TW202008895A - Method for preparing health-care material by compounding fermented and lyophilized extract of Taiwan sea bream and extract of monostroma nitidum oligosaccharides for providing a composite material rich in high-quality active peptides with low molecular weight - Google Patents

Abstract

The present invention relates to a method for preparing health-care material by compounding fermented and lyophilized extract of Taiwan sea bream and extract of monostroma nitidum oligosaccharides. The method is provided to extract the by-products remained from processing frozen Taiwanese sea bream fillets by hot extraction, and then to use two kinds of lactic acid bacteria to perform fermentation and lyophilizing treatment on the extract for a predetermined period of time so as to obtain fermented and lyophilized extract of Taiwan sea bream. For monostroma nitidum, after thermal extraction, the extracts are hydrolyzed and lyophilized with a microbial enzyme for a predetermined period of time to obtain extract of monostroma nitidum oligosaccharides. Finally, the fermented and lyophilized extract of Taiwan sea bream and the extract of monostroma nitidum oligosaccharides are compounded into a composite material at a predetermined weight percentage. As such, the composite material not only is rich in high-quality active peptides with low molecular weight to provide the effect of effectively suppressing the increase in blood pressure, but also has better ACE inhibitory efficiency than the fermented and lyophilized extract of Taiwan sea bream and the extract of monostroma nitidum oligosaccharides themselves.

Description

Method for preparing health-care material from Taiwan sea bream fermented freeze-dried extract and Qinghai vegetable oligosaccharide extract

The invention relates to a method for preparing an active peptide composite material, in particular to a method for preparing a composite material in which the active peptide extracted by fermentation and cultivation of by-products from Taiwan sea bream (Tilapia) is fermented and cultured to form a composite material. In order to make full use of Taiwan sea bream aquaculture operators based on years of rich and rigorous breeding knowledge, experience and hard work to develop Taiwan sea bream quality fish meat, and Taiwan sea bream processing industry based on years of rich and rigorous processing knowledge, experience and hard work It can cultivate high-quality by-products left after processing Taiwan sea bream (that is, the residuals of Taiwan sea bream frozen fish fillet processing, containing fish bones, fish skin, fish scales and residual meat... etc.), based on cultivation And the production of high-quality active peptides, coupled with the oligosaccharide extract of Qinghai vegetables, so that the composite materials can be synergistic due to the synergy between the active peptides in each other, so that the composite material has more Good health care effect and effect, so that it can be selected as the main component of high-quality cosmetics or health foods (to prevent diseases such as high blood pressure, heart failure, type 2 diabetes, and kidney disease) to protect consumers’ skin and The health of the body, in turn, enables the high-quality by-products remaining after the processing of Taiwan sea bream frozen fish fillets and the Qinghai vegetables readily available on the coast of Taiwan to be used for higher added value due to the present invention.

Press, peptide (from the Greek word "digestion"), also known as "peptide", also known as "peptide", is a small biological molecule in nature, between amines The substance between amino acid and protein. Among them, the amino acid has the smallest molecule and the protein has the largest molecule. The "peptide" is a short chain composed of amino acid monomers. It consists of a peptide (acylamine) Key connection. When the carboxyl group of one amino acid reacts with the amino group of another amino acid, a covalent chemical bond is formed. The short chain of peptides composed of amino acids is a precise protein fragment. Its molecules are only nano-sized, and are easily absorbed by the stomach, blood vessels and skin. Generally speaking, 2 peptides (referred to as 2 peptides) are protein fragments composed of two amino acids. Two or more amino acids are dehydrated and condensed to form several peptide bonds to form a peptide. Multi-level folding constitutes a protein molecule. Therefore, proteins are sometimes called "polypeptides"; common peptides include 2 peptides (dipeptide), 3 peptides (tripeptide), and even polypeptides (polypeptide). Among them, 2 ~20 peptides belong to oligo-peptides, 20~50 peptides belong to polypeptides, usually those with 10 peptides or less are more medically and commercially practical.

Check, "peptide" can be dietary protein (dietary protein) hydrolyzed by chemical methods, can also be obtained manually. As mentioned above, peptides are formed by the polymerization of two or more amino acids. Therefore, they are very important in the regulation of cell physiology and metabolic function. However, due to the nature of peptides It is not very stable, so if you want to store it for a long time, it should be placed in an environment with a moisture-proof mechanism and the temperature maintained below 4℃. In addition, according to many current studies, active peptides have a very close relationship with organisms' nutrition, hormones, enzyme inhibition, immunomodulation, antibacterial, antiviral, and antioxidant functions. As shown in Figure 1, the peptide is a chain molecule formed by dehydrating and condensing an amine group (-NH2) of an amino acid and a carboxyl group (-COOH) to form a peptide bond, wherein the peptide bond is composed of an amine group The -CO-NH- bond formed by the dehydration condensation reaction between the acid and the amine group of the next amino acid has the property of a double bond, and is in the same plane with a total of six adjacent atoms. Therefore, CN cannot rotate freely. Peptide bonds are the connecting bands that make up the protein architecture.

For peptides containing cysteine, methionine or tryptophan, deoxygenated buffers are essential for their dissolution. Such peptides are susceptible to oxidation in the air. Peptides containing glutamine or aspartame are also easily degraded. Peptides are very stable at -20°C, especially freeze-dried and stored in a desiccator. Before exposing them to air, freeze-dried polypeptides can be placed at room temperature.

In general, peptides are the most important "direct nutrients" of an organism. It can be said that "without peptides, there is no ecology!" ""Peptide" is a natural substance with a small molecular weight, which must be combined with amino acids. It has very strong physiological functions and activities in only a small amount, which is similar to the hormone function of living organisms. In general, when an organism decomposes the protein in food, it must be assisted by a variety of enzymes, so that the protein can be decomposed and absorbed. Now the structure of the "peptide" is considered, because its molecule is extremely small, it is easy to be completely The rate of absorption by the human body is 5 times that of ordinary protein. For example, insulin in the human body and enkephalin in the brain are a kind of peptide compound. Hormones in the human body are also composed of peptides, and branched chain amino acid peptides (BCAA) and casein phosphopeptides ( CPP), Glutamine Peptide, etc. are all peptides. There are more than 10,000 kinds of peptides in the human body. Each of these peptides is synthesized by the human body (not secreted) after digestion, hydrolysis, absorption and utilization of general food. Directly supplement the required peptides! Although, in the human body and many animals, pepsin and trypsin secreted by the stomach and pancreas can break down ingested proteins, thereby forming peptide substances. However, the number of peptides that can be directly ingested from food is extremely small. The important reason is that the human body and animal body are specific to the required enzymes. This also causes the human body and many animal bodies to be unable to produce sufficient functions on their own. A variety of functional peptides, based on this, no matter in quantity or in type, it proves that the foods of modern people are really lacking in the variety of peptides required by the human body. In addition, the experiments and tests of modern science also prove that the absorption mechanism of peptides has the following characteristics: 1. Peptides can be directly absorbed without the digestion of human and animal bodies; 2. The peptides of human and animal bodies The absorption rate is extremely fast; 3. When the human body and animal body absorb the peptide, the peptide will not be destroyed; 4. The peptide has the characteristics of 100% absorption by the human body and animal body; 5. The peptide has the initiative to be absorbed Absorption characteristics of human body and animal body; 6. Peptide has the characteristic of being preferentially absorbed by human body and animal body; 7. Absorption of peptide by human body and animal body has no need to consume energy of human body and animal body and will not increase digestive tract at all The characteristics of the burden, especially the feature that it does not increase the burden of gastrointestinal function at all; 8. Peptide shows the role of a carrier in human and animal physical performance, and can absorb the nutrients ingested by ordinary people, especially calcium, etc. The beneficial trace elements of the animal body are adsorbed, adhered, and carried on the carrier; and 9. Peptide can play a role as a means of transportation in the human body and animal body.

According to the history of peptides, the earliest is the growth factor isolated from human plasma in the early 1980s, which is actually a peptide 3 fragment of a protein. Until July 2, 2002, in the 20th Dermatology Congress held in Paris, it was confirmed through research and experiments that compared with vitamins A and C, 5 peptides belonging to oligopeptides have more significant wrinkle-reducing effect . Since then, peptides have become the mainstream ingredient in anti-aging skin care products in Europe and America. Not only is the Da Vinci code yet to be explained, in recent years, peptide care products have also appeared with digital codes! In fact, peptides are composed of a small piece of protein, which is composed of different numbers and types of amino acids. The different numbers represent several amino acids, which are composed of amino acid sequences. Different, can correspond to different needs of the skin. Relative to cosmetic components with large molecules such as collagen, oligopeptide components composed of ten or less amino acids have molecules equivalent to nanometers, so they are easily absorbed by the skin. For example: 5 peptides are composed of 5 different amino acids. At present, the number of peptides is from 2 to 100, and the peptides that have been used in maintenance products are 2, 3, 4, 5, 6, and 7. , 9 and other peptide components; many people think that the larger the peptide number, the better the effect. In fact, this is a wrong concept. In fact, the meaning of the number is that the smaller the number, the easier it is to be absorbed by the skin. The size of the number also represents the difference in the depth and depth of the skin. For example, peptides such as 2, 3, 4, 5, and 6 all have the function of promoting collagen hyperplasia, and the focus is on fibroblasts, while the molecules The larger peptides 7 and 9 are mainly for anti-free radicals, whitening, moisturizing and moisturizing. It acts just right on the epidermis, and it is less meaningful to act on fibroblasts.

