CN113491331B - Algal polysaccharide composition and compound fruit juice mixed fermentation powder as well as preparation method and application thereof - Google Patents
Algal polysaccharide composition and compound fruit juice mixed fermentation powder as well as preparation method and application thereof Download PDFInfo
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- CN113491331B CN113491331B CN202110745091.1A CN202110745091A CN113491331B CN 113491331 B CN113491331 B CN 113491331B CN 202110745091 A CN202110745091 A CN 202110745091A CN 113491331 B CN113491331 B CN 113491331B
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- polysaccharide
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- fruit juice
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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 a frequently encountered disease which currently endanger 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 effects, high treatment cost, different degrees of 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 in the conventional fruit and vegetable fermentation process, a carbon source such as glucose and the like is usually required to be additionally added 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 invention provides a preparation method of seaweed polysaccharide composition and composite fruit juice mixed fermentation powder, which 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 algal polysaccharide composition is one or more of a composition in which the mass ratio of fine ascophyllum polysaccharide powder to fine ascophyllum polysaccharide powder is 1 to 4, a composition in which the mass ratio of fine ascophyllum polysaccharide powder to fine ascophyllum polysaccharide powder is 1 to 10 to 12, a composition in which the mass ratio of fine ascophyllum polysaccharide powder to fine ascophyllum polysaccharide powder is 1 to 7 to 8, a composition in which the mass ratio of fine ascophyllum polysaccharide powder to fine ascophyllum polysaccharide powder is 1 to 2, a composition in which the mass ratio of fine ascophyllum polysaccharide powder to fine ascophyllum polysaccharide powder is 1 to 10 to 15, a composition in which the mass ratio of fine ascophyllum polysaccharide powder to fine ascophyllum polysaccharide powder is 1 to 10, and a composition in which the mass ratio of fine ascophyllum polysaccharide powder to fine ascophyllum polysaccharide powder is 1 to 2.
Further, the preparation of the seaweed polysaccharide (one of the fucus polysaccharide fine powder, the sargassum fusiforme polysaccharide fine powder, the undaria polysaccharide fine powder, the gulfweed polysaccharide fine powder, the leafy seaweed polysaccharide fine powder and the kelp 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 (4) passing the extracting solution 2 obtained in the step (3) through an ultrafiltration membrane, taking a component 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 mu m.
Further, in the step (1) of preparing the algal polysaccharide, 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 2-6h;
further, in the step (2) of preparing the algal polysaccharide, the time of soaking the seaweed after ethanol treatment in water is 3-5h;
further, in the step (2) of preparing the algal polysaccharides, the mass ratio of the seaweed after ethanol treatment to water in the paste is 1;
further, in the step (2) of preparing algal polysaccharides, the mass ratio of the seaweed after ethanol treatment to water in the mixed solution is 1;
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 2-8h.
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 50-80min;
further, in the step (3) of preparing the algal polysaccharide, the enzyme deactivation treatment is boiling water bath for 5-10min;
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 2-8wt%;
further, in the step (4) of algal polysaccharide preparation, the ultrafiltration membrane (hollow ultrafiltration membrane) has a molecular weight cut-off of 10kD;
preferably, in the step (4) of preparing algal polysaccharide, the extract 2 is repeatedly passed through the ultrafiltration membrane 4-8 times.
Further, in the step (4) of preparing the algal polysaccharide, the adding rate of the absolute ethyl alcohol is 3-8mL/min;
further, in the step (4) of preparing the algal polysaccharide, the solid content in the concentrated solution is 5-12wt%;
further, in the step (4) of preparing the algal polysaccharide, the volume ratio of the concentrated solution to the absolute ethyl alcohol is 1;
preferably, in the step (4) of preparing the algal polysaccharide, anhydrous ethanol is added to the concentrated solution by using a separating funnel.
Further, in the step (4) for preparing the algal polysaccharide, the standing temperature is 4 ℃, and the standing time is 12-16h.
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;
further, in the preparation process of the composite fruit juice, the mass ratio of the mixture to water is 1;
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 6-8h;
further, in the preparation process of the compound fruit juice, the rotation speed of the centrifugation is 8000-12000r/min, and the time of the centrifugation is 10-15min.
Further, the mass ratio of the algal polysaccharide composition to the compound fruit juice is 1;
further, the temperature of the heating treatment is 100-115 ℃, and the time of the heating treatment is 10-30s;
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 12-24h;
preferably, the fermentation treatment time is 24h.
