Chewable tablets for improving fetal alcohol syndrome and their uses

Abstract

The present invention relates to chewable tablets for improving fetal alcohol syndrome and their uses, belonging to the technical field of health foods. The technical problem solved by the present invention is to provide a chewable tablet that can improve fetal alcohol syndrome. The chewable tablet for improving fetal alcohol syndrome of the present invention has active ingredients composed of the following components in parts by weight: 0.5 to 65 parts of lactic acid bacteria and 0.10 to 12 parts of oleuropein. The chewable tablet of the present invention can effectively improve fetal alcohol syndrome, has a good taste, is easily acceptable to pregnant women, has a low cost, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to chewable tablets for improving fetal alcohol syndrome and their uses, belonging to the technical field of health foods. Background Art

[0002] Ethanol can freely cross the blood-brain barrier and the egg membrane of zebrafish embryos. Within 24 hours of development, 0.05% ethanol (lower than the upper limit of alcohol detection of 0.08% stipulated in the United States) has significantly affected the cognitive and memory abilities of zebrafish, affected the development of the cranial bones and facial nerves, and further affected the eyes, chin, skull width, etc. Clinically, if alcohol is contacted during pregnancy or fetal development, the offspring will develop fetal alcohol syndrome (FAS), or alcohol-related neurodevelopmental disorder (ARND), or fetal alcohol spectrum disorders (FASD), or fetal alcohol effects (FAE), manifested as microencephaly, thinning of the upper lip, short stature, mental retardation, abnormal movement, abnormal development of learning, memory and hearing nerves, and abnormal cardiovascular development. The occurrence probability of such diseases globally is approximately 1‰ - 9%.

[0003] Olive oil is rich in bioactive substances. Among them, polyphenolic compounds are the main effective active ingredients in olive oil, including secoiridoid compounds, such as oleuropein (OLE), hydroxytyrosol, etc. Among them, oleuropein and hydroxytyrosol are called "superfunctional foods" because of their strong antioxidant properties and physiological functions, and the European Food Safety Authority recommends regular consumption. Research shows that olive leaf extract can reduce the oxidation degree of low-density lipoprotein and prevent the occurrence of coronary heart disease, atherosclerosis and inflammation. Olive leaf extract also has pharmacological effects such as relaxing vascular smooth muscle, lowering blood pressure, reducing blood lipids, lowering blood sugar and inhibiting cancer. Olive leaf extract also has antibacterial, anti-inflammatory and antiviral functions. In addition, olive leaf extract can also protect the heart, brain, nerves, liver, kidneys and skin from the toxic effects of external chemicals. Previous studies have shown that olive leaf extract can improve ethanol-induced liver, gastric, intestinal mucosa and testicular damage by enhancing antioxidant capacity and has a protective effect on alcoholic digestive system damage. At the same time, animal experiments have shown that olive leaf extract is safe. After oral administration of olive leaf extract at a concentration of 1000 mg / kg bwt for 14 days or 23 weeks, no death or any signs of poisoning occurred in rats. Only high-dose (0.2 - 9%) olive leaf extract can cause hepatotoxicity in mice or rats.

[0004] The patent application with the application number CN201811486827.2 and the invention title "Application of Olive Leaf Extract in the Preparation of Drugs for Preventing and Treating Fetal Alcohol Syndrome" discloses that olive leaf extract has the effect of preventing and treating fetal alcohol syndrome. However, olive leaf extract has a bitter and pungent taste, which will affect the sensory quality of the product, and direct consumption by pregnant women may be more uncomfortable. Therefore, it is necessary to mask its unpleasant flavor when applying. Among them, microencapsulation is a commonly used embedding method. Commonly used wall materials for preparing microcapsules include chitosan, agar, sodium alginate, carrageenan, gum arabic, gum arabic powder, pectin, carboxymethyl cellulose, maltodextrin, dextrin, starch, resistant starch, modified starch, egg white, etc. In recent years, scientists at home and abroad have studied the preparation of microcapsules containing olive leaf extract using casein, sodium alginate and their composite wall materials, polylactic acid, and maltodextrin as wall materials. The patent applications with the application numbers CN20141064295.6 and the invention title "Preparation Method of an Oleuropein Liposome" and CN201710222330.9 and the invention title "Preparation Method of a Long-Circulating Liposome of an Olive Polyphenol Extract Rich in Hydroxytyrosol and Verbascoside" both disclose the preparation of microcapsules or nanoparticles of olive leaf extract using liposomes as wall materials. However, the cost of liposomes is relatively high.

[0005] Lactic acid bacteria (LAB) are a general term for a group of bacteria that can produce a large amount of lactic acid by fermenting sugars. Taxonomically, they can be divided into: Lactobacillus, Bifidobacterium, Streptococcus, Enterococcus, Lactococcus, Leuconostoc, Pediococcus, Atopobium, Sporolactobacillus, Brochothrix, Gemella, and Saccharococcus, etc.; morphologically, they are mainly divided into spherical and rod-shaped lactic acid bacteria. As probiotics, lactic acid bacteria directly participate in regulating the intestinal flora, inhibiting the reproduction of pathogenic bacteria under the established flora dominance, and maintaining the intestinal microecological balance, thereby improving gastrointestinal function, increasing food digestibility and nutrient absorption rate. Lactic acid bacteria can also indirectly reduce serum cholesterol, control endotoxin, promote animal growth, enhance the body's immunity, and have antioxidant effects by regulating relevant metabolic pathways in the human body. It has been found that lactic acid bacteria have a protective function against liver and gastric injuries caused by ethanol, but there is no relevant report on the protective effect of lactic acid bacteria on fetal alcohol syndrome. Summary of the Invention

[0006] The first technical problem to be solved by the present invention is to provide a chewable tablet that can improve fetal alcohol syndrome.

[0007] The chewable tablet for improving fetal alcohol syndrome of the present invention has an active ingredient composed of the following components in a weight ratio: 0.5 - 65 parts of lactic acid bacteria and 0.10 - 12 parts of oleuropein.

[0008] Preferably, the chewable tablet for improving fetal alcohol syndrome of the present invention has an active ingredient composed of the following components in a weight ratio: 0.6 - 60 parts of lactic acid bacteria and 0.11 - 11.1 parts of oleuropein.

[0009] Among them, the above-mentioned oleuropein can be replaced by an olive leaf extract with a corresponding oleuropein content; the above-mentioned oleuropein can also be replaced by an olive leaf extract microcapsule or an oleuropein microcapsule with a corresponding oleuropein content.

[0010] Further, the above-mentioned olive leaf extract microcapsule is preferably prepared by mixing an olive leaf extract and a wall material in a weight ratio of 1:0.8 - 2.5 and drying; among them, the wall material is at least one of maltodextrin, β-cyclodextrin, sodium alginate, and chitosan.

[0011] Furthermore, the above-mentioned olive leaf extract microcapsule is preferably prepared by mixing an olive leaf extract and a wall material in a weight ratio of 1:1 and drying, and the wall material is maltodextrin. As another preferred technical solution, the olive leaf extract microcapsule is prepared by mixing an olive leaf extract and a wall material in a weight ratio of 1:2 and drying, and the wall material is a mixture of maltodextrin and β-cyclodextrin, and the weight ratio of maltodextrin to β-cyclodextrin is 1:1.

[0012] The chewable tablets for improving fetal alcohol syndrome of the present invention further comprise pharmaceutically or food-acceptable excipient components; the excipients are preferably at least one of sweeteners, fillers, antioxidants, thickeners, disintegrants, binders, stabilizers, glidants, and flavor correctors.

[0013] As a more preferred technical solution, the chewable tablets for improving fetal alcohol syndrome of the present invention are prepared from the following components in the following weight ratios: 130-160 parts of olive leaf extract microcapsules, 45-55 parts of lactic acid bacteria, 90-110 parts of erythritol, 18-19 parts of sucrose, 4-6 parts of citric acid, 180-220 parts of starch, and 3-4 parts of magnesium stearate; the lactic acid bacteria are at least one of Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Leuconostoc, Pediococcus, and Sporolactobacillus that can be edible or medicinal.

[0014] Preferably, the lactic acid bacteria are at least one of Lactobacillus rhamnosus, Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir, Bifidobacterium lactis, Bifidobacterium longum, and Bifidobacterium infantis; more preferably, the lactic acid bacteria are a mixture of Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir, Bifidobacterium lactis, Bifidobacterium longum, and Bifidobacterium infantis in a weight ratio of 1:1:1:1:1:1 of the bacterial powder.

[0015] The chewable tablets for improving fetal alcohol syndrome of the present invention are most preferably prepared from the following components in the following weight ratios: 155 parts of olive leaf extract microcapsules, 50 parts of lactic acid bacteria, 100 parts of erythritol, 18.65 parts of sucrose, 5 parts of citric acid, 200 parts of starch, and 3.5 parts of magnesium stearate.

[0016] The chewable tablets for improving fetal alcohol syndrome of the present invention can be prepared by a conventional method for preparing chewable tablets. For example, it can be prepared by raw material mixing → dry granulation → sieving → adding lubricant → mixing → tabletting.

[0017] The second technical problem to be solved by the present invention is to provide the use of the above-mentioned chewable tablets for improving fetal alcohol syndrome in the preparation of drugs or foods for treating / preventing / improving fetal alcohol syndrome.

[0018] The third technical problem to be solved by the present invention is to provide the use of lactic acid bacteria in the preparation of drugs or foods for treating / preventing / improving fetal alcohol syndrome.

[0019] Among them, the lactic acid bacteria can be at least one of Lactobacillus, Bifidobacterium, Streptococcus, Enterococcus, Lactococcus, Leuconostoc, Pediococcus, Atopobium, Sporolactobacillus, Brochothrix, Gemella, and Saccharococcus.

[0020] Preferably, the lactic acid bacteria are at least one of Lactobacillus rhamnosus, Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir, Bifidobacterium lactis, Bifidobacterium longum, and Bifidobacterium infantis.

[0021] More preferably, the lactic acid bacteria are a mixture of Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir, Bifidobacterium lactis, Bifidobacterium longum, and Bifidobacterium infantis in a weight ratio of 1:1:1:1:1:1 of the bacterial powder.

[0022] The chewable tablets of the present invention can effectively improve fetal alcohol syndrome, have a good taste, are easily accepted by pregnant women, have a low cost, and have broad application prospects. Description of the Drawings

[0023] Figure 1 It is a result diagram of different concentrations of oleuropein (OLE) improving the morphological development distortion of zebrafish embryos treated with 1.2% ethanol (EtOH); A: Normal hatched larvae developed to 96 hpf; B: Malformed larvae exposed to 1.2% ethanol and developed to 96 hpf, where M: Small eyes, PE: Pericardial edema, SB: Underdeveloped swim bladder, AYS: Abnormal yolk; (C)-(F): Results of 0.0342 - 34.2 μM oleuropein (OLE) reducing the morphological development distortion of zebrafish embryos caused by 1.2% ethanol; (G) Embryos treated with 34.2 μM oleuropein (OLE) developed to normal hatched larvae at 96 hpf.

