained at 25 + -1 deg.C, the relative humidity is maintained at 55 + -5%, and the illumination is maintained for 12h and the darkness is maintained for 12h by controlling the illumination lamp. During the experiment, the animals were not restricted to food intake and water intake. All animals were randomized into groups of 4 animals per cage and 2 animals per group after 7 days of acclimatization. 8 SPF-grade db/m mice were enrolled as the NC group and were intervened with 1mL of saline daily during the experiment. 96 db/db mice were randomly divided into 12 groups of 8 mice each, registered as DC groups: during the experiment, 1mL of physiological saline was used for intervention every day; MET group: gavage 1mL of metformin solution daily during the experiment at an intervention dose of 200mg/kg body weight; SFP group: 1mL of sargassum fusiforme polysaccharide solution is gavaged every day during the experiment according to the intervention dosage of 800mg/kg of body weight; ANP group: 1mL of the Ascophyllum nodosum polysaccharide solution is gavaged every day during the experiment according to the intervention dosage of 800mg/kg of body weight; MPP group: 1mL of the kelp polysaccharide solution is gavaged every day during the experiment according to the intervention dosage of 800mg/kg of body weight; SCP group: 1mL of sargassum polysaccharide solution is intragastrically administered every day during the experiment period according to the intervention dosage of 800mg/kg body weight; USP group: 1mL of undaria pinnatifida polysaccharide solution is intragastrically infused every day during the experiment period according to the intervention dosage of 800mg/kg of body weight; FVCF group: 1mL of fruit and vegetable composite fermentation product solution is perfused every day during the experiment period according to the intervention dosage of 800mg/kg of body weight; ANP-SFP group: irrigating 1mL of the composition solution of the zostera marina polysaccharide and the sargassum fusiforme polysaccharide every day during the experiment according to the intervention dosage of 800mg/kg body weight; SCP-USP group: irrigating 1mL of composition solution of sargassum polysaccharide and undaria pinnatifida polysaccharide every day during the experiment according to the intervention dosage of 800mg/kg body weight; ANP-MPP group: irrigating 1mL of the composition solution of the ascophyllum nodosum polysaccharide and the macroalgae polysaccharide every day during the experiment according to the intervention dosage of 800mg/kg body weight; ASFM group: irrigating 1mL of a mixed solution of the composition of the ascophyllum nodosum polysaccharide and the sargassum fusiforme polysaccharide and the compound fruit juice fermentation product every day according to the intervention dosage of 800mg/kg of body weight during the experiment; SUFM group: irrigating 1mL of a mixed solution of sargassum polysaccharide and undaria pinnatifida polysaccharide composition and a compound fruit juice fermentation product every day according to an intervention dose of 800mg/kg body weight during an experiment; AMFM group: 1mL of the composition of the ascophyllum nodosum polysaccharide and the macroalgae polysaccharide and the mixed solution of the compound fruit juice fermentation product are irrigated with stomach every day during the experiment according to the intervention dosage of 800mg/kg body weight. After 56 days, all animals were fasted for 12 hours, bled by pulling the eye, and dissected for sampling after decapitation for subsequent analysis. The blood glucose of each mouse was measured using an ohilongglucometer HGM-114 and recorded.
There were no significant differences in initial blood glucose levels between diabetic mice in each experimental group (P < 0.05). After 8 weeks of test sample intervention, the blood glucose levels of the mice in each group are shown in fig. 1, and the data show that the fasting blood glucose values of the intervention groups are lower than those of the DC group, and have statistical difference (P < 0.05). In addition, blood glucose levels in animals in the ASFM, SUFM, AMFM groups were significantly lower than in the other test sample naive groups (P < 0.05). Specifically, fasting blood glucose levels were reduced by about 60.78% in the ASFM group compared to the DC group and by about 33.38% in the SHFM group compared to the MET group; the fasting blood glucose levels were reduced by about 61.23% for the SUFM group compared to the DC group and by about 34.08% for the SUFM group compared to the MET group; the fasting blood glucose levels were reduced by about 59.20% for the AMFM group compared to the DC group and by about 30.62% for the AMFM group compared to the MET group.