In addition, the famous la prairie laboratory also found that the first step in anti-aging is to solve the phenomenon of "Chronic Silent Inflammation" (CSI), which is the latest discovery of aging in the aesthetic medicine industry in the past two years Phenomenon, the average person will not be aware of this aging phenomenon, but it will directly invade the bottom of the skin, and everyone will be threatened by CSI. In particular, after the age of 20, under the influence of the interaction of the three reactions of carbonylation, saccharification, and oxidation, the CSI phenomenon will be triggered. 2 peptides are the behind-the-scenes pushers that can inhibit carbonylation, saccharification, and oxidation; 3 wins The function of the peptide is to promote the proliferation of fibroblast collagen. 3 peptides are the earliest peptide arrangement and combination method found in peptides. According to this, the industry's first maintenance product that incorporates peptides is "Lulu Tightening" "Rejuvenating Eye Cream", which contains the "active ketone" formula, refers to "3 peptides"; as for 4 peptides, it was isolated from the wound healing tissue in 1990, with anti-inflammatory and enhance skin resistance The effect of stress, promoting wound healing and hyperplasia. The conversion of youth hormones present in the body is called DHEA. This hormone will decrease with age. 4 peptides can just make up for DHEA that decreases with age, and can increase the body's changes to the external environment (such as pollution ) Defense ability; the function of 5 peptides is to stimulate collagen hyperplasia, and it is the earliest dermatology medical industry to use it with vitamin A and other anti-aging ingredients, advertised that it can directly act on the dermis layer to promote collagen hyperplasia , The ingredients that achieve the purpose of firming the skin, combined with other moisturizing ingredients, can still accelerate the effect of tightening and lifting; the function of 6 peptides is to soothe expression lines, also known as botulinum-like, with nerve suppression The excessive release of substances affects the transfer protein between the subcutaneous nerves and muscles. It has the best effect on slowing the dynamic lines such as crow's feet and decree lines. French dermatologists have studied that 6 peptides are particularly effective in eliminating wrinkles around the eyes. ; The function of 9 peptides is to reduce the synthesis and production of melanin. When the skin is exposed to external stimulation or ultraviolet radiation, the alpha-melanotropin located in the basal layer cells will combine with the receptor on the melanocytes to directly stimulate Melanocytes, which activate tyrosinase, begin to secrete melanin, 9 peptides can block the source of the formation of melanin, stop the transmission of α-melanotropin signal, and avoid its combination with the receptor on the melanocyte to reduce melanin Generated opportunities. Regarding the know-how of using peptides in makeup and maintenance, You Shanzhen, an assistant professor in the Department of Pharmaceutical Cosmetics of China Medical University, also pointed out that the peptide products acting on the "eyes" can be called the maintenance products with the highest return on investment, because, Compared to the face or other skin of the body, the fat layer and tissues around the eye are thinner, and the peptide molecule is small, the penetration is high, and the effect can be seen in a short time; in addition, in order to make it valuable Peptide ingredients can be absorbed more effectively. He also suggested that the face should be cleaned and applied before using peptide products to avoid possible bactericidal substances secreted by the skin and make the peptide ingredients change. Finally, the night is a period of active cells. Compared with the daytime, the effect of using peptide products at night will also increase.

The "active peptide" is the most basic element that constitutes a cell. It is equivalent to the commander-in-chief and general dispatcher of the cell. Where is the body defective? Or the cells are damaged? The commander-in-chief will immediately carry out regulation and take various nutrients to repair damaged or diseased cells. When the cells that make up these organs are all better, the health problems of organs and tissues will naturally be solved. According to this, when young, due to the sufficient manufacturing capacity of "active peptides", the body is healthy and full of energy. After 40 years of age, the body will lack the "active peptides" due to the sudden decline in the ability to manufacture "active peptides" Since then, various chronic diseases have followed! This is why supplementing with "active peptides" can make the three high (hypertension, hypertension and hyperlipidemia), cataracts, bones and joints and other diseases under good control, so that the patient's white hair turns black and energy changes An important reason for being abundant. What does "active peptide" do to the human body? In the life activities of the human body, mutations in cells cause abnormal phenomena in the cells. This is a natural phenomenon that often occurs. This is also the result of the intricate and comprehensive effect of various factors inside and outside the human body. Internally, it takes about hundreds of millions of cell divisions for a fertilized egg to change from a cell to hundreds of billions of cells. Each time a cell divides, each single-stranded DNA will be copied into another one, as long as there is a division, There will be a chance of error. Although the phenomenon of cell mutation and cell error is a natural phenomenon that often occurs, why do most of us still live normally from small to old? This is the function and function of "active peptide". "Active peptide" is the headquarters that controls human life activities, that is, the chief regulator of the human body building. It is precisely because of the existence of "active peptide" that the normal activities of various organizations of the living body are guaranteed. , The synthesis when the synthesis! Copy when copying! More critical is how to synthesize and copy? All need to be commanded and dispatched by "active peptide". Therefore, "active peptide" is actually the chief engineer of human cell regulation. The human body is composed of countless cells. Therefore, the health of cells naturally determines the health of organs and tissues. Zha, the Duke University School of Medicine, which represents the highest medical level in the United States, has launched a large-scale clinical experiment on "active peptides". Among them, the experimental team of Professor Kratz, in the experimental group of patients with stroke The experimental results of the control group clearly show that the patients in the group supplemented with "active multi-peptide" suffered from hypertension, coronary heart disease, diabetes and other diseases. The stability of the index and the health of the body have increased the survival rate of patients suffering from malignant tumors, cirrhosis, liver ascites and other diseases. Other patients suffering from chronic respiratory diseases, chronic gastrointestinal diseases and other diseases are After the "peptide" nutrition, there are signs of a substantial improvement!

Since "peptide" has many excellent functions that can not be replaced by other substances in skin beauty and body care, and "peptide" can be hydrolyzed by dietary protein by chemical methods, according to this, how to Choose a cheap, high-quality dietary protein from dietary protein? Based on this, the production of high-quality "active peptides" has become a common topic in the related peptide product industry in recent years, which has always drawn on the camp, continued exploration, and hard research and development in order to achieve breakthroughs. One of the most important topics to be discussed and overcome is urgently needed.

In view of the many excellent characteristics of the aforementioned "active peptides", after long-term hard research and experiment, the inventor finally developed and designed a "health material made from fermented freeze-dried extract of Taiwan sea bream and oligosaccharide extract of Qinghai vegetable into a health care material. With the proposal of the present invention, it is possible to effectively use the high-quality dietary protein in the by-products of cheap, high-quality and high-quality leftover from the processing of the Taiwan sea bream industry, by adding the Extract of Monostroma nitidum oligosaccharides , Hereinafter referred to as MO), after the two are combined, we can produce "active peptide" composite materials with excellent quality and reasonable price, so that Jiahui can provide the general public with an urgent need for high-quality "active peptide" products.

The main object of the present invention is to provide a method for preparing a health care material by compounding the fermented freeze-dried extract of Taiwan sea bream and the oligosaccharide extract of Qinghai vegetables. The method is a by-product remaining after processing the frozen fish fillet of Tilapia (for Taiwan sea bream frozen fish fillets after processing, such as: residues containing fish bones, fish skin, fish scales and residual meat...) After hot extraction, two lactic acid bacteria are used to extract Perform fermentation and lyophilization for a predetermined period of time to obtain Extraction of Fermented Tilapia (hereinafter referred to as EFT); after hot extraction of Qinghai vegetables (Monostroma nitidum), extract it with microbial enzymes Hydrolysis to obtain Extract of Monostroma nitidum oligosaccharides (hereinafter referred to as MO); finally, the Taiwan sea bream fermented freeze-dried extract (EFT) and Qinghai vegetable oligosaccharide extract (MO) are scheduled The weight percentage (such as: 1:1 or 1:2) is compounded into a composite material. The composite material has been confirmed by a series of experiments and analysis, not only the content of γ-aminobutyric acid (GABA) Much higher than the GABA content of EFT and MO itself, and analyzed by colloidal filtration chromatography, the molecular weight distribution of active peptides is about 90~2030Da, which can be divided into 6 peak areas (A~F), and the molecular weight The divided area C of 590-720Da has the highest effective inhibition percentage (IER) of ACE (the IER value is 178.3%/mg/mL); and the content of free amino acids and peptides in the composite material is also The free amino acid and peptide content values of EFT itself obtained through different fermentation periods are higher. The composite material is formed even after the composite material ₅₀ inhibition value IC ACE activity is also much higher than the activity of the EFT IC ACE inhibition MO ₅₀ itself is low, the apparent EFT MO compound will vary within each other between the activity of the peptide The resulting synergistic effect makes the composite material have better ACE inhibitory effect. According to this, the composite material is not only rich in high-quality and small molecular weight active peptides, but also has the effect of effectively suppressing the rise of blood pressure. It has better ACE inhibition efficiency than MO itself, so it can be selected as the main component of high-quality cosmetics or health foods (to prevent diseases such as hypertension, heart failure, type 2 diabetes, and kidney disease). Benefiting to ensure the health of consumers' skin and body, the high-quality by-products left after processing of Taiwan sea bream frozen fish fillets and Qinghai vegetables easily available along the coast of Taiwan can be utilized for higher added value due to the present invention.

