WO2024055983A1

WO2024055983A1 - Composition of pumpkin seed protein and use thereof

Info

Publication number: WO2024055983A1
Application number: PCT/CN2023/118501
Authority: WO
Inventors: 江龙; 吴婧; 廖琪林
Original assignee: 南京纽邦生物科技有限公司
Priority date: 2022-09-14
Filing date: 2023-09-13
Publication date: 2024-03-21
Also published as: CN117731007A

Abstract

A composition of a pumpkin seed protein or an isolated protein, protein hydrolysate or concentrated protein thereof, and the use thereof in the preparation of a drug, nutritional product, health-care product, food product, beverage and animal feed for regulating intestinal flora. The intestinal flora is selected from one or more of the following: Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum and Bifidobacterium lactis.

Description

Pumpkin seed protein composition and application thereof

Technical field

The invention belongs to the technical field of health food and dietary supplements, and specifically relates to compositions of pumpkin seed protein or its isolated protein, hydrolyzed protein, concentrated protein and its use in medicines, nutritional supplements, health products, food, beverages and animal feeds. Related applications.

Background technique

Research shows that human health is not only related to its own genome, but also inextricably linked to the environmental microorganisms in the intestines. Intestinal flora plays a very important role in the human body. It is regarded as another "hidden organ" of the human body and carries the human body's "second gene". Therefore, the human body is also described as a super organism. Intestinal flora regulate intestinal homeostasis through interaction with the host; imbalance of intestinal homeostasis caused by various reasons will affect human health and lead to various diseases.

Lactobacillus acidophilus mainly exists in the small intestine and releases lactic acid, acetic acid and some antibiotics that act on harmful bacteria. It is an intestinal probiotic; Streptococcus thermophilus is an oxygen-consuming Gram-positive bacterium that exists in the human body. A kind of beneficial bacteria in the intestines that can help people with lactose intolerance digest lactose; Bifidobacterium is an important intestinal beneficial microorganism, which has nutritional, anti-tumor and immune-enhancing effects on human health, plays a biological barrier, and improves gastrointestinal function. It has various physiological functions such as intestinal function, anti-aging, etc. Typical representatives include Bifidobacterium longum, Bifidobacterium lactis, etc. Escherichia coli is an opportunistic pathogen and is a gram-negative bacterium. Under certain conditions, it can cause gastrointestinal infection or urinary tract and other local tissue and organ infections in humans and animals.

Intestinal probiotics can synthesize a variety of vitamins (mainly vitamins K and B), participate in the digestion of food (such as carbohydrates), produce lactic acid, promote intestinal peristalsis, inhibit the growth of pathogenic bacteria and decompose harmful substances, and activate the immune system , maintaining the balance of intestinal flora is indispensable to human health. The regulation of intestinal flora and the improvement of intestinal microenvironment can be achieved through exogenous supplementation of probiotics; probiotics must reach a sufficient quantity to be effective, and have high requirements on preparation technology, storage conditions, price, etc. Pathogenic bacteria produce pathogenic substances and cause host infection. In order to maintain intestinal health, it is necessary to promote the proliferation of intestinal probiotics and reduce the number of intestinal pathogenic bacteria.

Pumpkin seeds contain a large amount of fat, protein, phytosterols, various minerals and other nutrients, and their health value has been recognized around the world. In recent years, people have begun to study the extraction, isolation and utilization of pumpkin seed protein from pumpkin seeds. Today, the development and application of pumpkin seed protein is mainly concentrated in the agricultural field. Studies have shown that the antifungal protein extracted from naked pumpkin seeds has significant growth inhibitory activity against common agricultural pathogenic bacteria; the activity of extracted from pumpkin seeds The protein has N-glycosidase activity and shows a strong inhibitory effect on the proliferation of human melanoma cells. These studies indicate that pumpkin seed protein has certain physiological activity.