The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.
Claims (10)
1. A preparation method of algal polysaccharide composition and composite fruit juice mixed fermentation powder is characterized by comprising the following steps:
adding the algal polysaccharide composition into the compound fruit juice, uniformly mixing, heating for heating to obtain a mixed solution, adjusting the pH of the mixed solution to be acidic, adding a lactobacillus rhamnosus microbial inoculum, performing fermentation treatment, sterilizing, concentrating, and drying to obtain the algal polysaccharide composition and compound fruit juice mixed fermentation powder.
2. The method for preparing the algal polysaccharide composition and composite fruit juice mixed baking powder according to claim 1, wherein the particle size of the algal polysaccharide composition is 2-20 μm; the composition of the algal polysaccharides comprises two or more than two of the following algal polysaccharides: fine micropowder of polysaccharide from Ascophyllum Nodosum, fine micropowder of polysaccharide from Cyrtymenia Sparsa, fine micropowder of polysaccharide from Fucus vesiculosus, fine micropowder of polysaccharide from Sargassum, fine micropowder of polysaccharide from Undaria pinnatifida, and fine micropowder of polysaccharide from Macrocystis.
3. The method for preparing the algal polysaccharide composition and composite fruit juice mixed fermentation powder according to claim 2, wherein the algal polysaccharide composition is one of a composition in which a mass ratio of fine powder of ascophyllum nodosum polysaccharide to fine powder of sargassum fusiforme polysaccharide is 1:1-4, a composition in which a mass ratio of fine powder of fucoidin to fine powder of ascophyllum nodosum polysaccharide is 1:10-12, a composition in which a mass ratio of fine powder of ascophyllum nodosum polysaccharide to fine powder of undaria pinnatifida polysaccharide is 1:7-8, a composition in which a mass ratio of fine powder of macroalgae polysaccharide to fine powder of undaria pinnatifida polysaccharide is 1:1-2, a composition in which a mass ratio of fine powder of fucoidin to fine powder of fucoidin is 1:10-15, a composition in which a mass ratio of fine powder of ascophyllum nodosum polysaccharide to fine powder of macroalgae is 1:8-10, and a composition in which a mass ratio of fine powder of ascophyllum nodosum polysaccharide is 1:1-2 And (4) performing the steps.
4. The method for preparing the algal polysaccharide composition and compound fruit juice mixed baking powder according to claim 2, wherein the preparation of algal polysaccharide comprises the following steps:
(1) cleaning seaweed, air drying, soaking in ethanol solution, heating for reflux extraction, filtering to obtain filter residue, and drying to obtain ethanol-treated seaweed;
(2) soaking the seaweed treated by the ethanol in the step (1) in water, pulping to obtain paste, adding water into the paste to obtain mixed solution 1, refrigerating, melting, leaching, centrifuging, and taking supernatant to obtain extract 1;
(3) adjusting the pH value of the extracting solution 1 in the step (2) to be acidic, adding papain to obtain a mixed solution 2, heating for oscillation treatment, then inactivating enzyme, centrifuging to obtain a supernatant, and concentrating to obtain an extracting solution 2;
(4) and (3) passing the extracting solution 2 obtained in the step (3) through an ultrafiltration membrane, taking components with the molecular weight of more than 10kD to obtain an extracting solution 3, concentrating the extracting solution 3 to obtain a concentrated solution, adding absolute ethyl alcohol into the concentrated solution, standing, centrifuging to obtain a precipitate, redissolving the precipitate, carrying out reduced pressure concentration to remove the ethyl alcohol, and carrying out freeze drying to obtain the algal polysaccharide with the particle size of 2-20 microns.