In order for your reviewing committee to have a better understanding and understanding of the purpose, technical features and efficacy of the present invention, the following examples and drawings are used to explain in detail:

〔Knowledge〕

None

〔this invention〕

200~207‧‧‧step

301~305, 401~405‧‧‧ steps

Figure 1 is a schematic diagram of the chain molecular structure of the peptide formed by the dehydration and condensation of the amino group (-NH2) of the amino acid and the carboxyl group (-COOH) to form a peptide bond; Figure 2 is the by-product of the invention after processing by Taiwan sea bream A schematic diagram of the manufacturing process for extracting the fermented freeze-dried extract of Taiwan sea bream by fermentation and cultivation; FIG. 3 is a schematic diagram of the manufacturing process for extracting oligosaccharides from Qinghai vegetables of the present invention; FIG. 4 is a fermented frozen sea bream of the present invention Colloid chromatogram of protein molecular weight of the dry extract; Figure 5 is the protein of the oligosaccharide extract MO and composite materials (ETF 30/MO) 1/1, (ETF 30/MO) 1/2 of the invention Colloid chromatography analysis spectrum of molecular weight; and Figure 6 is the reverse high-performance liquid chromatography spectrum of the colloidal chromatography partition C of the Taiwan sea bream fermentation lyophilized extract EFT (linear gradient concentration of cyanogen methane 0-100%) ).

In view of the fact that the related peptide product industry has been continuously exploring and studying how to select a cheap, high-quality dietary protein from many dietary proteins over the years. Based on the production of high-quality "active peptide" materials, the inventor is based on the rich practical experience in the cultivation, analysis and research of aquatic food ingredients for many years, thinking about what is left after processing with Taiwan sea bream (Tilapia) High-quality dietary protein in low-cost, high-quality by-products (ie, residues from processing of Taiwan sea bream frozen fish fillets, such as residues containing fish bones, fish skin, fish scales and residual meat...), And using Qinghai vegetables that are readily available along the coast of Taiwan, to produce high-quality and affordable "active peptides" and "Qinghai vegetables oligosaccharide extracts", respectively, and to synthesize "active wins" with better health effects and functions. "Peptide" composite materials, so that Canon can benefit the general public who have an urgent need for high-quality "active peptide" products.

According to the press, in the seventy years of the Republic of China, almost all of the cheap fish goods that people could buy in the vegetable market were "mixed Wu Guo fish" called "fushou fish". Even, the fish farming technology continued Under the progress, after the 1980s, the all-male "sexual Wu Guoyu" has appeared on the market. In the early years, due to the organic management of agriculture, fishery and animal husbandry, the cultivation method of "Wu Guoyu" caused the negative impression of "Wu Guoyu" eating and drinking "pig dung", "chicken dung" and "duck dung". Deeply planted in the minds of ordinary consumers, under the circumstances of the continuous improvement of marine aquaculture technology and the continuous progress and expansion of the ocean fish industry, other alternative fish species and imported fish species are constantly increasing, and with the gradual improvement of economic conditions Consumers’ demand for aquatic products is also changing day by day. As a result, "Wu Guoyu" has gradually been listed as a low-priced inferior fish species in the minds of domestic consumers. In the case of attention and counseling, the market price of "Wu Guoyu" has naturally been in a low and weak position for a long time.

Fortunately, with the long-term hard work of subsequent successors in the breeding industry, research, development and promotion, it gradually opened up the international market, making the "Wu Guoyu" farmed in Taiwan widely accepted by all parties. Since then, in order to establish Taiwan's own high-quality fishery brand, Taiwan's aquaculture industry has not only invested huge funds and efforts in seedling breeding technology, cultivation technology, feed formulation, processing technology and other aspects, but also experienced many difficulties and made breakthroughs one after another. , And finally succeeded in cultivating better quality "Wu Guoyu", and after officially named it as "Taiwan Snapper" (Tilapia), then entered the international market under the name of "Taiwan Snapper". Another new opportunity for Taiwan's fish farming. In recent years, after continuous efforts to improve and promote various aspects, the high-quality "Taiwan Snapper" variety has gradually become the main representative item of Taiwan’s aquatic products, not only widely recognized by domestic consumers, but also South Korea and other places have also repeatedly achieved good results in the export market. At present, the production area of "Taiwan Snapper" is mainly in Chiayi County in the southern region of Taiwan. Although there are a small number of farmed fish farms in the central and northern regions, the production output of Taiwan is still the largest in Yunnan and Jianan. 〉. The reason why "Taiwan sea bream" is widely favored by consumers at home and abroad is not only related to the high-quality meat quality cultivated by the rich farming experience and excellent farming technology of the Taiwan sea bream farming industry, but also to the excellence of the Taiwan sea bream frozen fish fillet processing industry. And the rigorous processing process has an inseparable relationship, all of which makes the Taiwan sea bream frozen fish fillet based on it can be widely praised by domestic and foreign consumers.

With regard to the entire industry of Taiwan seabream, Taiwan seabream aquaculture industry must be able to breed and develop through years of excellent farming techniques and rich farming experience, plus years of hard work and careful care of Taiwan sea bream farming. After harvesting such high-quality meaty Taiwan sea bream, in the end, it must be combined with the excellent, rigorous and complex processing operations of the Taiwan sea bream processing industry before it can be made into high-quality Taiwan sea bream frozen fish fillets. A large amount of high-quality by-products (such as fish bones, fish skins, and minced meat that has been trimmed... etc.) remaining after the processing of fish fillets can only be recycled to make inexpensive feed or fertilizer, Its utilization rate in value is obviously low. In addition to being a pity, it also quite lives up to the regret of the excellent breeding and processing technology and experience developed by Taiwan's breeding and processing industry for many years and the hard work of hard work.

In view of this, the inventors thought about the effective use of high-quality by-products left after processing of Taiwan sea bream (ie, the residues of Taiwan sea bream frozen fish fillet processing, containing fish bones, fish skin, fish scales and residual meat...etc. Residues), based on the cultivation and production of high-quality active peptides, and the high-quality by-products remaining after processing of Taiwan sea bream frozen fish fillets can be used for higher added value; the present invention is a fermented frozen sea bream from Taiwan The dry extract and the oligosaccharide extract of Qinghai vegetable are compounded into a health-care material. The method is to first process by-products remaining after the processing of Taiwanese sea bream (Tilapia) frozen fish fillet (the residue after processing of Taiwan sea bream frozen fish fillet, such as: (Residues containing fish bones, fish skin, fish scales and residual meat...) After hot extraction, the two lactic acid bacteria are used to perform fermentation and lyophilization on their extracts for a predetermined period of time to obtain Extract of fermented Tilapia (hereinafter referred to as EFT) rich in high-quality "active peptides"; secondly, after hot extraction of Qinghai vegetables (Monostroma nitidum), the extract is hydrolyzed by microbial enzymes To obtain Extraction of Monostroma nitidum oligosaccharides (hereinafter referred to as MO); finally, the Taiwan sea bream fermented freeze-dried extract (EFT) and Qinghai vegetable oligosaccharide extract (MO) according to a predetermined weight percentage (For example: 1:1 or 1:2) The composite becomes a composite material. Among them, as shown in Figure 2, the process of fermentation and freeze-drying extract (EFT) of Taiwan sea bream includes the following steps (press, the steps of the following processes are implemented by the inventor after multiple processes and repeated analysis And the best steps, data and conditions summarized after adjusting the details and conditions in each process step. Therefore, in order to simplify the description, the present invention uses only the best steps, data and conditions as examples in the subsequent description. Explain the subsequent process, but the actual implementation of the method of the present invention is not limited to this, its steps, data and conditions can also be fine-tuned according to actual needs, as long as the fine-tuning range of its data and conditions is as follows Within the range of ±5% of the listed data and conditions, it should still be able to achieve the main purpose of the invention, and obtaining the required EFT, MO and composite materials is also the scope of the right of the invention to claim protection; at the same time, the invention The raw materials used in the process are not limited to the residues of Taiwan sea bream frozen fish fillet processing, but can also use Taiwan sea bream whole fish or their fillets according to actual needs (such as during the overproduction or unsalable production of Taiwan sea bream). The replacement is also the scope of the rights claimed by the present invention here, and it is stated first. First of all, please refer to FIG. 2 for the method of preparing the fermented freeze-dried extract (EFT) of the Taiwan sea bream. , Including the following steps:

(200) Hot extraction treatment: After the by-products of Taiwan sea bream are minced evenly, take 2 kilograms (kg) and add 10 liters (L) of water to form a "by-product debris mixture", then cook on high heat Cook the "mixed liquid", turn it to medium heat as soon as it boils, and continue to cook until the "mixed liquid" has only 5 liters left, filter the "mixed liquid" with a filter to obtain the "clarified liquid" and "" "Fish bone residue"; where, continue to cook the "clear liquid" until only 2 liters remain; "fish bone residue" is to add 5 liters of water to form a "fish bone residue mixed liquid" and cook on high heat The "fish bone residue mixture", once boiled, turn to medium heat, and continue to cook until only 1 liter is left, then filter the "fish bone residue mixture" with a filter screen; finally, after mixing the filtrate and the former , The "crude extract" is obtained, and the "crude extract" is frozen at -20 degrees Celsius. After 24 hours, the frozen "crude extract" is degreased on the surface, and then freeze-dried. In order to form freeze-dried "fish crude extract powder".