Although pumpkin seed protein has high nutritional value and has some applications in other aspects, its development and utilization in health food and medicine are not enough. Protein, as an important nutrient, has received increasing attention for its impact on intestinal health. Intestinal flora can participate in the body's protein metabolism. Food protein will affect the composition and balance of the intestinal flora, and changes in the intestinal flora affect the body's utilization of protein. If pumpkin seed protein with the ability to regulate intestinal health can be extracted, the application scope of pumpkin seed protein can be expanded, and multifunctional peptide resources can be enriched, which is also crucial for improving intestinal health and/or preventing chronic diseases and complications.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention provides a culture medium that can promote the proliferation of intestinal probiotics (beneficial bacteria), reduce pathogenic bacteria (harmful bacteria), and effectively reduce the number of bacteria containing Lactobacillus acidophilus, Streptococcus thermophilus and Bifidobacterium The pH value promotes the production of short-chain fatty acids, thereby inhibiting the growth of spoilage bacteria, promoting gastrointestinal motility and the growth of probiotics, and regulating the composition and application of pumpkin seed protein for intestinal health. The pumpkin seed protein of the present invention can be added to various products according to different needs or compatible with other ingredients; the protein component is easy to use and has safe ingredients. The pumpkin seed protein can be isolated pumpkin seed protein, concentrated pumpkin seed protein or hydrolyzed pumpkin. Any one or more types of seed proteins.

In one aspect, the invention provides a method of increasing the level of a first intestinal microbiota in a subject, comprising: administering to the subject a composition containing pumpkin seed protein or its isolate, hydrolyzed protein, or protein concentrate. ; The composition effectively increases the level of the first intestinal microbiota in the subject; the first intestinal microbiota is selected from one or more of the following: Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium lactis.

In some embodiments, the above method further includes reducing the level of E. coli in the subject.

In some embodiments, the first intestinal microbiota are intestinal probiotics.

In some embodiments, the composition is formulated as a nutritional supplement, food, beverage, animal feed, or pharmaceutical.

In some embodiments, the compositions are prepared as solid or liquid formulations.

In some embodiments, the compositions are in the form of suppositories, tablets, pills, granules, powders, films, capsules, beverages, aerosols, spirits, tinctures, tonics, liquid suspensions, or syrups.

In some embodiments, the composition is administered to the subject at a dosage of 0.1-500g, preferably 1-400g, 2-300g, 3-200g, 5-100g per day. Alternatively, the composition can be administered to the subject at a dose of 0.1-10g/kg, 0.5-5g/kg, 1-3g/kg per day.

In another aspect, the present invention provides a composition containing pumpkin seed protein or its protein isolate, hydrolyzed protein, or concentrated protein in the manufacture of a nutritional supplement for increasing the level of the first intestinal microbiota in a subject, For use in food, beverages, animal feed or pharmaceuticals, the first intestinal microflora is selected from one or more of the following: Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum, and Bifidobacterium lactis. Preferably, the first intestinal microbiota are intestinal probiotics.

In some embodiments, the above uses further include reducing the level of E. coli in the subject.

In some embodiments, the composition is administered to the subject at a dose of 0.1-500g, preferably 1-400g, 2-300g, 3-200g, 5-100g per day; the composition is effective to increase the first intestinal tract in the subject level of the microbiota. Alternatively, the composition can be administered to the subject at a dose of 0.1-10g/kg, 0.5-5g/kg, 1-3g/kg per day.

The pumpkin seed protein of the present invention is easily absorbed directly by the intestinal tract, can promote the proliferation of intestinal probiotics, reduce the number of intestinal pathogenic bacteria, effectively reduce the pH value of the culture medium containing Lactobacillus acidophilus, Streptococcus thermophilus and Bifidobacterium, and promote The production of short-chain fatty acids, thereby regulating intestinal health and/or overall health; while discovering new functions and nutritional properties of pumpkin seed protein, increasing the added value of pumpkin seed protein utilization.

Description of drawings

Figure 1A is a graph showing the growth curves of Lactobacillus acidophilus and Streptococcus thermophilus in corresponding media with or without pumpkin seed protein.