[0024] Figure 2 It is a result diagram of different concentrations of oleuropein (OLE) improving the pericardial area (A), body length (B), eye diameter (C), head width (D) of zebrafish embryos treated with 1.2% ethanol (EtOH) at 96 hpf, as well as the number of spontaneous movements in 5 minutes at 24 hpf (E), and the number of heartbeats in 20 s at 48 and 72 hpf (F). The data differences between each treatment group were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the "ethanol" group, #p < 0.05, ##p < 0.01, p < 0.001.

[0025] Figure 3 It is a result diagram of different concentrations of oleuropein (OLE) improving the mortality rate (A), hatching rate (B), and malformation rate (C) of zebrafish embryos treated with 1.2% ethanol (EtOH).

[0026] Figure 4Results of different concentrations of oleuropein (OLE) improving the CAT activity (A) and SOD activity (B) of zebrafish embryos treated with 1.2% ethanol (EtOH) at 72 hpf. The data differences between treatment groups were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p<0.05, **p<0.01, ***p<0.001; compared with the "ethanol" group, #p<0.05, ##p<0.01.

[0027] Figure 5 Results of different concentrations of oleuropein (OLE) improving the GPX4 activity (A), GSH content (B), iron content (C) and MDA content (D) of zebrafish embryos treated with 1.2% ethanol (EtOH) at 72 hpf. The data differences between treatment groups were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p<0.05, **p<0.01; compared with the "ethanol" group, #p<0.05, ##p<0.01.

[0028] Figure 6 Results of different concentrations of oleuropein (OLE) improving the acridine orange fluorescence staining (A), relative fluorescence intensity (B), Caspase9 activity (C) and Caspase3 activity (D) of zebrafish embryos / larvae treated with 1.2% ethanol (EtOH) at 72 hpf. Among them, a in Figure A is 0.1% DMSO, b is 1.2% EtOH, c is 1.2% EtOH + 0.0342 μM OLE, d is 1.2% EtOH + 0.342 μM OLE, e is 1.2% EtOH + 3.42 μM OLE, f is 1.2% EtOH + 34.2 μM OLE, g is 34.2 μM OLE. The data differences between treatment groups were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p<0.05, **p<0.01; compared with the "ethanol" group, #p<0.05, ##p<0.01.

[0029] Figure 7 Results of different concentrations of oleuropein (OLE) improving the relative fluorescence intensity of ROS in zebrafish embryos / larvae treated with 1.2% ethanol (EtOH) at 72 hpf. The data differences between treatment groups were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p<0.05; compared with the "ethanol" group, #p<0.05.

[0030] Figure 8 Ventral view of alcian blue staining of the head cartilage of 96 hpf zebrafish larvae. Among them, ep: basihyal, ch: ceratohyal, hs: hyomandibular arch, mc: Meckel's cartilage, pq: palatoquadrate bone.

[0031] Figure 9 Graph showing the measurement results of oleuropein (OLE) in improving the angle between the ceratohyal and the basihyal (A), the length of the ceratohyal (B), the length of Meckel's cartilage (C), the length of the palatoquadrate (D), the length of the basihyal body (E), and the length of the hyomandibular arch (F) in zebrafish larvae at 96 hpf treated with 1.2% ethanol (EtOH). Among them, ep: basihyal body, ch: ceratohyal, hs: hyomandibular arch, mc: Meckel's cartilage, pq: palatoquadrate. The data differences between treatment groups were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01; compared with the "ethanol" group, #p < 0.05, ##p < 0.01.

[0032] Figure 10 Graph showing the results of Lactobacillus rhamnosus (LR) and combined lactic acid bacteria (cLAB, Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis = 1:1:1:1:1:1) separately and in combination in improving the hatching rate (A), mortality rate (B), and malformation rate (C) of zebrafish embryos treated with 1.2% ethanol (EtOH).

[0033] Figure 11 Graph showing the results of Lactobacillus rhamnosus (LR) and combined lactic acid bacteria (cLAB, Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis = 1:1:1:1:1:1) separately and in combination in improving the body length (A), eye diameter (B), pericardial area (C), heart rate at 20 s (D), yolk area (E) of zebrafish embryos at 72 hpf treated with 1.2% ethanol (EtOH), and the number of spontaneous movements in 5 min at 24 hpf (F). The data differences between treatment groups were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the "ethanol" group, #p < 0.05.

[0034] Figure 12 Graph showing the results of combined lactic acid bacteria (cLAB, Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis = 1:1:1:1:1:1) and oleuropein (OLE) in combination in improving the hatching rate (A), mortality rate (B), and malformation rate (C) of zebrafish embryos treated with 1.2% ethanol (EtOH).

[0035] Figure 13Results of the combined improvement of the body length (A), eye diameter (B), pericardial area (C), heart rate at 20 s (D), yolk area (E), and spontaneous movement frequency at 5 min after 24 hpf (F) of zebrafish embryos treated with 1.2% ethanol (EtOH) by a mixture of lactic acid bacteria (cLAB, Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis = 1:1:1:1:1:1) and oleuropein (OLE). The data differences between treatment groups were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the "ethanol" group, #p < 0.05, ##p < 0.01.

[0036] Figure 14 Results of the combined improvement of the CAT activity (A), GSH content (B), SOD activity (C), and MDA content (D) of zebrafish embryos treated with 1.2% ethanol (EtOH) at 72 hpf by a mixture of lactic acid bacteria (cLAB, Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis = 1:1:1:1:1:1) and oleuropein (OLE). The data differences between treatment groups were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the "ethanol" group, #p < 0.05, ##p < 0.01.

[0037] Figure 15 Results of the combined improvement of the relative fluorescence intensity of acridine orange fluorescence (A), relative fluorescence intensity of ROS (B), Caspase9 activity (C), and Caspase3 activity (D) of zebrafish embryos treated with 1.2% ethanol (EtOH) at 72 hpf by a mixture of lactic acid bacteria (cLAB, Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis = 1:1:1:1:1:1) and oleuropein (OLE). The data differences between treatment groups were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01; compared with the "ethanol" group, #p < 0.05, ##p < 0.01.

[0038] Figure 16 Results of the encapsulation efficiency of olive leaf extract microcapsules prepared with different wall materials. Among them, Figure A shows the encapsulation efficiency of microcapsules prepared with a single wall material, and Figure B shows the encapsulation efficiency of microcapsules prepared with a composite wall material.

[0039] Figure 17Results graphs of the particle sizes of microcapsules of olive leaf extract prepared with different wall materials. Among them, Figure A shows the particle size of the microcapsules prepared with maltodextrin as the wall material (maltodextrin:core material = 1:1, g:g); Figure B shows the particle size of the microcapsules prepared with β-cyclodextrin as the wall material (β-cyclodextrin:core material = 2:1, g:g); Figure C shows the particle size of the microcapsules prepared with sodium alginate as the wall material (sodium alginate:core material = 1:1, g:g); Figure D shows the particle size of the microcapsules prepared with chitosan as the wall material (chitosan:core material = 2:1, g:g); Figure E shows the particle size of the microcapsules prepared with maltodextrin and β-cyclodextrin as the composite wall material (maltodextrin:β-cyclodextrin:core material = 2:2:1, g:g:g); Figure F shows the particle sizes of the microcapsules prepared with different wall materials.

[0040] Figure 18 Appearance graphs of microcapsules of olive leaf extract prepared with different wall materials. Among them, A is olive leaf polyphenols; B is polyphenols:chitosan = 1:2; C is polyphenols:maltodextrin = 1:1; D is polyphenols:sodium alginate = 1:1; E is polyphenols:β-cyclodextrin = 1:2; F is polyphenols:maltodextrin:β-cyclodextrin = 1:1:1.

[0041] Figure 19 Results graphs of the stability of microcapsules of olive leaf extract. Among them, Figure A shows the changes in the polyphenol contents in the microcapsules and olive leaf extract during storage for 1 - 3 days under light and dark conditions; Figure B shows the changes in the polyphenol contents in the microcapsules and olive leaf extract at different temperatures.

[0042] Figure 20 Results graph of the antioxidant property of microcapsules of olive leaf extract in scavenging DPPH· free radicals.

[0043] Figure 21 Graph showing the effects of the chewing tablets of olive leaf extract with lactic acid bacteria added in 1.2% ethanol on the movement times (A) of 24hpf zebrafish embryos, the malformation rate (B) at 72hpf, the heart rate (C) at 72hpf, and the pericardial area (D) at 96hpf. The data differences between each treatment group were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the "ethanol" group, #p < 0.05, ##p < 0.01. Detailed implementation manners

[0044] The chewing tablets of the present invention for improving fetal alcohol syndrome have active ingredients composed of the following components in the following weight ratios: 0.5 - 65 parts of lactic acid bacteria and 0.10 - 12 parts of oleuropein.

[0045] Preferably, the chewable tablets for improving fetal alcohol syndrome according to the present invention have active ingredients composed of components in the following weight ratios: 0.6 to 60 parts of lactic acid bacteria, and 0.11 to 11.1 parts of oleuropein.

[0046] Among them, the above-mentioned oleuropein can be replaced by an olive leaf extract with a corresponding oleuropein content; the above-mentioned oleuropein can also be replaced by an olive leaf extract microcapsule or an oleuropein microcapsule with a corresponding oleuropein content.

[0047] Furthermore, the above-mentioned olive leaf extract microcapsule is preferably prepared by mixing an olive leaf extract and a wall material in a weight ratio of 1:0.8 to 2.5 and drying; among them, the wall material can be a conventional microcapsule wall material, such as at least one of plant gum, starch, cellulose, protein, polymer, wax, and lipid. The above-mentioned olive leaf extract microcapsule can be prepared by a conventional microcapsule preparation method.

[0048] As a preferred embodiment, the wall material can be at least one of maltodextrin, β-cyclodextrin, sodium alginate, and chitosan.

[0049] Even further, the above-mentioned olive leaf extract microcapsule is preferably prepared by mixing an olive leaf extract and a wall material in a weight ratio of 1:1 and drying, and the wall material is maltodextrin. The microcapsule prepared by this technical solution has the highest contents of oleuropein and hydroxytyrosol and the best stability.