(201) The first sterilization treatment: add 2% by weight of the "fish crude extract powder" (eg: 120 g) and 1% by weight of glucose (eg: 60 g) to a plastic bucket After 20 liters of pure water, shake the plastic bucket properly. Once the three are evenly mixed, cover the plastic bucket and put the plastic bucket into a sterilization kettle at 121 degrees Celsius. After 30 minutes of sterilization treatment, after completion, it is cooled to room temperature 25 degrees Celsius to form a "fish crude extract powder glucose mixed solution".

(202) Fermentation treatment: use the inoculation ring to aseptically operate from the frozen tubes of the lactic acid bacteria Enterococcus faecalis (BCRC 14046) and Lactobacillus rhamnosus (BCRC 10940) stored in the -80 degrees Celsius freezer, and pick a ring. And the three-division line is on MRSA medium (MRS arga is the medium for culturing and counting Lactobacillus), cultured at 37 degrees Celsius for 24 hours (referred to as the first activation); then, take one with the inoculation ring Isolate the colonies independently, and divide the three areas on the new MA medium at 37 degrees Celsius for 24 hours (referred to as the second activation); then, use the inoculation loop to capture the independent isolation of the second activation Inoculate the colonies in 60 ml of MRSB medium, shake them evenly by hand, and place them in an upright shaking incubator. Cultivate them to a colony density of OD 600nm equal to 0.8~1.0 under the environment of 37 degrees Celsius. Draw up 1% (60mL) of BCRC14046 and BCRC10940 lactic acid bacteria by electric pipette (Pipet-Aid), and add it to the cooled 20-liter "fish crude powdered glucose mixed solution" in the aforementioned plastic bucket, Under the environment of 37 degrees Celsius, after fermentation for 0, 10, 20 and 30 hours, a "fish crude extract powder glucose mixed fermentation broth" is formed.

(203) The second sterilization treatment: put the aforementioned plastic bucket containing "fish crude extract powder glucose mixed fermentation broth" into a sterilization kettle, sterilize it at 121 degrees Celsius for 30 minutes, stop the fermentation, and obtain The "Fish Crude Extract Powder and Glucose Mixed Fermentation Broth" that has been fermented and is numbered EFT0, EFT10, EFT20 and EFT30 according to the fermentation time of 0, 10, 20 and 30 hours, respectively.

(204) Centrifugal separation process: Aseptic operation, each of the "fermented fish crude powdered glucose mixed fermentation broth" that has been completely fermented is filled in batches into a sterilized centrifuge tube about 7 minutes full (eg: 300 ml) , Then put each centrifuge tube into a centrifuge that has been pre-cooled to 4 degrees Celsius, and let the centrifuge run at 14000 xg for 30 minutes to obtain the "supernatant" in each centrifuge tube. "section.

(205) Suction filtration treatment: Assemble a sterilized suction filtration device and a 2 liter serum bottle into one body, and filter the "supernatant" part obtained from each centrifuge tube. Among them, in order to prevent suction The 0.45μm filter membrane in the air filtration device cannot be smoothly carried out or is blocked during filtration. The "supernatant" after centrifuging the "fish crude extract powder glucose mixed fermentation broth" will be poured into 6.0μm. The suction filtration device of the filter membrane is used to filter, and then the "supernatant" is successively filtered through the 1.0μm and 0.5μm filter membranes; finally, the 0.45μm filter membrane is used to filter through the suction filter device. It should be specially stated here that in order to ensure the quality of the process of the present invention, the above filtration operations should be carried out under aseptic operation as much as possible.

(206) Plating freeze-drying treatment: the "supernatant" filtered by the aforementioned air filtration device is inverted and flattened on a stainless steel freeze-drying tray, each piece of freeze-drying tray can be covered with about 700~800 Ml of this "supernatant", the excess fermentation broth must be dispensed into a 2 liter serum bottle, and placed in a sterilization kettle, under the environment of 121 degrees Celsius, after 20 minutes of sterilization, wait for cooling It can be stored in a refrigerator at 4 degrees Celsius to a room temperature of about 25 degrees Celsius. It will be filtered by air suction and freeze-dried after the next time. The supernatants that have been spread out on each of the stainless steel freeze-drying plates are sequentially put into a freeze-drying machine and subjected to freeze-drying treatment for about 3 days in an environment of -80 degrees Celsius.

(207) Obtaining the fermented freeze-dried extract of Taiwan sea bream: After completing the aforementioned freeze-drying process, the sterilized medicine scoop is used to freeze-dry and form a powdered Taiwan sea bream fermented freeze-dried extract (Extraction of fermented Tilapia, (Referred to as EFT) quickly scraped into a centrifuge tube of known weight to avoid moisture absorption, weighed by an electronic balance, and marked as EFT0, EFT10, EFT20 and EFT0 according to 0, 10, 20 and 30 hours of fermentation period, respectively After EFT30 and other samples, store each centrifuge tube in a dry box and wait for subsequent experiments, analysis and use.

In addition, based on many years of research on Taiwan's coastal algae, the inventor knows that the algae body is yellow-green or egg yellow and the scientific name is abundant in the reef rocky areas of Taiwan, Japan, South Korea and China every December to April of the following year. "Monostroma latissimum" is a seaweed of the "Qinghaicai" family of reef membranes. Taiwan's annual output is about 76 metric tons. It is mainly produced from Penghu. In addition to being available for fresh food, it can also be made into processed foods, such as seaweed flake , Seaweed sauce, seaweed crisp, seaweed peanuts, etc., and the extract still contains many special ingredients, such as: sulfated polysaccharides or acidic polysaccharides, proteins, pigments, etc., not only can inhibit the formation of peroxides and free radicals (Mekideche and Briand 1994 reference), which can be used as an active substance for improving immunity and anti-cancer, and also has a cholesterol-lowering effect and effect (Maeda et al., 1991 reference). In addition, it can still be used to extract various algae, such as: Vegetables, alginic acid, carrageenan, etc., are widely used in the food industry to take advantage of their special gelation, viscosity and emulsification. Based on this, the inventor thought about extracting "Qinghai Vegetable Oligosaccharide" from the "Qinghai Vegetable" and adding it to the Taiwan Sea Bream Fermented Freeze-Dried Extract (EFT) to explore its composition and gelability The impact on the active peptide and its biochemical properties in the fermented freeze-dried extract (EFT) of Taiwan sea bream is expected to increase the application field of "Qinghai vegetables" and thus effectively increase the use value of "Qinghai vegetables".

In the present invention, referring to FIG. 3, the method for extracting Qinghai vegetable oligosaccharide from the "Qinghai vegetable" includes the following steps:

(301) Preparation of "MMB medium": First, prepare two 2-liter triangular conical flasks with a volume of pure water in advance; then, prepare the three conical flasks with the following table 1 The composition of the "MMB medium" is 1.3 liters. The hot extract in the "MMB medium" means "Qinghai vegetable hot extract", Algal powder means "Qinghai vegetable dried algae powder", and each layer is sealed with double aluminum foil The mouth of the triangular conical bottle is heated and stirred on the magnetic heating plate until it is boiled for a second time, and then placed in a sterilization kettle. Under the environment of 121 degrees Celsius, the sterilization process is performed for 15 minutes. After the sterilization process is completed After cooling to 25 degrees Celsius at room temperature, sample 10 ml from the triangular conical flask.