Figure 1B is a graph showing the growth curves of two bifidobacteria in corresponding media with or without pumpkin seed protein.

Figure 2 is a comparison chart of the log number of colonies of E. coli.

Figure 3 is a comparison chart of log colony values of Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum and Bifidobacterium lactis.

Detailed ways

Reference will now be made in detail to the preferred embodiments of the invention, examples of which will be further described. Although the invention will be described in conjunction with preferred embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the invention as defined by the claims. Additionally, in the detailed description of the invention, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without these specific details. In other cases, no details Well-known methods, procedures, components, and other features are described in detail so as not to unnecessarily obscure aspects of the invention.

As used herein, the term "or" is intended to include "and" and "or." In other words, the term "or" may also be replaced by "and/or".

As used herein, the singular forms "a/an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

As used herein, the term "comprises" or "includes" or variations thereof means where the term is used in its non-limiting sense to include the items following the word but not to exclude items not specifically mentioned. It also includes the more restrictive verbs 'consist essentially of' and 'consist of'.

As used herein, the terms "subject" or "individual" are used interchangeably to refer to any subject to whom the methods and compositions of the present disclosure may be applied or administered. It is possible for a subject to suffer from a disease or disorder, but the subject need not be ill to benefit from the methods and compositions of the present disclosure. A subject may need to improve his or her gut and/or overall health, but a subject may also have a generally healthy gut and wish to maintain or further improve his or her gut and/or overall health. Accordingly, any subject can take the disclosed compositions or be the recipient of the disclosed methods. As used herein, the term "subject" refers to animals (eg, birds, reptiles, and mammals). In certain embodiments, subjects may include non-primates (e.g., camels, donkeys, zebras, cows, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys, Chimpanzees, humans) mammals. In certain embodiments, the subject may be a non-human mammal. In other embodiments, the subject can be a human.

As used herein, the term "administration" refers to the process of delivering a disclosed composition or active ingredient to a subject. The compositions of the present invention may be administered in a variety of ways to achieve the desired effect, including orally, intragastric and parenterally (meaning intravenous and intraarterial as well as other suitable parenteral routes) and the like.

As used herein, the term "protein" when describing protein concentration, protein molecular weight distribution, protein solubility and protein amino acid content is defined as native protein structure or denatured protein structure or fragments thereof; shall be interpreted broadly and includes intact proteins, polypeptides , oligopeptides, amino acids and mixtures thereof. "Hydrolyzed protein (protein hydrolyzate)" refers to proteins that have been broken into shorter peptide fragments and amino acids by partial or complete acid hydrolysis or enzymatic hydrolysis. “Protein isolate (protein isolate)” means a protein obtained from natural sources and /or modified protein material obtained from natural sources. "Protein concentrate (protein concentrate)" means protein obtained from natural sources and /or modified protein material obtained from natural sources.

As used herein, the term "effective amount" refers to the amount required to achieve the effect as taught herein. Effective amounts herein include, but are not limited to, amounts necessary to increase intestinal probiotics in a subject; and/or to reduce intestinal pathogenic bacteria in a subject; and/or to improve intestinal health and/or overall The amount necessary for health. In accordance with the present disclosure, a suitable single dose size is one that achieves the above effects when administered one or more times over a suitable period of time.

As used herein, the term "pharmaceutically acceptable" means pharmaceutically, physiologically, dietaryally and/or nutritionally acceptable within the scope of sound medical judgment. Those compositions or agents, materials or combinations of compositions and/or dosage forms thereof which are suitable for contact with human and animal tissues, are compatible with the other ingredients of the composition, and do not exhibit undue toxicity, irritation, allergic reaction or Other problems or complications, commensurate with a reasonable benefit/risk ratio.

The present application found that administration of pumpkin seed protein significantly increased the levels of the first intestinal microbiota in the subjects while reducing the levels of E. coli in the subjects. To this end, the present invention and embodiments relate to methods and compositions using "pumpkin seed protein-containing" compositions and/or "pumpkin seed protein enhanced" compositions, wherein the pumpkin seed protein-containing compositions comprise pumpkin seed protein-containing compositions Or any combination of protein isolate, protein hydrolyzate, or protein concentrate.

