Composition for simulating whole breast milk oligosaccharide and application thereof

Abstract

The invention belongs to the technical field of nutritious food, and discloses a composition for simulating whole breast milk oligosaccharide and application of the composition. In the composition for simulating the whole breast milk oligosaccharide, the mass ratio of 2 '-fucosyllactose to lactose-N-neotetraose to 3'-sialyllactose is (3.9-8.1): (0.8-1.2): (0.8-1.2), and the breast milk oligosaccharide 2 '-fucosyllactose, the lactose-N-neotetraose and the 3'-sialyllactose are mutually matched to take effect in a synergistic manner, so that the whole breast milk oligosaccharide is obtained. The composition can improve the memory ability and cognitive ability of an eating object, can be added into infant food (including infant formula powder, complementary food and nutritional supplements) and nutritional supplements or food of children over 3 years old, teenagers and aged people, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of nutritional foods, and in particular to a composition simulating whole breast milk oligosaccharides and applications thereof. Background Art

[0002] Human milk oligosaccharides (HMOs) are the third largest component in human breast milk after lactose and fat, with a content of up to 22-25 g / L in colostrum and 12-13 g / L in mature milk. HMOs have important biological functions such as preventing intestinal diseases, antiviral, antibacterial, and regulating immunity, and have an important impact on the growth and development of infants and young children. Breast milk composition is the gold standard for the preparation of infant formula, so using a certain proportion of functional oligosaccharides to imitate the composition ratio of oligosaccharides in breast milk is a necessary prerequisite for the development of more nutritious and healthy infant formula.

[0003] HMOs are mainly composed of five core monomer structures: glucose, sialic acid, fucose, N-acetylglucosamine and galactose. Since HMOs with different structures will have different fucosylation and sialylation, human milk oligosaccharides are divided into three categories: (1) fucosyl oligosaccharides, represented by 2'-fucosyl oligosaccharides (2'-FL) and 3'-fucosyl oligosaccharides (3-FL); (2) sialic acid oligosaccharides, represented by 3'-sialyl lactose (3'-SL) and 6'-sialyl lactose (6'-SL); (3) oligosaccharides formed by core sugar chain structures without fucosyl or sialic acid groups, represented by lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

[0004] In recent years, many studies have found that various oligosaccharides can also promote brain development. Mice supplemented with 2'-FL showed vagal-mediated improvements in learning and memory, and mice supplemented with sialyllactose showed improved stress behavioral responses. Various oligosaccharides have been shown to be able to change brain or cognitive development in some way, but there are few studies on their effects on brain development after mixed use and the mixing ratio. Chinese patent application CN112384081A discloses a nutritional composition of human milk oligosaccharides (HMO) that supports neural development in mammals, especially compositions that improve, enhance, promote or regulate the central nervous system (CNS) related to or affect the function of the neurotransmitter serotonin (5-hydroxytryptamine or 5HT) and other related cognitive benefits, but the patent application focuses on the effects of a single category of breast milk oligosaccharides.

[0005] The types and concentrations of oligosaccharides in breast milk of different mothers vary, and oligosaccharides from a single source may not meet the needs of the comprehensive development of infants. There are many types of HMO, and it is very difficult to achieve complete breast milkization of infant formula. Moreover, currently only galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), 2'-fucosyllactose (2'-FL), and lactose-N-neotetraose (LNnT) are allowed to be added to infant formula, which cannot completely replace the role played by breast milk oligosaccharides. Therefore, the mixed use of different types and concentrations of breast milk oligosaccharides to better promote the development of infant brains is a hot topic in current research. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology and provide a composition for simulating whole breast milk oligosaccharides and its application. The present invention uses 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyllactose to represent three types of oligosaccharides, namely, salinosylated neutral oligosaccharides, non-fucosylated neutral oligosaccharides and sialylated oligosaccharides, respectively, and compound the three types of oligosaccharides in breast milk according to the proportion of these three types of oligosaccharides to simulate the composition of whole breast milk oligosaccharides, thereby improving the cognitive memory function of the consuming object.

[0007] To achieve the purpose of the present invention, the composition of simulated whole breast milk oligosaccharides of the present invention comprises 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyl lactose.