[0050] As another preferred technical solution, the above-mentioned olive leaf extract microcapsule is prepared by mixing an olive leaf extract and a wall material in a weight ratio of 1:2 and drying, and the wall material is a mixture of maltodextrin and β-cyclodextrin, and the weight ratio of maltodextrin to β-cyclodextrin is 1:1. The microcapsule prepared by this technical solution has the highest embedding rate, a relatively large increase in the particle size of the prepared microcapsule, and the contents of oleuropein and hydroxytyrosol in the microcapsule are slightly lower than those of the microcapsule using maltodextrin alone as the wall material.

[0051] The chewable tablets for improving fetal alcohol syndrome according to the present invention further include pharmaceutically or food-acceptable excipient components. The excipients can be conventional pharmaceutically or food-acceptable excipients, such as at least one of, including but not limited to, sweeteners, fillers, antioxidants, thickeners, disintegrants, binders, stabilizers, glidants, and flavoring agents.

[0052] Among them, in order to improve the taste of chewable tablets and increase their sweetness, sweeteners are added to different types of wines on the market, including natural sweeteners (stevioside, glycyrrhizinate, granulated sugar, rock sugar, honey, etc.) and artificial sweeteners (acesulfame potassium, sodium cyclamate, aspartame, sodium saccharin, sucralose, high fructose corn syrup, etc.). Although artificial sweeteners have high sweetness, low addition amount, and low calories, long-term use also has potential safety issues such as increasing the risk of diabetes, cancer, and neurological diseases, which has attracted increasing attention from scholars at home and abroad. Whether sweeteners will exacerbate alcohol toxicity has not been reported yet. The research experiments of this invention have shown that compared with sweeteners such as honey and rock sugar, sucrose can significantly promote the growth and development of zebrafish embryos inhibited by alcohol without causing teratogenesis to zebrafish embryo development. Therefore, the chewable tablets for improving fetal alcohol syndrome of this invention preferably use sucrose as the sweetener.

[0053] As a more preferred technical solution, the chewable tablets for improving fetal alcohol syndrome of this invention are prepared from the following components in the following weight ratios: 130 - 160 parts of olive leaf extract microcapsules, 45 - 55 parts of lactic acid bacteria, 90 - 110 parts of erythritol, 18 - 19 parts of sucrose, 4 - 6 parts of citric acid, 180 - 220 parts of starch, and 3 - 4 parts of magnesium stearate.

[0054] The chewable tablets for improving fetal alcohol syndrome of this invention are most preferably prepared from the following components in the following weight ratios: 155 parts of olive leaf extract microcapsules, 50 parts of lactic acid bacteria, 100 parts of erythritol, 18.65 parts of sucrose, 5 parts of citric acid, 200 parts of starch, and 3.5 parts of magnesium stearate.

[0055] According to needs, the chewable tablets for improving fetal alcohol syndrome of this invention can be made into conventional sizes. When the addition amount of olive leaf extract microcapsules in the chewable tablets is 155 mg / tablet and the addition amount of lactic acid bacteria powder is 50 mg / tablet, taking one tablet each time can prevent and protect against fetal alcohol syndrome.

[0056] Among them, the lactic acid bacteria described in this invention can be conventional edible or medicinal lactic acid bacteria, such as: at least one lactic acid bacteria selected from the group consisting of Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Leuconostoc, Pediococcus, and Sporolactobacillus.

[0057] The experimental research of the present invention proves that using Lactobacillus rhamnosus alone and mixed lactic acid bacteria can both significantly reduce the embryonic malformation rate, and have an improvement effect on the increase in the embryonic malformation rate of zebrafish caused by ethanol. Therefore, as a preferred embodiment, the lactic acid bacteria described in the present invention can be Lactobacillus rhamnosus, or Lactobacillus bulgaricus, or Streptococcus thermophilus, or Kefir, or Bifidobacterium lactis, or Bifidobacterium longum, or Bifidobacterium infantis, or a mixed bacterium of the above various lactic acid bacteria. For example, the lactic acid bacteria can be a mixture of Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir, Bifidobacterium lactis, Bifidobacterium longum, and Bifidobacterium infantis in a weight ratio of 1:1:1:1:1:1 of the bacterial powder.

[0058] The following further describes the specific implementation manners of the present invention in conjunction with embodiments, and the present invention is not limited to the scope of the described embodiments.

[0059] Example 1 Protective effect of oleuropein (OLE) on the toxic effect of ethanol on zebrafish embryo development

[0060] 1. Collect zebrafish embryos: Adult male and female zebrafish were placed in a mating box filled with breeding water at a ratio of 1:2 the night before the experiment, separated by a partition, and left overnight in the dark. In the morning of the experimental day, the partition was removed, and the male and female fish were chased by light stimulation to start in vitro spawning and fertilization. Embryos were collected within 30 minutes. The embryos were washed 2-3 times with embryo culture medium (5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, 0.16 mM MgSO4, pH 6.7), cultured at 28.5 ± 0.5 °C, and normal fertilized eggs that had divided were selected under a stereomicroscope at 4-5 hpf (hours post-fertilization) for standby.

[0061] 2. Treat zebrafish embryos with 1.2% ethanol and different concentrations of oleuropein (OLE), and observe the changes in embryonic morphological development:

[0062] The established treatment groups included the DMSO group (control group containing 0.1% DMSO), 1.2% EtOH group (1.2% ethanol), 1.2% EtOH + 0.0342 μM OLE group, 1.2% EtOH + 0.342 μM OLE group, 1.2% EtOH + 3.42 μM OLE group, 1.2% EtOH + 34.2 μM OLE group, and 34.2 μM OLE group. At 6 hpf, 30 embryos were placed in each well of a six-well plate, and 5 mL of the treatment solution was added to each well. After 24 h of treatment, all were replaced with embryo culture medium until 96 hpf. Every 24 h, the embryo hatching rate, mortality rate, malformation rate, heart rate, and number of spontaneous movements were counted, and the eye diameter, head width, body length, and pericardial area were measured. At the same time, fresh culture medium was replaced, and the whitened and dead embryos were picked out. This was repeated three times. The data differences between the treatment groups were statistically analyzed using the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the "ethanol" group, #p < 0.05, ##p < 0.01, p < 0.001. The results are shown in Figures 1 to 3 .

[0063] Figure 1 The morphological status of hatched larvae at 96 hpf after 6 hpf embryos were exposed to 1.2% ethanol and different concentrations of oleuropein (OLE) for 24 h. Among them, A: Hatched larvae with normal development; B: Malformed larvae at 96 hpf after the embryos were exposed to 1.2% ethanol for 24 h. Among them, M: Small eyes, PE: Pericardial edema, SB: Developmentally retarded swim bladder, AYS: Abnormally developed yolk; (C)-(F): The morphological status of hatched larvae of zebrafish embryos at 96 hpf after co-treatment with 1.2% ethanol and 0.0342 - 34.2 μM oleuropein (OLE) for 24 h; (G) Normal hatched larvae at 96 hpf after treatment with 34.2 μM oleuropein (OLE) for 24 h. Compared with the control, alcohol treatment led to malformations such as pericardial edema, small eyes, short body, abnormal development of yolk and swim bladder in embryo development. Adding 0.0342 - 34.2 μM oleuropein (OLE) could reduce or eliminate the pericardial edema of embryos caused by ethanol, increase the eyes, body length or yolk, and the swim bladder returned to normal, indicating that oleuropein could reduce the morphological development distortion caused by alcohol and had an improvement effect on fetal alcohol syndrome.

[0064] Figure 2Results of pericardial area (A), body length (B), eye diameter (C), head width (D), number of spontaneous movements (E), and heart rate (F) of hatched larvae measured at 96 hpf after embryos at 6 hpf were exposed to 1.2% ethanol and different concentrations of oleuropein (OLE) for 24 h. Compared with the control, alcohol treatment significantly increased the pericardial area, decreased the body length, reduced the eye size, and increased the number of spontaneous movements of the embryos. Oleuropein (OLE) at concentrations of 0.0342 - 34.2 μM significantly reduced the increase in pericardial area caused by ethanol, increased the decrease in body length and eye diameter caused by ethanol, and decreased the increase in the number of spontaneous movements of the embryos caused by ethanol. With the increase in the concentration of oleuropein (OLE), embryonic development gradually returned to the normal state. There were no differences in the head width and heart rate of hatched larvae at 96 hpf among the control, alcohol treatment, and combined treatment of alcohol and oleuropein (OLE). There were no differences in the head width and heart rate of hatched larvae at 96 hpf among the control, alcohol treatment, and combined treatment of alcohol and oleuropein (OLE).

[0065] Figure 3 Results of the mortality (A), hatching rate (B), and malformation rate (C) of zebrafish embryos during development to 96 hpf after embryos at 6 hpf were exposed to 1.2% ethanol and different concentrations of oleuropein (OLE) for 24 h. As can be seen from Figure A, within 96 hpf, the mortality of embryos in all treatments was within 10%, which was not significant; as can be seen from Figure B, compared with the control, 1.2% ethanol decreased the embryonic hatching rate, and oleuropein (OLE) at concentrations of 0.0342 - 34.2 μM significantly increased the hatching rate of zebrafish embryos with the increase in concentration, showing an improvement effect on the decrease in the hatching rate of zebrafish embryos caused by ethanol; as can be seen from Figure C, compared with the control, 1.2% ethanol significantly increased the embryonic malformation rate, and oleuropein (OLE) at concentrations of 0.0342 - 34.2 μM significantly decreased the malformation rate of zebrafish embryos with the increase in concentration, showing an improvement effect on the malformation of zebrafish embryos caused by ethanol.

[0066] 3. Zebrafish embryos were treated with 1.2% ethanol and different concentrations of oleuropein (OLE), and the changes in the activities of embryonic antioxidant enzymes were measured:

[0067] Seven treatment groups were set up, including the DMSO group (control group containing 0.1% DMSO), 1.2% EtOH group (1.2% ethanol), 1.2% EtOH + 0.0342 μM OLE group, 1.2% EtOH + 0.342 μM OLE group, 1.2% EtOH + 3.42 μM OLE group, 1.2% EtOH + 34.2 μM OLE group, and 34.2 μM OLE group. At 6 hpf, 100 normally dividing embryos were placed in each culture dish (8.8 cm * 8.8 cm) and 20 mL of the treatment solution was added. After 24 h of treatment, all were replaced with embryo culture medium until 72 hpf. Fresh culture medium was changed every 24 h, and the whitened and dead embryos were picked out. 80 larvae hatched at 72 hpf were randomly selected from each treatment group and rinsed three times with pre-cooled PBS buffer (pH 7.4). Then the embryos were placed in 2 mL centrifuge tubes, and 9 times the volume of pre-cooled PBS buffer was added. After freezing in a tissue grinder (Scientz-48, Ningbo Xinzhi Biotechnology Co., Ltd.) for 60 s, they were ground at 4°C and 60 - 65 HZ for 15 - 30 s. The homogenate was centrifuged at 12,000 rpm and 4°C for 10 min, and the supernatant was immediately used to measure the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) using a kit (Nanjing Jiancheng Bioengineering Institute). This was repeated three times. The data differences between the treatment groups were statistically analyzed using the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the "ethanol" group, #p < 0.05, ##p < 0.01. The results are shown in Figure 4 .