The methods of the present invention comprise the daily administration of at least 0.1 g, typically 0.1-500 g, 1-400 g, preferably 2-300 g, 3-200 g, 5-100 g, of a pumpkin seed protein-containing composition, depending on the specific formulation and form . The amount to be administered can also be 0.1-10g/kg, 0.5-5g/kg, 1-3g/kg of subject's body weight per day, depending on factors such as subject's sensitivity, age, gender and weight, idiosyncratic reactions, etc. Variety. One or more doses may be administered one or more times daily or at a suitable frequency for any period of time. For example, an effective dose may be administered daily for one day, several days, multiple days, or indefinitely.

The pumpkin seed protein-containing composition of the present invention may be formulated as a nutritional or dietary supplement, a (medical) food, an animal feed or a pharmaceutical composition, in liquid or solid form, and may optionally also include nutritional or a pharmaceutically acceptable carrier. For example, when in solid form, the compositions may be formulated as snack bars, yogurt, lozenges, tablets or capsules. On the other hand, when in liquid form, the compositions may be formulated as tinctures, soft gel capsules, liquid capsules, syrups, carbonated beverages, brewed beverages (eg, coffee or tea), juices, energy drinks, sports drinks, or flavored waters. Although special consideration is given to nutritional and pharmaceutical groups for human use compounds, it should be understood that pumpkin seed protein-containing compositions and formulations may also be used for veterinary purposes (e.g., animal feed for domestic companion animals ("pets") or animal feed for farm animals. In other considerations In terms of aspects, the pumpkin seed protein-containing composition may also be provided in the form of a bulk product (for example, in an amount equal to or greater than 100 g, equal to or greater than 1000 g, or equal to or greater than 10 kg) for the production of nutritional supplements, (medical) Food, animal feed or pharmaceuticals.

To further enhance the intestinal health of a subject, contemplated methods of administering a pumpkin seed protein-containing composition include administering at least one probiotic microorganism simultaneously with or subsequent to administration of a pumpkin seed protein-containing composition. In addition, the composition can also be combined with arabinoxylan (AX), arabinoxylan oligosaccharide (AXOS), xylo-oligosaccharide (XOS), fructooligosaccharide (FOS), galacto-oligosaccharide (GOS), inulin and/or one or a combination of two prebiotics in pectin.

The pumpkin seed protein-containing compositions of the present invention may be combined with health additives to increase the breadth of health benefits. Health additives may be added to the pumpkin seed protein-containing composition in any combination depending on the form of the composition and considerations of desired health effects and product cost. Exemplary additives that may be combined with pumpkin seed protein-containing compositions include superoxide dismutase (SOD), compositions containing activators of SOD, foods containing bioavailable SOD, or extracts thereof (e.g., sprouted wheat, Wheatgrass, encapsulated (lipid and/or protein encapsulated) cantaloupe, rye, barley, barley grass, broccoli sprouts, kale, Brussels sprouts and curcuminoids (e.g. turmeric), copper iodide (CuI ), selenium (Se), fulvic acid, foods containing fulvic acid or their extracts (e.g. potatoes, radish, beets, carrots, root vegetables, blackstrap molasses and shilajit), coenzyme Q10 (ubiquinone) or pyrrolo Quinoline quinone (PQQ).

The following examples are illustrative of selected embodiments of the invention and are not meant to limit the scope of the invention.