Further, the sterilization treatment is boiling water bath for 15-20min;
further, the concentration is carried out until the content of the solid matters is 15-20wt%.
The invention provides a seaweed polysaccharide composition prepared by the preparation method and composite fruit juice mixed fermentation powder.
The seaweed polysaccharide composition and the composite fruit juice mixed fermentation powder provided by the invention can be applied to the preparation of health-care foods, foods 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 various algal polysaccharide compositions with more remarkable alpha-glucosidase inhibitory activity than that of single algal polysaccharide in vitro, and the optimal combination proportion range is limited through process optimization, so that the harm of the algal polysaccharide composition and the compound fruit juice mixed fermentation powder to patients by reducing blood sugar fluctuation caused by eating is ensured.
(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 included to further illustrate the practice of the invention, but are not intended to limit the practice or protection of the invention. 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/L 2 CO 3 The 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 micro powder:
cleaning Sargassum (Sargassum is one of Ascophyllum nodosum, cyrtymenia Sparsa, fucus vesiculosus, sargassum, thallus laminariae, 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 after ethanol treatment in water (the mass of the water is 6 times of that of the seaweed after ethanol treatment), soaking for 4 hours, pulping to obtain paste, and then adding water into the paste to obtain a mixed solution 1, wherein in the mixed solution 1, the mass ratio of the seaweed after ethanol treatment to the water is 1; 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 extract S1;
adjusting the pH value of the extracting solution S1 to 6.0, adding 1wt% of papain, oscillating in a water bath shaker at 55 ℃ for 60min, then inactivating enzyme in a boiling water bath for 10min, cooling to 25 ℃, centrifuging to obtain a supernatant, and concentrating the solid content to 6wt% 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 extracting solution S3 under reduced pressure until the solid content is 10wt% 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 4 mL/min), standing for 16h at 4 ℃, centrifuging to obtain a precipitate, adding water for redissolution, concentrating under reduced pressure to remove ethanol, and after freeze drying, preparing the algal polysaccharide fine powder with the particle size of 2 mu m, wherein the mark is respectively: fucus vesiculosus fine powder (FVP), cyrtymenia Sparsa Fine Powder (SFP), undaria pinnatifida fine powder (USP), sargassum fine powder (SCP), ascophyllum nodosum fine powder (ANP), and Macrocystis giganteum 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 rate 50 ) Values, and 0.125 times IC were selected respectively 50 0.25 times IC 50 0.5 times IC 50 0.75 times IC 50 ，IC 50 1.25 times IC 50 And 1.5 times IC 50 The concentration of the polysaccharide composition is mixed in pairs according to the ratio of 1IC of individual polysaccharides 50 And 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.
IC 50 The value refers to half dosage concentration, IC, of the antagonist in inhibiting enzyme activity and cell receptor 50 The 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 combination 50 Is obviously reduced. Specifically, IC of FVP combined with SFP, ANP, MPP, SCP, USP 50 The reduction is 42.86%, 52.38%, 38.10%, 80.95% and 42.86% respectively; IC of SFP combined with FVP, ANP, MPP, SCP 50 Respectively reduced by 49.55%, 65.10%, 55.15% and 56.24%; IC of ANP combined with FVP, SFP, MPP, SCP, USP 50 The reduction is respectively 62.77%, 66.67%, 63.48%, 65.60% and 20.21%; IC of MPP combined with FVP, SFP, ANP, SCP, USP 50 Respectively reduced by 46.66%, 53.33%, 59.92%, 41.03% and 71.90%; IC of SCP combined with FVP, SFP, ANP, MPP, USP 50 The reduction is 86.48%, 65.56%, 71.43%, 55.36% and 78.06% respectively; IC of USP combined with FVP, ANP, MPP, SCP 50 The 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 alpha-glucosidase inhibition
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 respectively 7.812 and 12.573.