(302) Inoculation of "microbial enzymes": use the inoculation loop to aseptically operate and save the frozen tubes of P.vesicularis MA103 and A. salmonicida MAEF108 "microbial enzymes" stored in the -80 degrees Celsius freezer, respectively. One ring, and the three-zone line on the MA medium, cultured at 26 degrees Celsius for 24 hours (the first activation); then, use the inoculation ring to pick an independent colony and the three-zone line on the new MA medium Above, incubate for 24 hours at a temperature of 26 degrees Celsius (secondary activation); then, inoculate the isolated secondary isolated colonies with secondary activation by inoculation loop into 13 ml of MB medium, shake gently with your hands evenly , Placed in an upright shaking incubator, shaking culture at a speed of 90rpm under the environment of 26 degrees Celsius, when the culture is reached to a colony density OD 600nm equal to 0.8~1.0, aseptic operation method, using electric pipette (Pipet-Aid ) Draw 1% by weight of MA103 and MAEF108 (eg: 13 ml) microbial enzymes, and add them to the cooled MMB medium in each Erlenmeyer flask with a capacity of 2 liters An appropriately sized silicon stopper is attached to the mouth of the triangular conical bottle.

(303) Hydrolysis treatment to produce "crude enzyme solution": Place 2 bottles of 2 liter triangular conical flasks into a vertical vertical shaking incubator, set at 26 degrees Celsius, shaking culture at a speed of 90rpm 48 Hour to induce microbial enzymes such as MA103 and MAEF108 to produce "crude enzyme solution" in MMB medium.

(304) Centrifugal separation and sterilization treatment: the "crude enzyme solution" induced by MA103 and MAEF108 in MMB medium in aseptic operation mode is filled in batches into sterilized centrifuge tubes for about 7 minutes (for example: 300 Ml), then put each centrifuge tube into a centrifuge that has been pre-cooled to 4 degrees Celsius, centrifuge at 14000 xg for 30 minutes, then you can use the sterilized medicine dipper and the appropriate funnel, Obtain the "supernatant" portion from each centrifuge tube.

(305) Suction filtration treatment: Assemble a sterilized suction filtration device and a 2 liter serum bottle into one body, and filter the "supernatant" part obtained from each centrifuge tube. Among them, in order to prevent this The 0.45μm filter membrane in the suction filtration device cannot be smoothly carried out or is blocked during filtration. The "supernatant" part of the "crude enzyme solution" induced by MA103 and MAEF108 after centrifugation will be poured into the Put the suction filter device with a 1.0μm filter membrane to filter, and then continue to filter the liquid with a 0.5μm filter membrane; finally, the 0.45μm filter membrane is used to filter through the suction filter device for filtration. Filter out the macromolecular substances and toxins, and sample 10 ml from the "crude enzyme solution" induced by MA103 and MAEF108.

(401) Preparation of "Qinghai Vegetable Hot Extract": Take a 20-liter plastic bucket, wash it and fill it with 6 liters of pure water, and add 15% by weight of Qinghai Vegetable to the plastic bucket ( Such as: 0.6kg of wet algae), shake the plastic bucket properly, and mix the Qinghai vegetable with the once pure water to form a "Qinghai vegetable mixed solution", put on the lid of the plastic bucket, and put it into a sterilization kettle. Under the environment of 121 degrees Celsius, sterilize for 30 minutes. Once the sterilization process is completed, wait for the plastic barrel to cool to 25 degrees Celsius at room temperature, and then form the "Qinghai Vegetable Hot Extract" in the plastic barrel.

(402) Preparation of "Qinghai Vegetable Hydrolysate": Add the "crude enzyme liquid" induced by MA103 and MAEF108, which has been sampled and filtered, in a proportion of 10% by weight (eg 0.6 liter) To 6 liters of the "Qinghai Vegetable Hot Extract", 7.2 liters of "Qinghai Vegetable Hydrolysate" was formed.

(403) Perform hydrolysis treatment on "Qinghai Vegetable Hydrolysate": place 7.2 liters of "Qinghai Vegetable Hydrolysate" on a rotary oscillator, and set the rotary oscillator at a speed of 90 rpm after strengthening and fixing After shaking, after the enzyme hydrolysis reaction between the "crude enzyme solution" induced by MA103 and MAEF108 and the seaweed in the "Qinghai vegetable hydrolysate" has occurred for 48 hours, 7.2 liters of "Qinghai vegetable polysaccharide hydrolysate" will be formed; Then, dispense 7.2 liters of the "Qinghai Vegetable Polysaccharide Hydrolysate" into 1 liter serum bottles in sequence, each bottle can be divided into approximately 750 ml, and after the completion of the packaging, put each serum bottle into In a sterilization kettle, perform sterilization for 15 minutes in an environment of 121 degrees Celsius, and after stopping the enzyme hydrolysis reaction, sample the "Qinghai Vegetable Polysaccharide Hydrolysate".

(404) Plate freeze-drying treatment: Pour the sampled "Qinghai Vegetable Polysaccharide Hydrolysate" into the sterilized and covered with two layers of aluminum foil aseptically (and paste imported freeze-resistant tape to avoid gaps in the aluminum foil Infiltrate the air) one of the stainless steel freeze-drying trays, each of which can be filled with about 700-800 ml of the "Qinghai Vegetable Polysaccharide Hydrolysate", and put each of the stainless steel freeze-drying trays into a freeze dryer, in Perform lyophilization for 3 days at -60 degrees Celsius.

(405) Obtain Qinghai Vegetable Oligosaccharide Extract: Once the freeze-drying process is completed, a freeze-dried Qinghai Vegetable Polysaccharide Hydrolysate powder will be formed in each stainless steel freeze-drying tray, this is Qinghai Vegetable Oligosaccharide Extract (MO ), 嗣, aseptically (with sterilized medicine scoop), quickly scrape MO into a sterilized centrifuge tube of known weight to avoid moisture absorption, and then weigh it with an electronic balance and mark it as MO , It can be stored in a dry box, waiting for subsequent use.

In order to confirm the chemical composition and biochemical characteristics of EFT, MO and the composite materials (EFT/MO) formed at different compounding ratios of EFT, MO and the two, the fermented freeze-dried extracts EFT0, EFT10, EFT20, EFT30 and the Qinghai vegetable oligosaccharide extract (MO) are mixed according to different weight percentages (for example: 1:1 or 1:2), so that the two are combined into a composite material (EFT/MO) 1/1 and (EFT/MO)1/2, and for each of the Taiwan sea bream fermented freeze-dried extracts EFT0, EFT10, EFT20 and EFT30, the Qinghai vegetable oligosaccharide extract (MO) and each composite material (EFT/ MO)11, (EFT/MO)12 carry out a series of experiments, analysis and research as shown below to confirm each of the fermented freeze-dried extracts of the Taiwan sea bream EFT0, EFT10, EFT20 and EFT30, the Qinghai vegetables oligosaccharide extract (MO) and the composite materials (EFT/MO) 1/1, (EFT/MO) 1/2, etc. Chemical composition and biochemical characteristics:

1. Determination of soluble protein content: The present invention uses the lowry kit sold by Sigma to measure the soluble protein content of each sample. The method is to first configure each sample powder into a sample solution with a concentration of 20 mg/ml , Then take 1 ml of the sample solution to 1 ml of Lowry Reagent, mix it uniformly, let it sit for 20 minutes, then add 0.5 ml of Folin & Ciocalteu's Phenol Reagent Working Solution, let it mix for 30 minutes, Take 1.5 ml and measure the absorbance at 750 nm with a spectrophotometer. In this way, the standard calibration curve obtained from the BSA standard substance can convert the soluble protein content of each sample, and the unit is mg/g.

2. Determination of free amino acid content (Doi et al., 1981 reference): The present invention uses the Ninhydrin method to measure the free amino acid content of each sample, and its method is to configure each sample powder to a concentration of 50 Mg/ml of sample solution, then 0.5 ml of sample solution was added to 1 ml of Cd-ninhydrin reagent (ie, 0.8 g of ninhydrin was added to a mixture of 80 ml of absolute ethanol and 10 ml of acetic acid, and finally, to 1 Grams of CdCl2. 1/2 H2O dissolved in a mixture of 1 ml of deionized water), after heating in a water bath at 84 degrees Celsius for 5 minutes, the absorbance at 507nm was measured with a spectrophotometer. The standard calibration line can convert the free amino acid content of each sample, and the unit is mg/g.

3. Determination of the content of peptides (Church et al., 1983 reference): The present invention first configures a mixed solution of o-Phthaldialdehyde. Its method is to take 25 ml of 100 mM sodium tetraborate and 2.5 ml of 20% sodium dodecyl sulphate (SDS ), 1 ml of OPA (that is, 40 mg of o-Phthaldialdehyde dissolved in 1 ml of methanol) and 100 μL of β-mercaptoethanol, and pour these agents into a brown quantitative bottle one by one, and set with deionized water Dissolve to 50 ml; then, take an appropriate amount of each sample, dissolve in deionized water and dissolve to 10 ml, filter through 0.2 μm and 5,000 Da (Millipore, Bedford, MA, USA) filter membrane and properly dilute, take 25 μL of the diluted solution was added to a 1.5 ml quartz tube containing 1 ml of o-Phthaldialdehyde mixed solution, and after uniform mixing and then allowed to stand at room temperature for 2 minutes, the absorbance at 340 nm was measured with a spectrophotometer. The standard calibration curve obtained by the Leu-Gly standard substance can convert the peptide content of each sample, and the unit is mg/g.