[0008] 2'-fucosyllactose (2'-FL) is a trisaccharide structure formed by fucose and lactose, and is a representative substance of fucosylated oligosaccharides. The commercially available substance is usually prepared by microbial fermentation and has the same structure as 2'-fucosyllactose found in human milk.

[0009] Lacto-N-tetraose (LNnT) is a tetrasaccharide structure formed by lactose, N-acetylglucosamine and galactose. It is the core sugar chain of human milk oligosaccharides and a representative substance of oligosaccharides without fucosyl or sialic acid groups. Most of the commercial lactose-N-tetraose in the prior art is prepared by microbial fermentation and has the same structure as the lactose-N-tetraose oligosaccharide found in human milk.

[0010] 3'-sialyllactose (3'-SL) is a trisaccharide structure formed by sialic acid and lactose, and is a representative substance of sialylated oligosaccharides. Most of the commercial 3'-sialyllactose in the prior art is prepared by microbial fermentation and has the same structure as 3'-sialyllactose found in human milk.

[0011] Furthermore, in some embodiments of the present invention, the mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyllactose in the composition of simulated whole human milk oligosaccharides is (3.9-8.1):(0.8-1.2):(0.8-1.2).

[0012] Furthermore, in some embodiments of the present invention, the mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyl lactose in the composition of simulated whole human milk oligosaccharides is (3.95-8.05):(0.95-1.05):(0.95-1.05).

[0013] Furthermore, in some embodiments of the present invention, the mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyllactose in the composition simulating whole human milk oligosaccharides is (4-8):1:1.

[0014] In another aspect, the present invention further provides an application of the aforementioned composition simulating whole breast milk oligosaccharides, wherein the application is to use the aforementioned composition simulating whole breast milk oligosaccharides in food to improve the cognitive behavioral ability of the consumer.

[0015] Furthermore, in some embodiments of the present invention, the edible objects are infants, children over 3 years old, teenagers or the elderly.

[0016] Furthermore, in some embodiments of the present invention, the food is infant formula powder, complementary food or nutritional supplement.

[0017] Furthermore, in some embodiments of the present invention, the food is a nutritional supplement.

[0018] Furthermore, in some embodiments of the present invention, the improvement of the cognitive behavioral ability of the edible object is specifically manifested in significantly increasing the exploration index of the edible object in a Y-maze test.

[0019] Furthermore, in some embodiments of the present invention, the improvement of the cognitive behavioral ability of the edible object is specifically manifested in significantly increasing the cognitive index of the edible object in a new object recognition test.

[0020] Furthermore, in some embodiments of the present invention, the improvement of the cognitive behavioral ability of the edible subject is specifically manifested in significantly increasing the memory ability of the edible subject in a water maze test.

[0021] Furthermore, in some embodiments of the present invention, the subject consumes the composition simulating whole human milk oligosaccharides at an amount of 300 to 600 mg / kg / d.

[0022] The composition of the present invention comprises specific human milk oligosaccharides 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyllactose, which work synergistically with each other, so that the composition can enhance the memory and cognitive abilities of the consumer, and can be added to infant foods (including infant formula powder, complementary foods, nutritional supplements), as well as nutritional supplements or foods for children over 3 years old, adolescents and the elderly, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the ratio of the distance that mice explore the new arm to the total distance in the animal experiment of the embodiment of the present invention;

[0024] Figure 2 This is a schematic diagram of the ratio of the movement time of mice exploring a new arm to the total time in the animal experiment of the embodiment of the present invention;

[0025] Figure 3 This is a schematic diagram of the ratio of the total time spent by mice exploring new objects to the exploration time in the animal experiment of the embodiment of the present invention;

[0026] Figure 4 This is a schematic diagram of the ratio of the number of times mice explored new objects to the total number of times they explored in the animal experiment of the embodiment of the present invention;

[0027] Figure 5 This is a schematic diagram of the ratio of the time that mice spend in the quadrant where the platform is located to the total movement time in a water maze test in an animal experiment in an embodiment of the present invention;

[0028] Figure 6 This is a result diagram of the number of times mice passed through the platform in the animal experiment of the embodiment of the present invention;

[0029] Figure 7 This is a schematic diagram of the ratio of the distance traveled by mice in the quadrant where the platform is located to the total movement distance in the water maze test in the animal experiment of the embodiment of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. It should be understood that the following description is only used to explain the present invention and is not intended to limit the present invention.