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-woven fabric CI value <1.1 shows that the action effect has an overlapping action; CI >1.1 indicates that the effect has antagonism, and the larger the value is, the more remarkable the antagonism is. 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 synergistic effect is more significant when the concentration is higher. By optimizing the combination proportion, the coordination and synergism of the compositions are 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 according to the mass ratio of 1; mixing the FVP and the ANP according to the mass ratio of 1; mixing the SCP and the USP according to the mass ratio of 1; mixing MPP and USP according to the mass ratio of 1-2 to obtain a seaweed polysaccharide composition, and recording as MPP-USP; mixing the FVP and the SCP according to the mass ratio of 1; mixing ANP and MPP according to the mass ratio of 1; and (3) mixing the ANP and the SCP according to the mass ratio of 1 to 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 papaya, kiwi fruit and guava respectively, cutting the cleaned papaya, kiwi fruit and guava into fruit fragments, adding water respectively, wherein the mass ratio of the fruit fragments to the water is 1;
washing pawpaw, kiwi fruit and guava respectively, cutting into pawpaw fragments, kiwi fruit fragments and guava fragments respectively, and mixing the pawpaw fragments: kiwifruit pieces: mixing the guava pieces at a mass ratio of 5. 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 after ethanol treatment in water (the mass of the water is 6 times of that of the seaweed after ethanol treatment), soaking for 4 hours, pulping to obtain paste, and then adding water into the paste to obtain a mixed solution 1, wherein in the mixed solution 1, the mass ratio of the seaweed after ethanol treatment to the water is 1; 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 an extracting solution S1;
adjusting the pH value of the extracting solution S1 to 6.0, adding 1wt% of papain, oscillating in a water bath shaker at 55 ℃ for 60min, then inactivating enzyme in a boiling water bath for 10min, cooling to 25 ℃, centrifuging to obtain a supernatant, and concentrating the solid content to 6wt% to obtain an extracting solution S2.
Separating the extractive solution S2 with hollow ultrafiltration membrane with molecular weight cutoff 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 10wt% 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 16 hr, centrifuging to obtain precipitate, adding water for redissolving, concentrating under reduced pressure to remove ethanol, and freeze drying to obtain 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 according to the mass ratio of 1.
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 the guava pieces at a mass ratio of 5. 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 37 ℃, and the inoculation amount is 0.1g/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 15wt%, 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 after ethanol treatment in water (the mass of the water is 6 times of that of the seaweed after 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 in the mixed solution 1, the mass ratio of the seaweed after ethanol treatment to the water is 1; placing at-80 deg.C, freezing for 3 hr, 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 1wt% 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 6wt% 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 10wt% 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 16 hr, centrifuging to obtain precipitate, adding water for redissolving, concentrating under reduced pressure to remove ethanol, and freeze drying to obtain fine powder of Sargassum polysaccharide (SCP) and Undaria pinnatifida polysaccharide (USP) with particle size of 2 μm. And mixing the SCP and the USP according to the mass ratio of 1.
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 milk and activated for 12 hours at 37 ℃, and the inoculation amount is 0.1g/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 15wt%, 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 air drying Ascophyllum nodosum and Macrocystis giganteum, respectively soaking in (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.
Adding the ethanol-treated seaweed, soaking in water (the mass of the water is 6 times of that of the ethanol-treated seaweed), soaking for 4 hours, pulping to paste to obtain paste, and then adding water into the paste to obtain a mixed solution 1, wherein in the mixed solution 1, the mass ratio of the ethanol-treated seaweed to the water is 1; placing at-80 deg.C, freezing for 3 hr, thawing at 60 deg.C, repeatedly freezing and thawing for 3 times; hot water leaching is carried out for 4 hours at the temperature of 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 1wt% 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 6wt% 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 extract S3 under reduced pressure until the solid content is 10wt% to obtain a concentrated solution, adding 4 times volume of anhydrous ethanol (the dropping flow rate of the separating funnel is controlled to be 4 mL/min) into the concentrated solution by using a separating funnel, standing for 16h at 4 ℃, centrifuging to obtain a precipitate, adding water for redissolving, concentrating under reduced pressure to remove ethanol, and freeze-drying to prepare seaweed polysaccharide fine powder with the particle size of 2 mu m, wherein the fine powder is respectively marked as Ascophyllan (ANP) and macroalgae polysaccharide (MPP). Mixing the ANP and the MPP according to the mass ratio of 1.