4. Determination of ACE inhibitory ability (referenced by Wu and Ding et al., 2002): According to, ACE can decompose hippuric acid-histidine-leucine (HHL) under in vitro conditions to produce hippuric acid (HA), HA There is an obvious absorption peak at 228nm. Therefore, the present invention uses the amount of HA produced to evaluate the inhibitory effect of each sample on ACE (Phensri T et al., 2005 reference). Its method is to use boric acid plus NaCl The buffer solution replaces the absorbance of each sample as the control group. The analysis of ACE's inhibitory ability was modified according to the method described in Wu and Ding (reference 2002). It was based on Luna C18 column (4.6mm×250mm, 5μm), 50% methanol (containing 0.1% trifluoro Acetic acid) was used as the mobile phase, with a flow rate of 0.8 mL/min, and its absorbance was measured at a wavelength of 228 nm, and then calculated to calculate the percentage of each sample that inhibited ACE activity.

Once the present invention completes the aforementioned measurements one by one, the chemical composition and various biochemical characteristics data of each sample shown in Table 1 below can be obtained.

The chemical composition and biochemical characteristics of each sample shown in Table 1 can clearly reflect the EFT0, EFT10, EFT20, EFT30, MO, (EFT30/MO) 1/1 and (EFT30 /MO)1/2 of the following chemical composition and various biochemical characteristics:

1. Soluble protein content: In each sample of the aforementioned experimental group of the present invention, please refer to Table 1, the soluble protein of Taiwan sea bream fermented freeze-dried extract EFT0~EFT10 made from 0 to 10 hours during fermentation The content was greatly increased from 53.9±0.5 to 134.9±0.1mg/g. However, please refer to Table 1 for more information. The soluble protein content of Taiwan sea bream fermented freeze-dried extract EFT30, which was made up to 30 hours during fermentation, increased only slightly to 140.6±0.6mg/g(p< 0.05), based on the changes in the aforementioned data, it should be clear that as the fermentation period increases, the longer the protease action time in the lactic acid bacteria, the freeze-dried extracts EFT0, EFT10, EFT20 and EFT30 of each Taiwan sea bream fermentation The content of soluble protein in the sample also obviously has an increasing trend; among them, the content of soluble protein in the samples of the fermented freeze-dried extracts of Taiwan sea bream EFT0 and EFT30 made from 0 to 30 hours during fermentation obviously increased from 53.9± 0.5 was greatly increased to 146.6±0.6mg/g (as shown in Table 1), however, the soluble protein of the samples of the fermented freeze-dried extracts of Taiwan sea bream EFT10, EFT20 and EFT30 prepared after fermentation was increased to 10 hours later The contents (ie, 134.9±0.1, 140.8±0.1, and 140.6±0.6 mg/g) did not increase significantly (as shown in Table 1).

2. Free amino acid content: In each of the samples of the aforementioned experimental group of the present invention, please refer to Table 1 for details. EFT0~EFT10 samples of Taiwan sea bream fermentation lyophilized extracts made from 0 to 10 hours during fermentation The content of free amino acid was greatly increased from 2.40±0.01 to 16.8±0.32mg/g. However, the free amino group of the EFT20 sample of Taiwan sea bream fermentation lyophilized extract made by prolonging the fermentation time to 20 hours Instead, the acid content decreased to 13.5±0.01mg/g (as shown in Table 1) with the increase of fermentation time. Please refer to Table 1 for more information, Taiwan bream made from 0 to 20 hours during fermentation Fermented freeze-dried extracts EFT0 and EFT20 samples, the free amino acid content can also be significantly increased from 2.40 ± 0.01 to 13.5 ± 0.01mg/g, however, increased to 30 hours during the fermentation of Taiwan sea bream fermentation frozen The dry extract sample EFT30, the content of free amino acid will not increase significantly with the increase of fermentation time, only 14.4 ± 0.15mg/g. The inventors speculate that this phenomenon is most likely caused by the rapid growth and proliferation of lactic acid bacteria that require the use of large amounts of amino acids as a nitrogen source (Chen et al., 2003; Doeven et al., 2005). Therefore, in the early stage of fermentation ( 10 to 20 hours) will cause a reduction in free amino acid content. Although, the EFT20 sample of Taiwan sea bream fermented freeze-dried extract prepared after 20 hours of fermentation had a free amino acid content of 13.5±0.01 mg/g; however, the Taiwan sea bream fermented frozen made after fermentation to 30 hours The dry extract EFT30 sample showed no significant (p<0.05) increase in free amino acid content of 14.4±0.15.

3. Peptide content: In each of the samples of the aforementioned experimental group of the present invention, please refer to Table 1 below. The samples of the fermented lyophilized extract of Taiwan sea bream made from 0 to 10 hours during the fermentation period have the best results. Although the peptide content will increase from 12.6±0.5 to 51.8±1.4mg/g. However, when the fermentation period was extended to 20 hours, the EFT20 sample of Taiwan sea bream fermented freeze-dried extract had a peptide content of only 53.2±0.6, which was not significantly different from that of EFT10 (p<0.05). However, with the increase in the fermentation period, the peptide content of the EFT30 sample of Taiwan sea bream fermentation lyophilized extract made to extend to 30 hours has a significant increase trend, of which, EFT30 has the highest The peptide content reaches 62.7±0.5mg/g. This result clearly shows that the increase in the fermentation period of lactic acid bacteria can significantly improve the peptide content and biological activity in the freeze-dried extract of Taiwan sea bream, such as: from 12.6±0.5 to 62.7±0.5mg/g ( p<0.05). In a similar study (Quirós et al., 2007 reference), it was also shown that the milk was fermented with Enterococcus faecalis CECT 5727 at 30 degrees Celsius for 0 to 48 hours, and its peptide content will increase from 0.5 to 3.0 mg/mL. However, the results of the experimental group of the present invention show that the peptide content of the EFT10 sample of fermented freeze-dried Taiwan sea bream produced during the fermentation to 10 hours has the highest peptide content of 12.6±0.5 mg/g. However, the samples of Taiwan’s sea bream fermented freeze-dried extracts EFT10, EFT20, and EFT30, based on which they were prepared, had their peptide content (51.8±1.4, 53.2±0.6, 62.7±0.5) but stagnated without significant increase. The inventors speculate that this phenomenon may be related to the difference in results caused by different protease activities (proteases, polypeptide enzymes, dipeptidases, and aminopeptidases, etc.) of different lactic acid bacteria (Chen, 2007 reference).

4, inhibition of vasopressin converting enzyme _IC50 values of IC: ACE inhibiting activity of the response curve can be obtained the IC ₅₀ value of each of the samples, the IC ₅₀ values based upon the desired activity to ACE inhibitory concentration 50% peptides, this The lower the value, the better the effect of suppressing ACE. Thus, during the fermentation from the Taiwan porgy 10-20 hours made of fermented extracts were lyophilized samples the EFT EFT 10 and 20, an IC ₅₀ values of 1.04 ± 0.08 and 1.40 ± 0.01mg / mL, with a marked increase in the phenomenon, In response to this, the inventors speculate that it may be due to the action of lactic acid protease that causes the hydrolysis of some of the peptides that have ACE inhibitory effect, resulting in the deterioration of the inhibitory effect. On the other hand, during the fermentation from the Taiwan porgy 10-30 hours made of fermented and lyophilized extracts EFT10 EFT30 sample, an IC ₅₀ value of 1.04 ± 0.08, respectively, and 1.12 ± 0.01mg / mL, two sets of statistical data There was no significant difference (p<0.05).

According to the above, in each sample of the aforementioned experimental group of the present invention, the peptide content of EFT 30 is significantly higher than that of the samples of EFT 0, EFT 10, and EFT 20. Therefore, the present invention selects EFT and When the MO sample is combined into an active peptide composite according to different weight percentages, EFT 30 is selected as the best sample, and the 1:1 and 1:2 weight percentages are respectively mixed with the MO sample uniformly. And the active peptide composite materials (EFT/MO) 1/1 and (EFT/MO) 1/2 are compounded; Si, for EFT 30, MO, (EFT/MO) 1/1 and (EFT/MO) 1 respectively /2 After analysis of ACE inhibitory activity, the analysis results showed that the IC ₅₀ value of EFT 30 was 1.12±0.01, the IC ₅₀ value of MO was 1.09±0.03 mg/mL, and the IC ₅₀ value of (EFT/MO) 1/1 The IC ₅₀ value of 1.08 and (EFT/MO) 1/1 is 0.80 mg/mL, which clearly shows that the IC ₅₀ value (0.8 mg/mL) of (EFT 30/MO) 1/2 is the lowest, This means that it has the highest inhibitory effect on ACE, and the inventor speculates that the reason is that the synergistic effect between the active peptides of EFT and MO has a better ACE inhibitory effect.