[0068] Figure 4 Results of the CAT activity (A) and SOD activity (B) measured when embryos at 6 hpf were exposed to 1.2% ethanol and different concentrations of oleuropein (OLE) for 24 h and developed to 72 hpf. Compared with the control, ethanol significantly decreased the CAT activity but had no effect on the SOD activity. However, after adding oleuropein, the CAT activity increased significantly and the SOD activity did not change significantly, indicating that oleuropein enhanced the antioxidant system by increasing the CAT activity and alleviated the toxic effects of alcohol-induced zebrafish embryos.

[0069] 4. Treatment of zebrafish embryos with 1.2% ethanol and different concentrations of oleuropein (OLE), and determination of the changes in embryonic ferroptosis:

[0070] Ferroptosis is dependent on divalent iron ions (Fe 2+) Reactive oxygen species stress caused by lipid peroxidation. The characteristic biochemical reaction of ferroptosis is the decrease in the activity of cellular glutathione peroxidase (GPXs), especially GPX4, and the content of reduced glutathione (GSH). Seven treatment groups were set up, including the DMSO group (control group containing 0.1% DMSO), 1.2% EtOH group (1.2% ethanol), 1.2% EtOH + 0.0342 μM OLE group, 1.2% EtOH + 0.342 μM OLE group, 1.2% EtOH + 3.42 μM OLE group, 1.2% EtOH + 34.2 μM OLE group, and 34.2 μM OLE group. At 6 hpf, 100 normally dividing embryos were placed in each petri dish (8.8 cm * 8.8 cm) and 20 mL of the treatment solution was added. After 24 h of treatment, all were replaced with embryo culture medium until 72 hpf. Fresh culture medium was changed every 24 h, and the whitened and dead embryos were picked out. For each treatment group, 80 larvae hatched at 72 hpf were randomly selected and rinsed three times with pre-cooled PBS buffer (pH 7.4). Then the embryos were placed in 2 mL centrifuge tubes, and 9 times the volume of pre-cooled PBS buffer was added. After being frozen in a tissue grinder (Scientz-48, Ningbo Xinzhi Biotechnology Co., Ltd.) for 60 s, they were ground at 4°C and 60 - 65 HZ for 15 - 30 s. The homogenate was centrifuged at 12,000 rpm and 4°C for 10 min, and the supernatant was immediately used to measure the GSH content, the content of lipid peroxidation product malondialdehyde (MDA), iron content, and GPX4 activity using a kit (Nanjing Jiancheng Bioengineering Institute). This was repeated three times. The data differences between the treatment groups were statistically analyzed using the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the "ethanol" group, #p < 0.05, ##p < 0.01. The results are shown in Figure 5 .

[0071] Figure 5 Results of GPX4 activity (A), GSH content (B), iron content (C), and MDA content (D) measured when embryos at 6 hpf were exposed to 1.2% ethanol and different concentrations of oleuropein (OLE) for 24 h and developed to 72 hpf. Compared with the control, ethanol significantly decreased GPX4 activity, decreased GSH content, and increased iron and MDA contents. After adding oleuropein, GPX4 activity was significantly increased, GSH content increased, and iron and MDA contents decreased, indicating that oleuropein alleviated the toxic effect of alcohol-induced zebrafish embryos by reducing ferroptosis

[0072] 5. Treat zebrafish embryos with 1.2% ethanol and different concentrations of oleuropein (OLE), and measure the changes in embryonic cell apoptosis:

[0073] Seven treatment groups were set up, including the DMSO group (control group containing 0.1% DMSO), 1.2% EtOH group (1.2% ethanol), 1.2% EtOH + 0.0342 μM OLE group, 1.2% EtOH + 0.342 μM OLE group, 1.2% EtOH + 3.42 μM OLE group, 1.2% EtOH + 34.2 μM OLE group, and 34.2 μM OLE group. At 6 hpf, 80 normally dividing embryos were placed in each petri dish (8.8 cm * 8.8 cm) and 20 mL of treatment solution was added. After 24 h of treatment, all were replaced with embryo culture medium until 72 hpf. Fresh culture medium was changed every 24 h, and the whitened and dead embryos were picked out. After the embryos were treated until 72 hpf, acridine orange (AO) fluorescence staining and apoptotic enzyme activity detection were performed respectively. This was repeated three times. The data differences between the treatment groups were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01; compared with the "ethanol" group, #p < 0.05, ##p < 0.01.

[0074] (1) Analysis of zebrafish cell apoptosis by acridine orange fluorescence staining: Twenty hatched larvae were selected from each treatment and placed in a six-well plate. 5 mL of 5 mg / L acridine orange staining solution was added, and staining was carried out in the dark at room temperature for 20 min. The larvae were washed 3 times with embryo culture medium for 5 min each time. The larvae were divided into two parts. Ten of the larvae were anesthetized with 0.08% ethylene glycol phenyl ether for 5 min and then observed and photographed under a fluorescence microscope. For the other ten larvae, the culture medium was aspirated, 150 μL of pre-cooled PBS buffer (pH 7.4) was added, and then ground at 4°C and 60 - 65 HZ for 15 - 30 s. The homogenate was centrifuged at 12,000 rpm at 4°C for 4 min. 100 μL of the supernatant was taken and added to a 96-well microplate, incubated at 37°C for 5 min, and the fluorescence intensity was measured (excitation wavelength 488 nm, emission wavelength 515 nm). The apoptosis of cells was calculated by comparing the fluorescence intensity of the treated embryos with that of the control group. The results are shown in Figure 6 (A) and (B) in

[0075] (2) Apoptosis enzyme activity assay: Randomly select 40 embryos from each treatment group, and rinse them three times with pre-cooled PBS buffer (pH 7.4). Then place the embryos in a 2 mL centrifuge tube, add 9 times the volume of pre-cooled PBS buffer, put them into a tissue grinder (Scientz-48, Ningbo Xinzhi Biotechnology Co., Ltd.), freeze for 60 s, and grind at 4°C and 60 - 65 HZ for 15 - 30 s. Centrifuge the homogenate at 12,000 rpm and 4°C for 10 min, and immediately use a kit (Nanjing Jiancheng Bioengineering Institute) to measure the activities of apoptosis enzymes Caspase9 and Caspase3 in the supernatant. Calculate the strength of apoptosis enzyme activity by comparing the fluorescence intensities of the treated embryos and the control group. The results are shown in Figure 6 (C) and (D) in

[0076] Figure 6 The results of OA fluorescence staining (A), relative fluorescence intensity (B), Caspase9 activity (C), and Caspase3 activity (D) of embryos at 6 hpf exposed to 1.2% ethanol and different concentrations of oleuropein (OLE) for 24 h and then developed to 72 hpf. Compared with the control, ethanol significantly enhanced the acridine orange fluorescence staining intensity and increased the activities of Caspase-9 and Caspase-3. After adding oleuropein, the fluorescence intensity of acridine orange staining decreased, and the activities of Caspase-9 and Caspase-3 decreased, indicating that oleuropein alleviates the toxic effect of alcohol on zebrafish embryo development by reducing cell apoptosis.

[0077] 6. Treat zebrafish embryos with 1.2% ethanol and different concentrations of oleuropein (OLE), and measure the changes in reactive oxygen species (ROS) in embryonic cells:

[0078] Seven treatment groups were set up, including the DMSO group (control group containing 0.1% DMSO), 1.2% EtOH group (1.2% ethanol), 1.2% EtOH + 0.0342 μM OLE group, 1.2% EtOH + 0.342 μM OLE group, 1.2% EtOH + 3.42 μM OLE group, 1.2% EtOH + 34.2 μM OLE group, and 34.2 μM OLE group. At 6 hpf, 20 normally dividing embryos were placed in each well of a six-well plate, and 5 mL of the treatment solution was added to each well. After 24 h of treatment, all were replaced with embryo culture medium until 72 hpf. Fresh culture medium was changed every 24 h, and the whitened and dead embryos were picked out. Ten 72-hpf hatched larvae were randomly selected from each treatment, washed 3 times with pre-cooled embryo culture medium, the culture medium was aspirated clean, 150 μL of pre-cooled PBS buffer (pH 7.4) was added for homogenization, and the homogenate was centrifuged at 12,000 rpm for 20 min at 4°C. 100 μL of the supernatant was taken and added to a 96-well microplate, incubated at room temperature for 5 min, 8.3 μL of the dichlorofluorescein diacetate (DCFH-DA) stock solution (10 mg / L, dissolved in DMSO) was added to each well, mixed evenly, incubated at 37°C for 30 min, and the fluorescence intensity was measured with a fluorescence microplate reader (excitation wavelength 485 nm, emission wavelength 530 nm). The relative fluorescence intensity of the embryo treatment group and the control group was used to represent the strength of ROS production. This was repeated three times. The data differences between each treatment group were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05; compared with the "ethanol" group, #p < 0.05. The results are shown in Figure 7 .

[0079] Figure 7 Results of the reactive oxygen species (ROS) levels in embryos at 6 hpf exposed to 1.2% ethanol and different concentrations of oleuropein (OLE) for 24 h and developed to 72 hpf. Compared with the control, ethanol significantly enhanced the fluorescence intensity of DCFH-DA staining, while the fluorescence intensity of DCFH-DA staining significantly decreased after adding oleuropein, indicating that oleuropein alleviates ethanol-induced oxidative stress by reducing the ROS level and protects embryo development from the toxicity of ethanol.