Example 1

Inoculate each activated strain into each culture medium with or without the addition of pumpkin seed protein or its isolated protein, hydrolyzed protein, or concentrated protein at an inoculum volume of 2%: Lactobacillus acidophilus (L. acid) 563 in MRS liquid The culture medium was statically cultured at 37°C; Streptococcus thermophilus (S.thermophilus) 176 was cultured statically in Eliker medium at 42°C; Bifidobacterium longum (Bifido.longum) BB536 and Bifidobacterium lactis ( Bifido. lactis) BB12 were cultured anaerobically at 37°C in mMRS medium (MRS medium containing 0.05% L-cysteine hydrochloride). The cultures were cultured to a certain point in the logarithmic phase, and then the pH values of the two fermentation broths in each group were measured, and viable bacteria were counted. The results are shown in Table 1.

Table 1

As shown in Table 1, compared with the control group, the pH of the protein groups all decreased, indicating that pumpkin seed protein effectively reduced the pH of culture media containing Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum, and Bifidobacterium lactis. pH, it can be seen that pumpkin seed protein promotes the production of short-chain fatty acids, thereby inhibiting the growth of spoilage bacteria, promoting gastrointestinal motility and the growth of probiotics; the number of viable bacteria in the protein group increased, indicating that pumpkin seed protein can effectively increase the growth rate of Lactobacillus acidophilus, Viable bacterial counts of Streptococcus thermophilus, Bifidobacterium longum, and Bifidobacterium lactis.

In addition, within 0 to 24 hours after cultivation of Lactobacillus acidophilus 563 and Streptococcus thermophilus 176, and within 0 to 30 hours after cultivation of two kinds of bifidobacteria, samples were taken every 2 hours to measure their OD value at 600 nm. The growth curve of probiotic bacteria was drawn according to the change of OD value with time, and the results are shown in Figure 1A and Figure 1B.

All experiments were performed in parallel three times, and the results were expressed as mean ± standard deviation.

Figure 1A is a growth curve diagram of Lactobacillus acidophilus 563 and Streptococcus thermophilus 176 in corresponding media with or without the addition of pumpkin seed protein or its isolated protein, hydrolyzed protein, or concentrated protein. Figure 1B is a graph showing the growth curves of Bifidobacterium longum BB536 and Bifidobacterium lactis BB12 in corresponding media with or without the addition of pumpkin seed protein or its isolated protein, hydrolyzed protein, or concentrated protein. As shown in Figure 1A, when pumpkin seed protein was added for culture, the OD values of Lactobacillus acidophilus 563 and Streptococcus thermophilus 176 changed compared to when pumpkin seed protein was not added. are faster and maintained at a higher level. As shown in Figure 1B, when pumpkin seed protein was added to culture, the OD value of Bifidobacterium longum BB536 changed faster than when pumpkin seed protein was not added, and the OD value of the culture group without pumpkin seed protein at 26 hours had reached a certain level. remained unchanged, while the culture group adding pumpkin seed protein still had an increasing trend; the OD value of Bifidobacterium lactis BB12 changed faster and maintained at a higher level earlier than when pumpkin seed protein was not added. It shows that pumpkin seed protein can effectively promote the proliferation of Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum and Bifidobacterium lactis.

Example 2

Twenty SPF male SD rats were divided into blank control group and pumpkin seed protein group. They were fed normal feed and had their own drinking water. Environmental parameters: temperature (23±1)℃, relative humidity (60±5)%, 12 hours Day and night alternate. The blank control group was fed normal feed and an equal volume of distilled water, and the pumpkin seed protein group was fed pumpkin seed protein in addition to normal feed. The pumpkin seed protein group was given about 1-3g/kg of pumpkin seed protein or its isolated protein, hydrolyzed protein, or concentrated protein every day.

After 30 days of feeding, the feces of the two groups of mice were collected for intestinal flora culture and analysis. Manually collect fresh fecal balls from the end of the rat rectum, dilute them to 10 ^-9 , spot them on various culture media, and culture them under appropriate conditions. After culturing, high-throughput sequencing and metabolomic analysis of 16S rDNA of intestinal flora were performed. The number of bacteria was counted in colony forming units lg (CFU/g), and the changes in bacterial colonies were comparatively analyzed. The results are shown in Figures 2 and 3.