5. The method for preparing algal polysaccharide composition and composite fruit juice mixed fermentation powder according to claim 4, wherein the seaweed in the step (1) is one of Ascophyllum nodosum, Cyrtymenia Sparsa, Fucus vesiculosus, Sargassum, Undaria pinnatifida, and Macrocystis japonica; the volume percentage concentration of the ethanol solution in the step (1) is 90-100%; the temperature of the reflux extraction treatment in the step (1) is 75-85 ℃, and the time of the reflux extraction treatment is 2-6 h; soaking the seaweed treated by the ethanol in water for 3-5 hours in the step (2); in the paste obtained in the step (2), the mass ratio of the seaweed treated by the ethanol to the water is 1: 3-8; in the mixed solution in the step (2), the mass ratio of the seaweed treated by the ethanol to the water is 1: 15-30; the temperature of the cold storage treatment is-80 to-40 ℃, and the time of the cold storage treatment is 2 to 5 hours; the temperature of the melting treatment in the step (2) is 60-80 ℃; the times of the cold storage treatment and the melting treatment in the step (2) are 2-3 times; the temperature of the leaching treatment in the step (2) is 80-100 ℃, and the time of the leaching treatment is 2-8 h.
6. The method for preparing algal polysaccharide composition and composite fruit juice mixed fermentation powder according to claim 4, wherein in the step (3), the pH of the extraction solution 1 is adjusted to 5.5-6.0; in the mixed solution 2 in the step (3), the enzyme activity of the papain is 50-200U/L; the temperature of the oscillation treatment in the step (3) is 55-65 ℃, and the time of the oscillation treatment is 50-80 min; the enzyme deactivation treatment in the step (3) is boiling water bath for 5-10 min; the solid content in the extracting solution 2 in the step (3) is 2-8 wt%; the molecular weight cut-off of the ultrafiltration membrane in the step (4) is 10 kD; the adding speed of the absolute ethyl alcohol in the step (4) is 3-8 mL/min; the solid content in the concentrated solution in the step (4) is 5-12 wt%; the volume ratio of the concentrated solution in the step (4) to the absolute ethyl alcohol is 1: 4; and (4) standing for 12-16h at 4 ℃.
7. The method for preparing the algal polysaccharide composition and compound fruit juice mixed baking powder according to claim 1, wherein the preparation of the compound fruit juice comprises:
cleaning pawpaw, kiwi fruit and guava, cutting, mixing to obtain a mixture, adding water, pulping, heating in a shaking table for extraction treatment, centrifuging to remove precipitates, filtering to obtain filtrate, and thus obtaining the compound fruit juice;
the mass ratio of the pawpaw to the kiwi fruit to the guava is 5:2: 6; the mass ratio of the mixture to the water is 1: 10-15; the temperature of the extraction treatment is 60-70 ℃, and the time of the extraction treatment is 6-8 h; the rotating speed of the centrifugation is 8000-12000r/min, and the time of the centrifugation is 10-15 min.
8. The method for preparing the algal polysaccharide composition and compound fruit juice mixed baking powder according to claim 1, wherein the mass ratio of the algal polysaccharide composition to the compound fruit juice is 1: 50-80; the temperature of the heating treatment is 100-115 ℃, and the time of the heating treatment is 10-30 s; the pH value of the mixed solution is adjusted to 3.0-4.0; the volume of the lactobacillus rhamnosus agent is 0.5-2% of the volume of the mixed solution; the temperature of the fermentation treatment is 37-40 ℃, and the time of the fermentation treatment is 12-24 h; the sterilization treatment is boiling water bath for 15-20 min; concentrating until the solid content is 15-20 wt%.
9. A seaweed polysaccharide composition prepared by the preparation method of any one of claims 1-8 and mixed fermentation powder of compound fruit juice.
10. The use of the algal polysaccharide composition of claim 9 in combination with a composite fruit juice fermentation powder for the preparation of health foods, foods and pharmaceuticals for diabetes and its complications.
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