[0080] 7. Treatment of zebrafish embryos with 1.2% ethanol and oleuropein (OLE), and determination of changes in embryonic craniofacial cartilage: At 6 hpf, 50 normally dividing embryos were placed in each well of a six-well plate, and 5 mL of treatment solution was added to each well, including 0.1% DMSO, 1.2% EtOH, 1 μM OLE + 1.2% EtOH, and 1 μM OLE. After exposure treatment at 28.5 °C for 24 h, all treatment solutions were replaced with embryo culture medium, and the culture was continued until 96 hpf. Fresh culture medium was replaced every 24 h, and during this period, the whitened dead embryos were picked out. The zebrafish larvae at 96 hpf were collected in 1.5 mL EP tubes, and 0.08% ethylene glycol phenyl ether anesthetic was added and soaked for 5 min; the anesthetic in the EP tube was aspirated, 1 mL of 4% paraformaldehyde was added, and the mixture was gently shaken and fixed on a shaker at room temperature for 2 h; the solution in the EP tube was aspirated, and the sample was washed twice with PBS buffer (pH 7.4). The sample was dehydrated by shaking at room temperature with 1 mL of 50% and 100% ethanol for 10 min each; the ethanol was aspirated, 1 mL of 0.1% alcian blue staining solution was added to the EP tube, and the mixture was gently shaken overnight at room temperature for 24 h of staining; the staining solution was aspirated, 1 mL of pure water was added and inverted and mixed, the liquid was aspirated, and the sample was washed 2 - 3 times. Then 1 mL of 1.5% H2O2 / 1% KOH bleaching solution was added, and the centrifuge tube cap was opened and placed at room temperature for 20 min; 1 mL of 1% trypsin was added and digested for 1 h to remove tissues other than cartilage, and the observation was continued under a stereomicroscope until the cartilage was clearly visible; the stained larvae were stored in 100% glycerol, observed under a stereomicroscope, photographed, and measured. Repeat three times. The data differences between treatment groups were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, **p < 0.01; compared with the "ethanol" group, ##p < 0.01. The results are shown in Figure 8 and Figure 9 .

[0081] Figure 8 and Figure 9 showed that alcian blue staining showed that the angle between the ceratohyal bone and the skull in the ethanol treatment group was larger than that in the normal control group, and the lengths of Meckel's cartilage, ceratohyal bone, palatoquadrate bone, hyomandibular arch of the hyoid bone, and the body of the hyoid bone were all shortened; while oleuropein (OLE) could significantly reduce the increase in the angle between the ceratohyal bones caused by ethanol, increase the lengths of Meckel's cartilage, ceratohyal bone, palatoquadrate bone, hyomandibular arch of the hyoid bone, and the body of the hyoid bone, and had an improving effect on the changes in embryonic skull cartilage development caused by ethanol.

[0082] Example 2 Protective effect of Lactobacillus rhamnosus (LR) and mixed lactic acid bacteria (cLAB, Lactobacillus bulgaricus: Streptococcus thermophilus: Kefir: Bifidobacterium lactis: Bifidobacterium longum: Bifidobacterium infantis = 1:1:1:1:1:1) on the developmental toxicity of zebrafish embryos caused by ethanol

[0083] Zebrafish embryos were treated with 1.2% ethanol, Lactobacillus rhamnosus (LR), and a mixed lactic acid bacteria (cLAB) to observe changes in embryonic morphological development:

[0084] The set treatment groups included a DMSO group (control group containing 0.1% DMSO), a 1.2% EtOH group (1.2% ethanol), a 1.2% EtOH + 10 3 cfu / mL LR group, a 1.2% EtOH + 10 3 cfu / mL cLAB group, a 1.2% EtOH + 10 3 cfu / mLLR + 10 3 cfu / mL cLAB group, 10 3 cfu / mL LR + 10 3 cfu / mL cLAB group, 10 3 cfu / mL LR group, and 10 3 cfu / mLcLAB group. At 6 hpf, 30 normally dividing embryos were placed in each well of a six-well plate, and 5 mL of the treatment solution was added to each well. After 24 h of treatment, all were replaced with embryo culture medium until 96 hpf. Every 24 h, the embryo hatching rate, mortality rate, malformation rate, heart rate, and number of spontaneous movements were counted, and the eye diameter, body length, pericardial area, and yolk area were measured. At the same time, fresh culture medium was replaced, and the whitened and dead embryos were picked out. This was repeated three times. The data differences between the treatment groups were statistically analyzed using the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the "ethanol" group, #p < 0.05. The results are shown in Figures 10 to 11 .

[0085] Figure 10 Results of the hatching rate (A), mortality rate (B), and malformation rate (C) of embryos at 96 hpf after 6 hpf embryos were exposed to 1.2% ethanol, Lactobacillus rhamnosus (LR), and a mixed lactic acid bacteria (cLAB) for 24 h. As can be seen from Figure A, ethanol decreased the embryo hatching rate, while Lactobacillus rhamnosus (LR) and the mixed lactic acid bacteria (cLAB), either alone or in combination, could significantly increase the embryo hatching rate, showing an improvement effect on the decrease in the hatching rate of zebrafish embryos caused by ethanol; as can be seen from Figure B, ethanol increased the embryo mortality rate, while Lactobacillus rhamnosus (LR) and the mixed lactic acid bacteria (cLAB), either alone or in combination, could significantly decrease the embryo mortality rate, showing an improvement effect on the increase in the mortality rate of zebrafish embryos caused by ethanol; as can be seen from Figure C, ethanol significantly increased embryo malformations, while Lactobacillus rhamnosus (LR) and the mixed lactic acid bacteria (cLAB), either alone or in combination, could significantly decrease the embryo malformation rate, showing an improvement effect on the increase in the malformation rate of zebrafish embryos caused by ethanol.

[0086] Figure 11 Results of body length (A), eye diameter (B), pericardial area (C), heart rate (D), yolk area (E), and spontaneous movement counts (F) of hatched larvae at 72 hpf after embryos at 6 hpf were exposed to 1.2% ethanol, Lactobacillus rhamnosus (LR), and a combined lactic acid bacteria (cLAB) for 24 h. Compared with the control, ethanol significantly reduced the body length and eye diameter of embryonic development, increased the pericardial area, slowed the heart rate, increased the yolk area, and decreased the number of spontaneous movements. However, Lactobacillus rhamnosus (LR) and the combined lactic acid bacteria (cLAB), either alone or in combination, significantly increased the body length and eye diameter of hatched larvae, decreased the pericardial area and yolk area, and restored the heart rate and spontaneous movement to normal levels, showing an improvement effect on the developmental toxicity of zebrafish embryos caused by ethanol in terms of body length, eyes, heart, etc.

[0087] Example 3 Combined protective effect of oleuropein (OLE) and combined lactic acid bacteria (cLAB, Lactobacillus bulgaricus: Streptococcus thermophilus: Kefir: Bifidobacterium lactis: Bifidobacterium longum: Bifidobacterium infantis = 1:1:1:1:1:1) on the developmental toxicity of zebrafish embryos caused by ethanol

[0088] 1. Treat zebrafish embryos with a combination of 1.2% ethanol, combined lactic acid bacteria (cLAB), and oleuropein (OLE), and observe the changes in embryonic morphological development:

[0089] The set treatment groups included a DMSO group (control group containing 0.1% DMSO), a 1.2% EtOH group (1.2% ethanol), a 1.2% EtOH + 10 3 cfu / mL cLAB + 0.342 μM OLE group, a 1.2% EtOH + 10 3 cfu / mL cLAB + 3.42 μM OLE group, a 1.2% EtOH + 10 3 cfu / mL cLAB + 34.2 μM OLE group. At 6 hpf, 30 normally dividing embryos were placed in each well of a six-well plate, and 5 mL of the treatment solution was added to each well. After 24 h of treatment, all were replaced with embryo culture medium until 96 hpf. The embryo hatching rate, mortality rate, malformation rate, heart rate, and number of spontaneous movements were counted every 24 h, and the eye diameter, body length, and pericardial area were measured. At the same time, fresh culture medium was replaced, and the embryos that turned white and died were picked out. Repeat three times. Use the one-way significant difference method in Excel to statistically analyze the differences in data between treatment groups. Compared with the "control" group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the "ethanol" group, #p < 0.05, ##p < 0.01. The results are shown in Figures 12 to 13 。

[0090] Figure 12 Results of the hatching rate (A), mortality rate (B), and malformation rate (C) of embryos at 96 hpf after 6-hpf embryos were exposed to 1.2% ethanol, combined lactic acid bacteria (cLAB), and oleuropein (OLE) for 24 h. Compared with the control, ethanol decreased the embryo hatching rate, increased the mortality rate, and increased the malformation rate, while the combined action of cLAB and oleuropein significantly increased the embryo hatching rate, decreased the embryo mortality rate and embryo malformation rate, and had a protective effect on the developmental toxicity of zebrafish embryos caused by ethanol.

[0091] Figure 13 Results of the body length (A), eye diameter (B), pericardial area (C), heart rate (D), yolk area (E), and spontaneous movement times (F) of hatched larvae at 72 hpf after 6-hpf embryos were exposed to 1.2% ethanol, combined lactic acid bacteria (cLAB), and oleuropein (OLE) for 24 h. Compared with the control, ethanol significantly decreased the body length and eye diameter of embryo development, increased the pericardial area, slowed down the heart rate, increased the yolk area, and decreased the spontaneous movement times, while the combined action of cLAB and OLE significantly increased the body length and eye diameter of hatched larvae, decreased the pericardial area and yolk area, and restored the heart rate and spontaneous movement to normal levels, and had an improvement effect on the developmental toxicity of zebrafish embryos such as body length, eyes, and heart caused by ethanol.

[0092] 2. Zebrafish embryos were treated with 1.2% ethanol, combined lactic acid bacteria (cLAB), and oleuropein (OLE), and the changes in embryo antioxidant biochemical indexes were measured: eight treatment groups were set up, including the DMSO group (control group containing 0.1% DMSO), 1.2% EtOH group (1.2% ethanol), 1.2% EtOH + 10 3 cfu / mL cLAB group, 1.2% EtOH + 0.342 μM OLE group, 1.2% EtOH + 10 3 cfu / mL cLAB + 0.342 μM OLE group, 10 3 cfu / mL cLAB + 0.342 μM OLE group, 10 3cfu / mL cLAB group, and 0.342 μM OLE group. At 6 hpf, 100 normally dividing embryos were placed in each petri dish (8.8 cm * 8.8 cm) and 20 mL of the treatment solution was added. After 24 h of treatment, all were replaced with embryo culture medium until 72 hpf. Fresh culture medium was changed every 24 h, and the whitish and dead embryos were picked out. 80 larvae hatched at 72 hpf were randomly selected from each treatment group and rinsed three times with pre-cooled PBS buffer (pH 7.4). Then the embryos were placed in 2 mL centrifuge tubes, and 9 times the volume of pre-cooled PBS buffer was added. After being frozen in a tissue grinder (Scientz-48, Ningbo Xinzhi Biotechnology Co., Ltd.) for 60 s, they were ground at 4 °C and 60 - 65 HZ for 15 - 30 s. The homogenate was centrifuged at 12,000 rpm and 4 °C for 10 min, and the supernatant was immediately used to measure the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD), the content of reduced glutathione (GSH), and the content of oxidation product malondialdehyde (MDA) using a kit (Nanjing Jiancheng Bioengineering Institute). This was repeated three times. The data differences between treatment groups were statistically analyzed using the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the "ethanol" group, #p < 0.05, ##p < 0.01. The results are shown in Figure 14 .