[0031] As used herein, the terms "comprises," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0032] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0033] When amount, concentration or other value or parameter is expressed as range, preferred range or a series of upper preferred value and lower preferred value limit range, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when disclosing range "1 to 5", described range should be interpreted as including range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0034] Singular forms include plural references unless the context clearly indicates otherwise. "Optional" or "either" means that the subsequently described event or incident may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0035] Approximate terms in the specification and claims are used to modify quantities, indicating that the present invention is not limited to the specific quantity, but also includes acceptable and modified parts close to the quantity without causing changes in the relevant basic functions. Accordingly, the use of "about", "approximately", etc. to modify a numerical value means that the present invention is not limited to the exact numerical value. In some examples, the approximate terms may correspond to the accuracy of the instrument for measuring the numerical value. In the specification and claims of this application, range limitations can be combined and / or interchanged, and if not otherwise stated, these ranges include all subranges contained therein.

[0036] The indefinite articles "a" and "an" before the elements or components of the present invention have no limitation on the quantity requirements (i.e. the number of occurrences) of the elements or components. Therefore, "a" or "an" should be interpreted as including one or at least one, and the elements or components in the singular form also include the plural form, unless the quantity obviously refers to the singular form only.

[0037] In addition, the descriptions of the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" described below mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0038] In some embodiments of the present invention, the mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyl lactose in the composition simulating whole human milk oligosaccharides is (3.9-8.1):(0.8-1.2):(0.8-1.2).

[0039] Furthermore, in some embodiments of the present invention, the mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyl lactose in the composition of simulated whole human milk oligosaccharides is (3.95-8.05):(0.95-1.05):(0.95-1.05).

[0040] Furthermore, in some embodiments of the present invention, the mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyllactose in the composition simulating whole human milk oligosaccharides is (4-8):1:1.

[0041] When and only when the mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyl lactose in the composition of simulated whole breast milk oligosaccharides is within the above-mentioned mass ratio range, they can cooperate with each other to take effect synergistically, so that the composition has the ability to enhance the memory and cognitive ability of the consumer, and the effect is equivalent to that of the oligosaccharides extracted from breast milk, and is significantly better than the blank control group, galacto-oligosaccharide group (GOS) and fructo-oligosaccharide group (FOS).

[0042] 1. Experimental Materials and Methods

[0043] The human milk oligosaccharide samples tested are as follows:

[0044] Chinese name English name Abbreviations 2'-fucosyllactose 2'-fucosyllactose 2'-FL Lactose-N-neotetraose Lacto-N-neotetraose LqCy 3'-Sialyl lactose 3'-sialyllactose 3'-SL Galacto-oligosaccharides Galactooligosaccharides GOS Fructo-oligosaccharides Fructooligosaccharides FOS Extraction of human milk oligosaccharides Extracted human milk oligosaccharides HMO

[0045] 2. Animal Experiments

[0046] In order to verify that the combination of human milk oligosaccharides 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyl lactose can improve the memory and cognitive abilities of the subjects, and play a role in promoting memory and cognitive abilities similar to oligosaccharides extracted from breast milk, the present invention uses mouse experiments for verification, and the specific scheme is as follows.

[0047] Experimental method: A total of 60 3-week-old growing male mice were equally divided into 5 groups, namely, blank control group, galacto-oligosaccharide group (GOS), fructo-oligosaccharide group (FOS), oligosaccharide composition group (the composition group of the present invention) and oligosaccharide group extracted from breast milk (HMO group) (the source of breast milk was mature milk (lactation period of more than 30 days) collected from 25-35 years old healthy volunteers by the laboratory research team). Each group had 12 mice, and each mouse was required to be healthy and without obvious adverse symptoms; the blank control group was given 200uL / d physiological saline by gavage, and the combined human milk oligosaccharide group of the present invention was given human milk oligosaccharide mixture by gavage at 300-600mg / kg / d, wherein the mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyllactose in Example 1 was 4:1:1, the mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyllactose in Example 2 was 6:1:1, and the mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyllactose in Example 2 was 8:1:1. The dosage of GOS, FOS and HMO groups was 300-600mg / kg / d. Cognitive function tests were performed after five consecutive weeks of gavage, including Y-maze test, novel object recognition (NOR test) and water maze test (MWM).