In addition, in order to determine the health effects and effects of each sample on the human body, the inventors specifically determined the GABA content of each sample EFT0, EFT10, EFT20, EFT30, MO (EFT/MO) 11, and (EFT/MO) 12 In the present invention, the OPA derivation method is used to analyze the content of γ-aminobutyric acid (γ-aminobutyric acid, hereinafter referred to as GABA) in each sample. The method is to add 100 ml of each sample solution to 500 ml of OPA working solution, even shaking, react at room temperature for 2 minutes, and filter with 0.22μm filter, then perform HPLC analysis-take 20μL into C18 column (5μm, 250mm×4.6mm); mobile phase A 0.1M sodium acetate (pH=6.7); mobile phase B is methanol; A is 90% at the beginning of the gradient; it drops to 40% after 10 minutes, and the gradient is maintained for 18 minutes; after 18 minutes, it is changed to B mobile phase 100 % Until the baseline is stable, and the flow rate is 0.8mL/min, the UV-visible light detector is detected at 340nm; and the data results shown in Table 2 below are detected. The results clearly show that with the fermentation time Increase, the GABA content in each EFT sample from 10 hours to 30 hours of fermentation, its content can be increased from 12.92mg/100g to 25.59mg/100g, an increase of up to 2 times; Among them, EFT30 has the highest GABA content; MO The GABA content is 20.03mg/100g, and its GABA content is relatively lower than EFT30; the GABA content of each composite (EFT/MO) 1/1, (EFT/MO) 1/2 is 25.79 and 24.46, respectively. mg/100g, of which (EFT30/MO)1/2 has a low GABA content, the inventor speculates that the reason is that the ratio of oligosaccharide content in the composite material is high, resulting in a decrease in GABA content by dilution.

Table 2. GABA content of Taiwan sea bream fermented freeze-dried extract, Qinghai vegetable oligosaccharide extract and their complex

According to GABA, an inhibitory nerve-conducting substance, glutamic acid can be converted to GABA by fermentation of lactic acid bacteria, and according to many studies, it is clearly and clearly pointed out that this substance has the effect of inhibiting the rise of blood pressure (Inoue et al., 2003 reference). In addition, Sun et al. (2009 reference) isolated lactic acid bacteria Lactobacillus helveticus ND01 fermented skim milk from Xinjiang fermented milk (Xinjiang koumiss) for 24 hours, which had an ACE inhibition rate of 67.18% and a GABA content of 165.11 mg/L. In addition, more studies have pointed out that, in the case of primary hypertensive rats, GABA with 0.5 mg/kg body weight was administered by tube feeding, and the systolic blood pressure decreased by 20.8 mm Hg on average 4 hours after the feeding (Hayakawa et al., 2002). Lb.casei and Lc.lactis fermented milk (with a GABA concentration of 10-12mg/100mL) was given to mildly hypertensive patients for 12 weeks. At 8 weeks, the systolic blood pressure decreased by an average of 17.4±4.3mm Hg (Refer to Inoue et al., 2003). Although, the experimental data results shown in Table 2 clearly show that the GABA content of the samples of the Taiwan sea bream fermented freeze-dried extracts EFT 0, EFT 10, EFT 20 and EFT 30 and the MO sample of Qinghai vegetable oligosaccharide extract are relatively low For fermented dairy products, the inventor reasonably speculates that this phenomenon may be the result of different factors caused by different factors such as fermentation raw materials and fermentation conditions.

Finally, in order to determine the protein molecular weight distribution range of each sample of the present invention, in order to discuss the distribution of peptide molecular weight, colloidal chromatography was carried out on the protein molecular weight of each sample, and its method was to prepare each sample as The sample solution of appropriate concentration (50mg/mL) was filtered through 0.2μm and 5000Da filters in sequence, and then subjected to Sephadex G-25 colloid chromatography, in which, EFT 0, EFT 10, EFT 20, EFT 30 and other samples The results of colloidal chromatography show that the molecular weight range of EFT 10 is between 2140 and 150 Da; the molecular weight range of EFT 20 is between 1840 and 90 Da; and the molecular weight range of EFT 30 is between 2030 and 100 Da. In addition, please refer to the EFT 10, EFT 20, and EFT 30 chromatograms shown in Figure 4. After comparing the spectra, you can still find that the peaks of the spectra have shifted to the right. This phenomenon indicates the molecular weight There is a downward trend. The inventors speculate that this trend is most likely due to the protease secreted by the lactic acid bacteria during the fermentation process that hydrolyzes large-molecule proteins or peptides, resulting in an increase in the peptide content of small molecules. Cha, Sinha et al. used Sephadex G-75 colloidal chromatography to analyze the changes of protein in hydrolysate and found that the proportion of macromolecular protein was the largest when it was not hydrolyzed. However, as the hydrolysis time increased , The peak of the protein map shifts to the right, showing that the content of small molecule protein increases with the increase of hydrolysis time (Sinha et al., 2007 reference), the inventor also deeply agrees; for MO, (EFT 30/ MO) 1/1 and (EFT 30/MO) 1/2 colloidal chromatography results, please refer to the MO, (EFT 30/MO) 1/1 and (EFT 30/MO) 1/2 layers shown in Figure 5 The analysis of the results shows that the molecular weight range of the protein in MO is between 2030 and 330 Da, and the molecular weight range of the protein in (EFT 30/MO) 1/1 is between 2140 and 110 Da, (EFT 30/MO) 1/2 The molecular weight range of the protein is between 2030~90Da, and can be divided into six divided regions A to F as shown in Table 3 below. The molecular weight distribution of each divided region is 2030~1150 and 1100~760 respectively. , 720 ~ 590, 560 ~ 390, 370 ~ 230 and 140 ~ 90Da; and the peptide concentration of each of the divided areas is 1.2, 0.2, 0.3, 0.9, 0.2 and 0.1 mg/mL.

Table 3. The molecular weight, peptide concentration and ACE of each division of composite material (EFT 30/MO) 1/2

According to press, angiotensin converting enzyme (ACE for short) is an angiotensin converting enzyme, which mainly has the following two major functions: (1) catalyze the conversion of angiotensin I to angiotensin II; (2) inactivate bradykinin.

ACE is the ideal target for the treatment of hypertension, heart failure, type 2 diabetes and diabetic nephropathy due to the two functions listed above. Angiotensin-converting enzyme inhibitors can reduce the production of angiotensin II and increase bradykinin activity. After analyzing each sample by colloidal chromatography in the present invention, please refer to Table 3 above. For composite materials (EFT 30/MO) 1/2, six divided regions (A to F) can be chromatographed, and their molecular weights The distribution ranges are 2030-1150, 100-760, 720-590, 560-390, 370-230 and 140-90Da; the peptide concentrations are 1.2, 0.2, 0.3, 0.9, 0.2 and 0.1mg/mL; and The inhibition capacity of ACE is 83.5, 27.2, 58.9, 1.3, 26.2 and 7.6%; the effective inhibition percentage {IER=Inhibition(%)/Peptide concentration(mg/mL)} is 71.2, 156.5, 178.3, 1.41, 122.9 And 57.3%, as shown in Table 3 above, in which the IER value of the partition C is the highest among the partitioned areas, therefore, the partition C is collected for further RP-HPLC purification analysis, please refer to Figure 6 , Which can be used as a reference basis for screening and identifying peptide sequences with inhibitory ACE activity.

However, in the aforementioned measurement, analysis, and study of the present invention, the composite material with the highest ACE inhibition ability of each of the samples was (EFT 30/MO) 1/2, and the composite material (EFT 30/MO) 1/2 After colloidal chromatography analysis, six divided regions (A~F) can be chromatographed, and their ACE inhibition capabilities are 83.5, 27.2, 58.9, 1.3, 26.2, and 7.6%, respectively, and the effective inhibition percentage {IER=Inhibition( %)/Peptide concentration (mg/mL)} is 71.2, 156.5, 178.3, 1.41, 122.9 and 57.3%, which means that in the divided area of the composite material (EFT 30/MO) 1/2, it is The IER value of the C partition is the highest, so if you want to study the sequence of the active peptide contained in it, you only need to collect the components of the C partition area for further RP-HPLC purification analysis, which can be used as Reference basis for screening and identifying peptide sequences with inhibitory ACE activity.

Although the foregoing experimental results show that the Taiwan sea bream fermented crude extract fermented freeze-dried powders EFT0, EFT10, EFT20 and EFT30 made in the present invention have a lower GABA content than fermented dairy products, this phenomenon is most likely due to the fermentation raw materials and The different results produced by different factors such as fermentation conditions, but the results shown in Table 2 can clearly reflect, regardless of the length of fermentation, the samples made by the experimental group of the present invention EFT0, EFT10, EFT20, EFT30, MO, ( The content of GABA in EFT 30/MO) 1/1 and (EFT 30/MO) 1/2 has undoubtedly made it have the health care effect and effect of effectively suppressing the rise of blood pressure.