[0093] Figure 14 Embryos at 6 hpf were exposed to 1.2% ethanol, 10 3 cfu / mL of mixed lactic acid bacteria (cLAB), and 0.342 μM oleuropein (OLE) for 24 h. The results of CAT activity (A), GSH content (B), SOD activity (C), and MDA content (D) were measured when they developed to 72 hpf. Compared with the control, ethanol significantly decreased CAT and SOD activities, decreased GSH content, and increased MDA content. After adding mixed lactic acid bacteria and oleuropein, CAT and SOD activities were significantly increased, GSH content increased, and MDA content decreased, and the combined effect of mixed lactic acid bacteria and oleuropein was more significant, indicating that the combination of lactic acid bacteria and oleuropein alleviated the toxic effect of alcohol-induced zebrafish embryos by enhancing the antioxidant system.

[0094] 3. Co-treat zebrafish embryos with 1.2% ethanol, mixed lactic acid bacteria (cLAB), and oleuropein (OLE), and measure the changes in embryonic cell apoptosis and reactive oxygen species (ROS): Set 8 treatment groups, including the DMSO group (control group containing 0.1% DMSO), 1.2% EtOH group (1.2% ethanol), 1.2% EtOH + 10 3cfu / mL cLAB group, 1.2% EtOH + 0.342 μMOLE group, 1.2% EtOH + 10 3 cfu / mL cLAB + 0.342 μM OLE group, 10 3 cfu / mL cLAB + 0.342 μM OLE group, 10 3 cfu / mL cLAB group, and 0.342 μM OLE group. At 6 hpf, 80 normally dividing embryos were placed in each petri dish (8.8 cm * 8.8 cm) and 20 mL of the treatment solution was added. After 24 h of treatment, all were replaced with embryo culture medium until 72 hpf. Fresh culture medium was changed every 24 h, and the whitened and dead embryos were picked out. After the embryos were treated until 72 hpf, acridine orange (AO) and dichlorofluorescein diacetate (DCFH-DA) fluorescence staining, as well as apoptotic enzyme activity detection were performed respectively. This was repeated three times. The data differences between the treatment groups were statistically analyzed by the one-way significant difference method in Excel. Compared with the "control" group, *p < 0.05, **p < 0.01; compared with the "ethanol" group, #p < 0.05, ##p < 0.01.

[0095] (1) Detection of zebrafish cell apoptosis by acridine orange (OA) fluorescence staining: For each treatment, 10 hatched larvae were selected and placed in a six-well plate, and 5 mL of 5 mg / L acridine orange staining solution was added. Staining was performed in the dark at room temperature for 20 min, and the embryos were washed 3 times with embryo culture medium for 5 min each time. After sucking out the culture medium, 150 μL of pre-cooled PBS buffer (pH 7.4) was added, and then ground at 4°C and 60 - 65 HZ for 15 - 30 s. The homogenate was centrifuged at 4°C and 12,000 rpm for 4 min. 100 μL of the supernatant was taken and added to a 96-well microplate, incubated at 37°C for 5 min, and the fluorescence intensity was measured (excitation wavelength 488 nm, emission wavelength 515 nm). The apoptosis production was calculated by comparing the fluorescence intensity of the treated embryos with that of the control group. The results are shown in Figure 15 Figure (A).

[0096] (2) Fluorescent analysis of dichlorofluorescein diacetate (DCFH-DA) to determine the level of reactive oxygen species (ROS) in embryonic cells: Randomly select 10 hatched larvae at 72 hpf for each treatment, wash them 3 times with pre-cooled embryo culture medium, suck out the culture medium completely, add 150 μL of pre-cooled PBS buffer (pH 7.4) for homogenization, and centrifuge the homogenate at 4°C and 12,000 rpm for 20 min. Take 100 μL of the supernatant and add it to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, incubate at room temperature for 5 min, add 8.3 μL of the dichlorofluorescein diacetate (DCFH-DA) stock solution (10 mg / L, dissolved in DMSO) to each well, mix well, incubate at 37°C for 30 min, and measure the fluorescence intensity with a fluorescence microplate reader (excitation wavelength 485 nm, emission wavelength 530 nm). The relative fluorescence intensity of the embryo treatment group and the control group is used to represent the strength of ROS production. The results are shown in Figure 15 Figure (B) in

[0097] (3) Measurement of apoptotic enzyme activity: Randomly select 40 embryos for each treatment group, and rinse them three times with pre-cooled PBS buffer (pH 7.4). Then place the embryos in a 2 mL centrifuge tube, add 9 volumes of pre-cooled PBS buffer, place them in a tissue grinder (Scientz-48, Ningbo Xinzhi Biotechnology Co., Ltd.), freeze for 60 s, and grind at 4°C and 60 - 65 HZ for 15 - 30 s. Centrifuge the homogenate at 12,000 rpm and 4°C for 10 min, and immediately use a kit (Nanjing Jiancheng Bioengineering Institute) to measure the activities of apoptotic enzymes Caspase9 and Caspase3 in the supernatant. The strength of apoptotic enzyme activity is calculated by comparing the fluorescence intensity of the treated embryos and the control group. The results are shown in Figure 6 Figure (C) and (D) in

[0098] Figure 15 Results of the relative fluorescence intensity of acridine orange (OA) (A), DCFH-DA relative fluorescence intensity (B), Caspase9 activity (C), and Caspase3 activity (D) of embryos at 6 hpf after exposure to 1.2% ethanol, combined lactic acid bacteria (cLAB), and oleuropein (OLE) for 24 h and developed to 72 hpf. Compared with the control, ethanol significantly enhanced the fluorescence staining intensity of acridine orange and DCFH-DA and increased the activities of Caspase9 and Caspase3. After adding combined lactic acid bacteria (cLAB) and oleuropein, whether alone or in combination, the fluorescence intensity of acridine orange and DCFH-DA staining was significantly reduced, and the activities of Caspase9 and Caspase3 were reduced, indicating that combined lactic acid bacteria (cLAB) and oleuropein, either alone or in combination, alleviated ethanol-induced oxidative stress by reducing apoptosis and the level of reactive oxygen species (ROS), protecting embryonic development from the toxicity of ethanol.

[0099] Preparation of Olea europaea Leaf Polyphenol Microcapsules Rich in Oleuropein

[0100] 1. Preparation of Olea europaea leaf polyphenols: Fresh Olea europaea leaves were taken and air-dried naturally in a cool, dry and well-ventilated place. After air-drying, they were dried at 40 °C until constant weight and then crushed through a 60-mesh sieve. A certain amount of Olea europaea leaf powder was weighed, and 60% ethanol was used as the extraction agent at a solid-liquid ratio of 1:35 (g:mL). The extraction was carried out by heating in a water bath at 80 °C for 105 min, and then centrifuged at 5000 rpm for 10 min. The supernatant was collected and rotary evaporated and concentrated to about 100 mL at 40 - 50 °C, and then the concentrated solution was freeze-dried to obtain Olea europaea leaf polyphenols.

[0101] 2. Preparation of microcapsules: Weigh 2.5 g of Olea europaea leaf polyphenols as the core material, and weigh an appropriate amount of maltodextrin, β-cyclodextrin, sodium alginate or chitosan as the wall material according to a certain ratio. The core material and the wall material were mixed, added with 250 mL of ddH2O to dissolve, and stirred at 350 rpm for 30 min. The obtained solution was spray-dried (SD-1010, Tokyo Rika Co., Ltd.). The inlet temperature of spray drying was 150 °C, and the outlet temperature was 90 °C. The prepared Olea europaea leaf polyphenol microcapsules were analyzed and detected for indexes such as encapsulation efficiency, particle size, oleuropein and hydroxytyrosol content, sensory evaluation, and stability.

[0102] (1) Encapsulation efficiency: After crushing the microcapsules, accurately weigh 100 mg of microcapsules and dissolve them in 200 mL of ddH2O. Release them at a speed of 100 rpm in a constant temperature water bath oscillator at 40 °C for 48 h, and then centrifuge the sample at 10,000 rpm for 5 min. Measure the absorbance of the supernatant at 300 nm. According to the standard curve y = 0.0023x + 0.0049, R 2 = 0.9878, the concentration of Olea europaea leaf extract was obtained. According to the formula encapsulation efficiency = mass of Olea europaea leaf extract in microcapsules / mass of Olea europaea leaf extract put in × 100%, the encapsulation efficiency can be calculated. The results are shown in Figure 16 .

[0103] From Figure 16 (A), it can be seen that the encapsulation efficiency of Olea europaea leaf extract microcapsules prepared with four common wall materials, namely maltodextrin, β-cyclodextrin, sodium alginate and chitosan, from high to low is: maltodextrin > β-cyclodextrin > sodium alginate > chitosan. Therefore, maltodextrin and β-cyclodextrin were selected as the wall materials to prepare a composite wall material. Taking the mass ratios of maltodextrin:β-cyclodextrin as 1:1, 1:2, 1:4, 2:1, 4:1 (g:g) as variables, microcapsules with composite wall materials were prepared, and the changes in the encapsulation efficiency of microcapsules caused by different ratios of the two wall materials were tested. The results are shown in Figure 16 (B). From Figure 16(B) It can be seen that when other conditions are the same, the microcapsule embedding rate is the highest when maltodextrin:β-cyclodextrin=1:1, total wall material mass: core material mass=2:1.

[0104] (2) Particle size: According to the results of embedding rate, the microcapsules prepared with different wall materials with better embedding rate were selected as samples for particle size detection, namely maltodextrin: core material = 1:1, β-cyclodextrin: core material = 2:1, sodium alginate: core material = 1:1, chitosan: core material = 2:1, maltodextrin: β-cyclodextrin: core material = 1:1:1, a total of 5 samples. The specific angle light energy was analyzed by a laser particle size analyzer (Bettersize2600, Dandong Better Instrument Co., Ltd.) to obtain the product particle size distribution. The results are shown in Figure 17 .

[0105] from Figure 17 It can be seen that the particle sizes of microcapsules prepared with maltodextrin, β-cyclodextrin, sodium alginate and chitosan as single wall materials are in the order of β-cyclodextrin < chitosan < sodium alginate < maltodextrin; when a composite material of β-cyclodextrin and maltodextrin is used as the wall material, the particle size of the prepared microcapsules increases significantly.