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 the crushed guava blocks in a mass ratio of 5. 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 milk and activated for 12 hours at 37 ℃, and the inoculation amount is 0.1g/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 15wt%, 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, composition of algal polysaccharide 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 period according to the intervention dosage of 800mg/kg body weight; SCP group: 1mL of sargassum polysaccharide solution is gavaged every day during the experiment period according to the intervention dosage of 800mg/kg body weight; USP group: performing intragastric administration on 1mL of undaria pinnatifida polysaccharide solution 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 intragastrically infused every day during the experiment period according to the intervention dosage of 800mg/kg body weight; ANP-SFP group: irrigating 1mL of composition solution of the ascophyllum nodosum polysaccharide and the sargassum fusiforme polysaccharide every day during the experiment according to the intervention dose of 800mg/kg body weight; SCP-USP group: 1mL of sargassum polysaccharide and undaria pinnatifida polysaccharide composition solution is intragastrically injected 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: performing intragastric administration of 1mL of a mixed solution of the sargassum polysaccharide and undaria pinnatifida polysaccharide composition and the compound fruit juice fermentation product every day according to the intervention dosage of 800mg/kg of body weight during the experiment period; 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 to remove the neck to sacrifice the animals for subsequent analysis. The blood glucose of each mouse was measured using an ohron glucometer 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% in the SUFM group compared to the DC group and by about 34.08% in the SUFM group compared to the MET group; fasting blood glucose levels were reduced by about 59.20% in the AMFM group compared to the DC group and by about 30.62% in 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 mixed fermentation powder of the algal polysaccharide composition and the compound fruit juice;
the seaweed polysaccharide composition is more than one of a composition with a mass ratio of the fine polysaccharide micro powder of the ascophyllum nodosum to the fine polysaccharide micro powder of the sargassum fusiforme of 1 to 4, a composition with a mass ratio of the fine polysaccharide micro powder of the fucoidin to the fine polysaccharide micro powder of the ascophyllum nodosum of 1 to 10 to 12, a composition with a mass ratio of the fine polysaccharide micro powder of the Sargassum to the fine polysaccharide micro powder of the undaria pinnatifida of 1 to 7 to 8, a composition with a mass ratio of the fine polysaccharide micro powder of the kelp to the fine polysaccharide micro powder of 1 to 2, a composition with a mass ratio of the fine polysaccharide micro powder of the fucoidin to the fine polysaccharide micro powder of the Sargassum of 1 to 10 to 15, a composition with a mass ratio of the fine polysaccharide micro powder of the ascophyllum nodosum to the fine polysaccharide micro powder of the kelp of 1 to 8 to 10, and a composition with a mass ratio of the fine polysaccharide micro powder of the ascophyllum nodosum of 1 to 2.
2. The method for preparing the algal polysaccharide composition and complex fruit juice mixed baking powder of claim 1, wherein the particle size of the algal polysaccharide composition is 2-20 μm.
3. The method for preparing the algal polysaccharide composition and compound fruit juice mixed baking powder according to claim 1, 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 (4) passing the extracting solution 2 obtained in the step (3) through an ultrafiltration membrane, taking a component 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 mu m.
4. The method for preparing algal polysaccharide composition and composite fruit juice mixed fermentation powder according to claim 3, 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 2-6h; 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 1 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 2-8h.
5. The method for preparing the seaweed polysaccharide composition and composite fruit juice mixed baking powder as claimed in claim 3, wherein in the step (3), the pH of the extract 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 50-80min; the enzyme deactivation treatment in the step (3) is boiling water bath for 5-10min; the solid content in the extracting solution 2 in the step (3) is 2-8wt%; the molecular weight cut-off of the ultrafiltration membrane in the step (4) is 10kD; the adding speed of the absolute ethyl alcohol in the step (4) is 3-8mL/min; the solid content in the concentrated solution in the step (4) is 5-12wt%; the volume ratio of the concentrated solution obtained in the step (4) to the absolute ethyl alcohol is 1; and (4) standing for 12-16h at 4 ℃.
6. 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, the kiwi fruit and 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 6-8h; the rotating speed of the centrifugation is 8000-12000r/min, and the time of the centrifugation is 10-15min.
7. 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 10-30s; the pH value of the mixed solution is adjusted to 3.0-4.0; the volume of the lactobacillus rhamnosus microbial inoculum 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 12-24h; the sterilization treatment is boiling water bath for 15-20min; concentrating until the solid content is 15-20wt%.
8. A seaweed polysaccharide composition prepared by the preparation method of any one of claims 1-7 and a composite fruit juice mixed fermentation powder.
9. The use of the algal polysaccharide composition of claim 8 in combination with a complex fruit juice fermentation broth for the preparation of a health food for the adjuvant treatment of blood glucose.
10. The use of the algal polysaccharide composition of claim 8 in combination with a complex fruit juice fermentation broth for the preparation of a hypoglycemic medicament.
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