In summary, the fermented freeze-dried extracts of Taiwan sea bream made by the present invention, EFT0, EFT10, EFT20, EFT30, MO, (EFT 30/MO) 1/1 and (EFT 30/MO) 1/2, Many experiments have clearly confirmed that not only is it rich in high-quality, small-molecular-weight active peptides, but also has the effect of effectively suppressing blood pressure rise, it still has excellent ACE inhibition efficiency, so it can be selected as high-quality cosmetics or Ideal ingredients for health foods (used to prevent diseases such as hypertension, heart failure, type 2 diabetes, and kidney disease), and can benefit consumers to ensure the health of consumers' skin and body, so that the quality of Taiwan sea bream frozen fish fillets remaining after processing By-products and Qinghai vegetables, which are readily available along the coast of Taiwan, can be used for higher added value due to the present invention.

As mentioned above, the above is only one of the best embodiments of the present invention, but the structural features of the present invention are not limited to this, any person familiar with the art can easily think of changes or modifications in the field of the present invention , Can be covered in the patent scope of the following case.

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Claims (7)

A method for preparing a health-care material from the fermented freeze-dried extract of Taiwan sea bream and oligosaccharides from Qinghai vegetables. The method uses the by-products remaining after processing of Taiwan sea bream frozen fish fillets, and includes the following steps: First, the by-products are extracted by heat After extraction, two kinds of lactic acid bacteria were used for fermentation and lyophilization of their extracts for a predetermined period of time to obtain fermented freeze-dried extracts of Taiwan sea bream; secondly, after hot extraction of Qinghai vegetables, they were extracted using microbial enzymes The material is subjected to hydrolysis and lyophilization for a predetermined period of time to obtain the Qinghai vegetable oligosaccharide extract; and finally, the Taiwan sea bream fermented lyophilized extract is compounded with the Qinghai vegetable oligosaccharide extract at a predetermined weight percentage to form a compound Materials, the content of γ-aminobutyric acid in the composite material is much higher than the content of γ-aminobutyric acid in the fermented freeze-dried extract of Taiwan sea bream and the oligosaccharide extract of Qinghai vegetables, and analyzed by colloidal filter chromatography The molecular weight distribution of active peptides in the composite material is about 90~2030Da, which can be divided into 6 peak regions, and the effective inhibition percentage of ACE is highest in the divided region with a molecular weight of 590~720Da; and in the composite material The content of free amino acids and peptides is also higher than that of the fermented freeze-dried extract of Taiwan sea bream obtained during different fermentation periods, and the IC ₅₀ value of the composite material to inhibit ACE activity is also after much higher than that obtained during the fermentation of different fermentation Taiwan porgy and the extracts were freeze-dried oligosaccharide extracts were Qinghai dish itself IC ACE inhibiting activity of ₅₀ is low. The manufacturing method according to claim 1, wherein the by-product is the residue after processing of Taiwan sea bream frozen fish fillets. The preparation method as described in claim 1, wherein, before hot extracting the Qinghai vegetable, an MMB medium needs to be prepared. The preparation procedure includes the following steps: first prepare a triangle with a capacity of 2 liters washed with pure water Two Erlenmeyer flasks; heirs, each prepared 1.3 liters of MMB medium with predetermined ingredients in each triangular Erlenmeyer flask, respectively prepared 1.3 liters of MMB medium with predetermined ingredients in each triangular Erlenmeyer flask, wherein, the The predetermined ingredients include 40.3 grams of Marine broth 2216, 1.0 grams of Tris, 6.0 grams of NaCL, 1.0 grams of NaNO3, 0.1 grams of KH2PO4, 1.5 grams of Algal Powder and 15 milliliters of hot extract, and the hot extract is Qinghai Vegetable hot extract, the Algal powder is dried algae powder of Qinghai vegetables; the mouth of each triangular conical bottle is sealed with double-layer aluminum foil paper; and after heating and stirring to the second boiling, it is placed in a sterilization kettle. Under the environment of 121 degrees Celsius, the sterilization process is performed for 15 minutes. After the sterilization process is completed, the preparation of the MMB medium is completed when it is cooled to 25 degrees Celsius at room temperature. According to the manufacturing method described in claim 3, before the hot extraction treatment of the Qinghai vegetable, it is necessary to inoculate the MMB culture medium with microbial enzymes. The inoculation procedure includes the following steps: use the inoculation ring in a sterile manner and store it in Celsius- The freezing tubes of the P. vesicularis MA103 and A. salmonicida MAEF108 microbial enzymes in the 80-degree freezer are respectively ticked, and the three areas are lined on the MA medium, and cultured at 26 degrees Celsius for 24 hours, As the first activation; heir, and then use the inoculation loop to pick an isolated colony and three-division line on the new MA medium, in the environment of 26 degrees Celsius, culture for 24 hours, as the second activation; Then, Then use the inoculation loop to pick up the second activated independent colonies and inoculate them into 13 ml of MB medium. After shaking gently with your hands, place them in an upright shaking incubator at 90 rpm at 90 rpm. At a speed of shaking; and once cultured to a colony density of OD 600nm equal to 0.8 to 1.0, use a motorized pipette to aseptically absorb 1% by weight of MA103 and MAEF108 microbial enzymes and add them to a volume of 2 liters, respectively In each of the triangular conical flasks, the MMB medium has been cooled, and a silicon cork of appropriate size is placed on the mouth of each triangular conical flask. According to the manufacturing method described in claim 4, before performing hot extraction treatment on the Qinghai vegetable, it is still necessary to perform hydrolysis treatment on the MMB medium to produce a crude enzyme solution. The hydrolysis treatment procedure includes the following steps: Place the shaped bottle into a vertical vertical shaking incubator, set at 26 degrees Celsius and shake at 90rpm for 48 hours to induce the microbial enzymes of MA103 and MAEF108 to produce crude enzyme solution in MMB medium; Aseptic operation method, the crude enzyme solution induced by MA103 and MAEF108 in MMB medium is filled in batches into sterilized centrifuge tubes for about 7 minutes, and then each of the centrifuge tubes is sequentially placed until it has been pre-cooled to In a centrifuge at 4 degrees Celsius, after centrifugation at 14000xg for 30 minutes, the sterilized drug dipper and appropriate funnel can be used to obtain the supernatant portion from each centrifuge tube; and the heir, the sterilized one The air filter device is assembled with a 2 liter serum bottle to filter the supernatant part obtained from each centrifuge tube. Among them, in order to prevent the 0.45 μm filter membrane in the air suction filter device from filtering smoothly If it is clogged, the crude enzyme solution induced by MA103 and MAEF108 will be centrifuged into the supernatant to the suction filter device with a 1.0μm filter membrane for filtration, heir, and then continue. The liquid is filtered with a 0.5 μm filter membrane; finally, the 0.45 μm filter membrane is used to pass through the suction filter device to perform filtration to filter out the macromolecular substances and toxins therein. The preparation method as described in claim 5, wherein the hot extraction process of the Qinghai vegetable includes the following steps: take a 20-liter plastic bucket, and after washing, fill it with 6 liters of pure water, and put it in the plastic Add 15% by weight of Qinghai vegetables to the barrel, shake the plastic barrel properly, and mix the Qinghai vegetables with pure water once to form a mixture of Qinghai vegetables, then put the lid on the plastic barrel and put it into a sterilization kettle. Sterilize for 30 minutes in an environment of 121 degrees Celsius. After the sterilization process is completed, wait for the plastic barrel to cool to 25 degrees Celsius at room temperature. That is, the hot extract of Qinghai vegetables is prepared in the plastic barrel; and The crude enzyme solution was added to 6 liters of the hot extract of Qinghai vegetables at a ratio of 10% by weight to form 7.2 liters of Qinghai vegetables to be hydrolyzed. The manufacturing method according to claim 6, wherein the hydrolysis and freeze-drying of the Qinghai vegetable includes the following steps: placing 7.2 liters of the Qinghai vegetable to be hydrolyzed on a rotary oscillator and strengthening After that, the rotary oscillator was set to oscillate at a speed of 90 rpm. After the crude enzyme solution induced by MA103 and MAEF108 and the algae in the hydrolyzate of Qinghai vegetable had undergone an enzyme hydrolysis reaction for 48 hours, 7.2 liters of Qinghai Vegetable Polysaccharide Hydrolysate; Si, divide 7.2 liters of Qinghai Vegetable Polysaccharide Hydrolysate into 1 liter serum bottles in sequence, each bottle can be divided into approximately 750 milliliters. Put the serum bottle into a sterilization kettle, perform sterilization for 15 minutes under the environment of 121 degrees Celsius, and after the enzyme hydrolysis reaction is terminated, sample the polysaccharide hydrolysate of Qinghai vegetable; sample the Qinghai vegetable The polysaccharide hydrolysate is aseptically poured into a sterilized stainless steel freeze-dried pan covered with two layers of aluminum foil. Each pan can be filled with 700~800 ml of the Qinghai vegetable polysaccharide hydrolysate, and each Put the stainless steel freeze-drying trays into a freeze-drying machine and perform the freeze-drying treatment for 3 days under the environment of -60 degrees Celsius; and once the freeze-drying treatment is completed, it will be formed in each of the stainless steel freeze-drying trays The freeze-dried Qinghai vegetable polysaccharide hydrolysate powder is the extract of Qinghai vegetable oligosaccharide.

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