[0106] (3) Oleuropein and hydroxytyrosol content: Determined by HPLC. Weigh 70 mg and 4 mg of oleuropein and hydroxytyrosol standard respectively, dissolve in methanol and dilute to 10 mL, prepare 7 g / L and 0.4 g / L mother liquor respectively, dilute 2-2000 times and 2-800 times respectively, filter through 0.22 μm, and detect with Shimadzu LC20 high performance liquid chromatograph, SinoChron ODS-BP column (250×4.6 mm, 5 μm), DAD detector, detection wavelength 230 nm, flow rate 1.0 mL / min, injection volume 10 μL, mobile phase: water (A) + methanol (B), gradient elution: 0-5 min, 30% B; 5-20 min, 30% B-52% B; 20-25 min, 52% B-50% B. Inject three times and take the average of the peak area. The horizontal axis is the concentration of the standard product, and the vertical axis is the peak area. The linear equations are Y=1443691X+273739, R 2 = 0.9948 and Y = 1523853X-12697, R 2= 0.9998. Weigh maltodextrin:core material = 1:1, β-cyclodextrin:core material = 2:1, sodium alginate:core material = 1:1, chitosan:core material = 2:1, maltodextrin:β-cyclodextrin:core material = 1:1:1. A total of 5 microcapsule samples and 0.08 g of unembedded oleuropein extract were placed in a stoppered conical flask, 10 mL of methanol was added, the flask was stoppered tightly, weighed, ultrasonically extracted (30 °C, 125 W, 1 h). After extraction, it was left to room temperature, weighed again, the lost weight was made up with methanol, shaken well, filtered through a 0.45 μm filter membrane, and then detected. The determination results are shown in Table 1. The content of oleuropein in the microcapsules prepared with maltodextrin as the wall material is the highest, followed by the microcapsules prepared with chitosan and composite wall materials; the content of hydroxytyrosol in the microcapsules prepared with maltodextrin as the wall material is the highest, followed by the microcapsules prepared with β-cyclodextrin and composite wall materials. Therefore, the contents of oleuropein and hydroxytyrosol in the microcapsules prepared with maltodextrin as the wall material are the highest, followed by the microcapsules prepared with the composite wall material (maltodextrin and β-cyclodextrin).

[0107] (4) Sensory evaluation: According to the results of the encapsulation rate, samples with better encapsulation rates among the microcapsules prepared with different wall materials were selected for sensory evaluation, namely core material:maltodextrin = 1:1, core material:β-cyclodextrin = 1:2, core material:sodium alginate = 1:1, core material:chitosan = 1:2, core material:maltodextrin:β-cyclodextrin = 1:1:1, and a sample prepared by spray drying unembedded oleuropein polyphenols, a total of 6 samples. The results are shown in Table 1 and Figure 17 . Among them, the contents of oleuropein and hydroxytyrosol in the microcapsules prepared with maltodextrin as the wall material are the highest, followed by the microcapsules prepared with maltodextrin and β-cyclodextrin as the composite wall material; the microcapsules prepared with maltodextrin and β-cyclodextrin as the composite wall material have better sensory evaluation, followed by the microcapsules prepared with maltodextrin as the wall material.

[0108] Comprehensive evaluation, it is preferred to prepare oleuropein extract microcapsules with maltodextrin as the wall material, and secondly, to prepare oleuropein extract microcapsules with maltodextrin and β-cyclodextrin as the composite wall material.

[0109] Table 1 Contents of oleuropein and hydroxytyrosol in the prepared microcapsules (mg / g microcapsule) and sensory evaluation

[0110]

[0111] (5) Polyphenol content: The Folin-Ciocalteu method was used. Respectively, 0 mL, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of 0.1 g / L gallic acid standard solution were measured into 6 50-mL volumetric flasks. Then, 2 mL of Folin-Ciocalteu reagent and 8 mL of 10% Na2CO3 solution were added successively. The volume was made up to the mark with ddH2O, and the mixture was allowed to stand and react for 20 min. Using ddH2O as the control, the absorbance was measured at 680 nm. With the gallic acid concentration and absorbance value as the abscissa and ordinate respectively, a standard curve was plotted, and the linear equation y = 0.0485x - 0.0233, R 2 = 0.9802 was obtained. 10 mg of the sample was dissolved in 10 mL of ddH2O. After sufficient shaking, 2 mL of the solution was taken, and 2 mL of Folin-Ciocalteu reagent and 8 mL of 10% Na2CO3 solution were added successively. The volume was made up to 50 mL with ddH2O, and the mixture was allowed to stand and react for 20 min. Using ddH2O as the control, the absorbance was measured at 680 nm, and the polyphenol content was calculated according to the formula.

[0112] (6) Stability: 50 mg of olea europaea leaf extract and microcapsules prepared with maltodextrin (containing an equal amount of olea europaea leaf polyphenols) were weighed separately into test tubes and stored at room temperature under natural light conditions for 72 h. The polyphenol content was measured to study the effect of different storage conditions on the stability of polyphenols. The results are shown in Figure 19 (A); 10 mg of olea europaea leaf extract and microcapsules prepared with maltodextrin as the wall material (containing an equal amount of olea europaea leaf polyphenols) were weighed separately into test tubes and heated at 30 - 90 °C for 30 min. The polyphenol content was measured to study the effect of temperature on the stability of polyphenols. The results are shown in Figure 19 (B).

[0113] From Figure 19 (A), it can be seen that within three days of storage, the decline rate of the polyphenol content encapsulated by the microcapsules prepared with maltodextrin as the wall material is lower than that of the unencapsulated polyphenol content; from Figure 19 (B), it can be seen that as the temperature increases, the polyphenol content in the sample gradually decreases. However, compared with the unencapsulated olea europaea leaf extract, the polyphenol content in the olea europaea leaf extract encapsulated by the microcapsules prepared with maltodextrin as the wall material decreases more slowly.

[0114] It can be Figure 18 seen that the microcapsules have good protective performance on the polyphenols of olea europaea leaf extract.

[0115] (7) Antioxidant activity: The DPPH· scavenging method was used. 20 mg of DPPH powder was accurately weighed and made up to 250 mL with absolute ethanol to obtain 2×10 -4mol / L DPPH· solution. Take 2 mL of each of the sample solutions of olea europaea leaf extract at 1 mg / mL, 2 mg / mL, and 3 mg / mL and microcapsules prepared with maltodextrin as the wall material (containing the same amount of olea europaea leaf polyphenols), and mix them with 2 mL of 2×10 -4 mol / L DPPH· solution, let stand for 30 min, use absolute ethanol as the reference solution, and measure its absorbance A i at 517 nm. At the same time, measure the absorbance A0 of the mixture of 2 mL of 2×10 -4 mol / L DPPH· solution and 2 mL of absolute ethanol, and the absorbance A j of the mixture of 2 mL of the sample solution and 2 mL of absolute ethanol. Calculate the inhibition rate of the sample solution on the DPPH· solution according to the following formula: Inhibition rate = [1 - (A i - A j ) / A0] × 100%, where A0: absorbance without adding the sample solution; A j : absorbance without adding DPPH·; A i : absorbance when adding the sample solution and DPPH·. The results are shown in Figure 20 .

[0116] From Figure 20 it can be seen that the ability of olea europaea leaf extract and olea europaea leaf extract microcapsules prepared with maltodextrin as the wall material to scavenge DPPH free radicals increases with the increase of the sample concentration; due to the influence of the wall material on the release of the core material, the antioxidant property of the microcapsules decreases, and the inhibition rate of the 1 - 3 mg / mL microcapsules on DPPH· decreases by 8.64 - 13.6% compared with that of the olea europaea leaf extract.

[0117] Example 5 Preparation of Olea Europaea Leaf Extract Chewable Tablets

[0118] 1. Process flow for preparing chewable tablets: Weigh olea europaea leaf extract microcapsules, lactic acid bacteria powder, and excipients → mix → dry granulation → sieving → add lubricant → mix → tabletting → sensory evaluation.

[0119] 2. Formulation Design and Experiments: Dose Conversion Formula between Zebrafish and Humans: Zebrafish (mg / L) = [Human (g / day) × 1000] / 6. Using oleuropein and mixed lactic acid bacteria as the landmark components, determine the addition amounts of olive leaf extract microcapsules and lactic acid bacteria in the chewable tablets. Taking the microcapsules prepared with maltodextrin and β-cyclodextrin as the composite wall materials as an example, since the content of oleuropein in the microcapsules is 21.83 mg / g of microcapsules, the addition amount of olive leaf extract microcapsules in each chewable tablet is 5.08 - 508 mg; the viable count of the mixed lactic acid bacteria dry powder is 108 cfu / g of the bacterial powder, so the addition amount of lactic acid bacteria powder in each chewable tablet is 0.6 - 60 mg. According to the results of the preliminary experiments, finally determine that the addition amount of olive leaf extract microcapsules in the chewable tablets is 155 mg / tablet, and the addition amount of lactic acid bacteria powder is 50 mg / tablet. Take one tablet each time to prevent and protect against fetal alcohol syndrome. The excipients in the chewable tablets include the sweetener sucrose, the flavoring agent erythritol and citric acid, the binder starch, and the lubricant magnesium stearate. The addition amount of magnesium stearate is determined to be 3.5 mg / tablet, and the addition amounts of other excipients should be as small as possible. Study the effects of the addition amount of erythritol (A), the addition amount of sucrose (B), the addition amount of citric acid (C), and the addition amount of soluble starch (D) on the chewable tablets through orthogonal experiments. According to the combinations in the L9(3 4 ) orthogonal experiment table (Table 3), prepare 9 groups of chewable tablet samples. According to the sensory evaluation criteria of the chewable tablets in Table 4, select 10 food professionals to evaluate each group. Considering various indicators comprehensively, obtain the optimal ratio, and the results are shown in Table 5.

[0120] Table 3 Orthogonal Experiment Factor-Level Table (%)

[0121]

[0122] Table 4 Sensory Evaluation Indexes of Chewable Tablets

[0123]

[0124] Table 5 Orthogonal Experiment Results and Range Analysis Table

[0125]

[0126] 3. Results: As can be seen from Table 5, the order of the range R values is: C > A = B > D. Among them, for factor A, K2 > K1 > K3; for factor B, K2 > K1 > K3; for factor C, K2 > K3 > K1; for factor D, K1 > K3 > K2. Therefore, the optimal preparation plan is A2B2C2D1, that is, each chewable tablet contains 155 mg of olive leaf extract microcapsules, 50 mg of lactic acid bacteria powder, 100 mg of erythritol, 18.65 mg of sucrose, 5 mg of citric acid, 200 mg of starch, and 3.5 mg of magnesium stearate. Under these conditions, a verification experiment was carried out three times. The chewable tablets prepared had good taste, hardness, appearance, friability, etc., and were stable.