Figure 2 is a comparison chart of the log number of colonies of E. coli in the blank group and the pumpkin seed protein treatment group. As shown in Figure 2, compared with the blank group, the E. coli in the pumpkin seed protein group decreased by 12.07%, indicating that pumpkin seed protein can effectively reduce the content of E. coli.

Figure 3 is a comparison chart of the logarithmic colony values of Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum and Bifidobacterium lactis in the blank group and the pumpkin seed protein treatment group. As shown in Figure 3, compared with the blank group, Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum and Bifidobacterium lactis increased by 4.97%, 11.66%, 5.65% and 5.58% respectively in the pumpkin seed protein group. , indicating that pumpkin seed protein can effectively promote the in vivo proliferation of Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum and Bifidobacterium lactis.

In the present invention, after pumpkin seed protein treatment, the content of other intestinal probiotics can also be increased, such as Lactobacillus rhamnosus, Bifidobacterium adolescentis, Lactobacillus bulgaricus, etc.; the content of other intestinal pathogenic bacteria can be reduced, Such as Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, etc. This allows the levels of gut microbiota to be maintained and/or restored to near-normal levels, which is highly beneficial for maintaining gut health and/or overall health.

The pumpkin seed protein of the present invention can promote the proliferation of intestinal probiotics, reduce the number of pathogenic bacteria, effectively reduce the pH value of the culture medium containing Lactobacillus acidophilus, Streptococcus thermophilus and Bifidobacterium, promote the production of short-chain fatty acids, and thereby regulate the intestinal tract. Tao Health can be combined with different ingredients according to different needs. It can not only meet the needs of vegetarians but also regulate intestinal health and/or overall health; it provides a basis for the future development and utilization of pharmaceuticals and health products containing pumpkin seed protein and corresponding peptides. and laid the foundation for the production and application of various products.

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any skilled person familiar with the art can make various changes to these embodiments without departing from the principles and purposes of the present invention. Modifications, substitutions and variations, the scope of the present invention is defined by the claims and their equivalents.

Claims

A method of increasing the level of a first intestinal microbiota in a subject, the method comprising: administering to the subject a combination containing pumpkin seed protein or its isolate, hydrolyzed protein, or concentrated protein composition; the composition effectively increases the level of the first intestinal microbiota in the subject; the first intestinal microbiota is selected from one or more of the following: Lactobacillus acidophilus, Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium lactis.
The method of claim 1, further comprising reducing the level of E. coli in the subject.
The method according to claim 1 or 2, characterized in that the first intestinal microflora is intestinal probiotics.
The method according to any one of claims 1 to 3, characterized in that the composition is formulated as a nutritional supplement, food, beverage, animal feed or medicine.
The method according to any one of claims 1 to 4, characterized in that the composition is prepared into a solid preparation or a liquid preparation.
The method according to any one of claims 1 to 5, characterized in that the composition is in the form of suppositories, tablets, pills, granules, powders, films, capsules, beverages, aerosols, spirits, In the form of tinctures, tonics, liquid suspensions or syrups.
The method of any one of claims 1-6, wherein the composition is administered to the subject at a dose of 0.1-500 g per day.
A composition containing pumpkin seed protein or its protein isolate, hydrolyzed protein, or protein concentrate in the manufacture of a nutritional supplement, food, beverage, animal feed, or drug for increasing the level of a first intestinal microbiota in a subject The purpose is characterized in that the first intestinal microorganism The group of organisms is selected from one or more of the following: Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium lactis.
The use according to claim 8, further comprising: reducing the level of E. coli in the subject.
The use according to claim 8 or 9, characterized in that the composition is administered to the subject at a dose of 0.1-500g per day; the composition effectively increases the first Levels of gut microbiota.
The use according to any one of claims 8-10, characterized in that the composition is prepared into a solid preparation or a liquid preparation.
The use according to any one of claims 8-11, characterized in that the composition is in the form of suppositories, tablets, pills, granules, powders, films, capsules, beverages, aerosols, spirits, In the form of tinctures, tonics, liquid suspensions or syrups.