[0127] Example 6 Preparation of Chewable Tablets with the Function of Improving Fetal Alcohol Syndrome of the Present Invention

[0128] By weight, olive leaf extract dry powder, maltodextrin, and β-cyclodextrin were weighed according to the ratio of olive leaf extract: maltodextrin: β-cyclodextrin = 1:1:1, dissolved in water and mixed evenly, and spray-dried at an inlet air temperature of 150 °C and an outlet air temperature of 90 °C to prepare olive leaf extract microcapsules with maltodextrin and β-cyclodextrin as wall materials. According to the formula of 29.1% microcapsules, 9.4% lactic acid bacteria, 18.8% erythritol, 3.5% sucrose, 0.9% citric acid, and 37.6% soluble starch, the materials were weighed, mixed, passed through a 100-mesh sieve, granulated by a granulator, mixed with 0.6% magnesium stearate, and pressed into tablets by a tableting machine. The prepared chewable tablets were slightly yellowish-green in color, had a good taste, and moderate hardness and friability.

[0129] Example 7 Preparation of Chewable Tablets with the Function of Improving Fetal Alcohol Syndrome of the Present Invention

[0130] By weight, olive leaf extract dry powder and maltodextrin were weighed according to the ratio of olive leaf extract: maltodextrin = 1:1, dissolved in water and mixed evenly, and spray-dried at an inlet air temperature of 150 °C and an outlet air temperature of 90 °C to prepare olive leaf extract microcapsules with maltodextrin as the wall material. According to the formula of 23.7% microcapsules (oleuropein content was 6.84 mg / g), 23.3% erythritol, 1.3% citric acid, 4.4% sucrose, and 46.6% starch, the materials were weighed, mixed, passed through a 100-mesh sieve, and granulated by a granulator. 0.7% magnesium stearate was added and mixed, and tablets were pressed by a tableting machine to obtain lactic acid bacteria chewable tablets. The prepared chewable tablets were slightly yellowish-green in color, slightly bitter, had a cool feeling, and had good other sensory evaluations.

[0131] Example 8 Preparation of Chewable Tablets with the Function of Improving Fetal Alcohol Syndrome of the Present Invention

[0132] Weigh the dry powder of olea europaea leaf extract and maltodextrin according to the ratio of olea europaea leaf extract:maltodextrin = 1:1 by weight, dissolve and mix them with water, and perform spray drying at an inlet air temperature of 150 °C and an outlet air temperature of 90 °C to prepare olea europaea leaf extract microcapsules with maltodextrin as the wall material. According to the formula of 23.7% microcapsules (oleuropein content is 6.84 mg / g), 10.1% lactic acid bacteria (Lactobacillus bulgaricus: Streptococcus thermophilus: Kefir bacteria: Bifidobacterium lactis: Bifidobacterium longum: Bifidobacterium infantis = 1:1:1:1:1:1, 10 8 cfu / g bacterial powder), 20.2% erythritol, 3.8% sucrose, 1.1% citric acid and 40.4% soluble starch, weigh the materials, mix them, pass through a 100-mesh sieve, granulate with a granulator, add 0.7% magnesium stearate and mix, and press tablets with a tableting machine to obtain lactic acid bacteria + olea europaea leaf extract chewable tablets. The prepared chewable tablets are light yellow, slightly bitter, have a cooling sensation, and have good other sensory evaluations.

[0133] Example 9 Preparation of chewable tablets of the present invention for improving fetal alcohol syndrome

[0134] By weight, according to the formula of 10.1% mixed lactic acid bacteria powder (Lactobacillus bulgaricus: Streptococcus thermophilus: Kefir bacteria: Bifidobacterium lactis: Bifidobacterium longum: Bifidobacterium infantis = 1:1:1:1:1:1, 10 8 cfu / g bacterial powder), 27.5% erythritol, 1.5% citric acid, 5.2% sucrose, 40.0% starch and 15.0% maltodextrin, weigh the materials, mix them, pass through a 100-mesh sieve, and granulate with a granulator. Add 0.7% magnesium stearate and mix, and press tablets with a tableting machine to obtain lactic acid bacteria chewable tablets. The prepared chewable tablets are light yellow, slightly sweet, have a cooling sensation, and have good other sensory evaluations.

[0135] Example 10 Protective effect of chewable tablets of the present invention for improving fetal alcohol syndrome on the toxicity of ethanol-induced zebrafish embryo development

[0136] 1. Preparation of alcohol extract of chewable tablets

[0137] Weigh 1 g of the olea europaea leaf extract chewable tablets prepared in Example 8, the lactic acid bacteria olea europaea leaf extract prepared in Example 9, and the lactic acid bacteria chewable tablets prepared in Example 10 respectively, grind them, pass through a 200-mesh sieve, add 50 mL of 60% ethanol, extract in a water bath at 30 °C for 1 h, centrifuge at 4500 rpm·min-1 for 15 min, perform alcohol extraction and centrifugation on the precipitate once again according to the above steps, combine the supernatant, and concentrate at 40 °C on a rotary evaporator. Freeze-dry the obtained concentrated solution, and place the obtained freeze-dried powder in a desiccator for storage and standby.

[0138] 2. Treat zebrafish embryos with the combination of 1.2% ethanol and the freeze-dried powder of chewable tablets, and observe the changes in embryonic morphological development:

[0139] The set treatment groups included the DMSO group (control group containing 0.1% DMSO), 1.2% EtOH group (1.2% ethanol), 10 μg / mL lactic acid bacteria chewable tablet group, 10 μg / mL olive leaf extract chewable tablet group, 10 μg / mL lactic acid bacteria olive leaf extract group, 1.2% EtOH + 10 μg / mL lactic acid bacteria chewable tablet group, 1.2% EtOH + 10 μg / mL olive leaf extract chewable tablet group, 1.2% EtOH + 10 μg / mL lactic acid bacteria olive leaf extract group. At 6 hpf, 30 normally dividing embryos were placed in each well of a six-well plate, and 5 mL of the treatment solution was added to each well. After 24 h of treatment, all were replaced with embryo culture medium until 96 hpf. Every 24 h, the embryo hatching rate, mortality rate, malformation rate, heart rate, and number of spontaneous movements were counted, and the pericardial area was measured. At the same time, fresh culture medium was replaced, and the whitened and dead embryos were picked out. Repeat three times. Use the one-way significant difference method in Excel to statistically analyze the differences in data between treatment groups. Compared with the "control" group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the "ethanol" group, #p < 0.05, ##p < 0.01. The results are shown in Figure 21 .

[0140] From Figure 21 It can be seen that compared with the control, lactic acid bacteria chewable tablets, olive leaf extract chewable tablets, and lactic acid bacteria olive leaf extract chewable tablets had no effect on embryonic development, while ethanol stimulated increased embryonic movement, increased embryonic developmental malformations, and increased embryonic heart rate and pericardial area; adding lactic acid bacteria chewable tablets, olive leaf extract chewable tablets, and lactic acid bacteria olive leaf extract chewable tablets could significantly reduce the increased embryonic movement, increased malformation rate, increased heart rate, and increased pericardial area caused by ethanol, and had a protective effect on the developmental toxicity of zebrafish embryos caused by ethanol.

Claims

1. Chewable tablets for improving fetal alcohol syndrome, characterized in that Its active ingredient is a component with the following weight ratio: 0.5 - 65 parts of lactic acid bacteria and 0.10 - 12 parts of oleuropein; the lactic acid bacteria is a mixture of Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir bacteria, Bifidobacterium lactis, Bifidobacterium longum, and Bifidobacterium infantis in a weight ratio of 1:1:1:1:1:1 of the bacterial powder.

2. The chewable tablet for improving fetal alcohol syndrome according to claim 1, characterized in that Its active ingredient is a component with the following weight ratio: 0.6 - 60 parts of lactic acid bacteria and 0.11 - 11.1 parts of oleuropein.

3. The chewable tablet for improving fetal alcohol syndrome according to claim 1, characterized in that The oleuropein described above is replaced by an olea europaea leaf extract with a corresponding oleuropein content.

4. The chewable tablet for improving fetal alcohol syndrome according to claim 1, characterized in that The oleuropein described above is replaced by a microcapsule of olea europaea leaf extract with a corresponding oleuropein content or a microcapsule of oleuropein.

5. The chewable tablet for improving fetal alcohol syndrome according to claim 4, wherein: The microcapsule of olea europaea leaf extract is prepared by mixing an olea europaea leaf extract and a wall material in a weight ratio of 1:0.8 - 2.5 and drying; wherein, the wall material is at least one of maltodextrin, β-cyclodextrin, sodium alginate, and chitosan.

6. The chewable tablet for improving fetal alcohol syndrome according to claim 5, characterized in that: The microcapsule of olea europaea leaf extract is prepared by mixing an olea europaea leaf extract and a wall material in a weight ratio of 1:1 and drying, wherein the wall material is maltodextrin. Or the microcapsule of olea europaea leaf extract is prepared by mixing an olea europaea leaf extract and a wall material in a weight ratio of 1:2 and drying, wherein the wall material is a mixture of maltodextrin and β-cyclodextrin, and the weight ratio of maltodextrin to β-cyclodextrin is 1:

1.

7. The chewable tablet for improving fetal alcohol syndrome according to claim 1, characterized in that: The chewable tablets described above also include pharmaceutically or food-acceptable excipient components.

8. The chewable tablet for improving fetal alcohol syndrome according to claim 7, characterized in that: The excipient is at least one of a sweetening agent, a filler, an antioxidant, a thickening agent, a disintegrant, a binder, a stabilizer, a glidant, and a flavoring agent.

9. The chewable tablet for improving fetal alcohol syndrome according to claim 7, wherein The chewable tablets are prepared from components with the following weight ratio: 130 - 160 parts of microcapsule of olea europaea leaf extract, 45 - 55 parts of lactic acid bacteria, 90 - 110 parts of erythritol, 18 - 19 parts of sucrose, 4 - 6 parts of citric acid, 180 - 220 parts of starch, and 3 - 4 parts of magnesium stearate.

10. The chewable tablet for improving fetal alcohol syndrome according to claim 9, characterized in that, The chewable tablets are prepared from components with the following weight ratio: 155 parts of microcapsule of olea europaea leaf extract, 50 parts of lactic acid bacteria, 100 parts of erythritol, 18.65 parts of sucrose, 5 parts of citric acid, 200 parts of starch, and 3.5 parts of magnesium stearate.

11. Use of the chewable tablets for improving fetal alcohol syndrome according to any one of claims 1 - 10 in the preparation of a medicament for treating / preventing / improving fetal alcohol syndrome.

12. Use of lactic acid bacteria in the preparation of a medicament for treating / preventing / improving fetal alcohol syndrome; the lactic acid bacteria is a mixture of Lactobacillus bulgaricus, Streptococcus thermophilus, Kefir bacteria, Bifidobacterium lactis, Bifidobacterium longum, and Bifidobacterium infantis in a weight ratio of 1:1:1:1:1:1 of the bacterial powder.

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