[0048] The Y-maze test is used to measure the memory of animals based on their tendency to explore new environments. Animals generally prefer to explore new mazes rather than return to previously familiar mazes. This test involves several brain regions including the hippocampus, septum, forebrain, and prefrontal cortex. This test is used to study the exploration and memory abilities of animals.

[0049] The present invention adopts a modified version of the Y-maze test, and the equipment is composed of three arms made of black acrylic of equal size (10×40×16 cm) and connected to each other at 120°. Before all experiments are carried out, the experimental mice are accustomed to adapt for 2 days, during which they can freely visit all arms for 10 minutes. In the sample phase test, each animal is placed in the maze separately, and one of the three arms is closed. Allow the animal to freely explore the other two arms for 10 minutes. A 5-minute test phase test is carried out 24 hours after the sample phase test. In the test phase test, the previously closed arm is opened and defined as a "new arm". The latency of each mouse to explore the new arm for the first time, the proportion of the time the mouse explores the new arm to the total time, and the proportion of the distance explored in the new arm to the total distance are recorded.

[0050] The NOR test is based on the experimental animal's tendency to novelty. After being exposed to an object, the experimental animal shows greater interest in a new element than an object that has already been explored, indicating that it can recognize and remember familiar objects. This test evaluates the animal's cognitive memory ability through the behavioral method of how long the experimental animal explores familiar objects and new unfamiliar objects.

[0051] The NOR test was performed in an open black plastic room (40 cm × 40 cm × 50 cm). Three days before training and testing, the experimental mice were stroked for 1 min every day to avoid stimulating the mice so that they would eliminate the unfamiliarity with the tester. 24 hours before testing or training, the animals were placed in the test room to adapt to the test environment. At the beginning of training, objects A and B were placed at the left and right ends of one side wall. The mice were placed in the venue with their backs facing the two objects, and the distance between the tip of the mouse's nose and the two objects should be consistent. The mice were placed in the venue for 10 minutes, and the video recording equipment was turned on immediately after placement. The experimenter immediately left the test room and recorded the contact between the mice and the two objects, including the number of times the nose or mouth touched the objects and the time spent exploring within 2-3 cm of the objects (the front paws on the objects, the nose sniffing the objects, licking the objects, etc. are all considered to be exploring the objects. Posing or climbing on the objects without moving cannot be considered to be exploring new objects). After 10 minutes, the mice were immediately placed back in the original mouse box. After the mice rested for 1 hour, the test began. At this time, object B in the field was replaced with object C. The mice were still facing the two objects with their noses at the same distance from the two objects, and observed for 5 minutes. The key parameter considered in this test is the time the experimental mice spend exploring each novel or familiar object.

[0052] Morris water maze (MWM) is an experiment that forces experimental animals to swim and learn to find a platform hidden in the water. It is widely used in the study of learning and memory, Alzheimer's disease, hippocampus / exohippocampus research, intelligence and aging, etc. The experiment tests the learning and memory ability of mice in terms of spatial position and direction (spatial positioning) through two parts: the navigation test and the spatial exploration test.

[0053] The Morris water maze (MWM) experiment was completed in a pool with a diameter of 120 cm. The milky white water in the pool submerged the platform by 0.5-1 cm, and the water temperature was maintained at 22±2℃; 4 logos of different shapes and colors were posted in the axial direction of the pool wall (circle, triangle, square, polygon, etc., with obvious distinction). The experiment lasted for 6 days, and 4 training sessions were scheduled at fixed time periods every day. In order to make the mice familiar with the environment, a round of learning can be done first, and the mice are placed on the platform in turn to observe the water maze environment for 30 seconds. Training was carried out for 5 consecutive days, 4 times a day (4 starting points were randomly placed), each training lasted 1 minute, and the interval between each training was >10 minutes. The platform was removed on the sixth day. The original location of the platform was marked with a circular ring on the computer screen, so that the number of times the original platform location was crossed could be recorded. Then the mice were placed in the water at the same entry point in each quadrant, and the swimming path of the mice within 60 seconds was recorded, and the number of times the mice crossed the target quadrant platform was recorded, and the spatial positioning ability of the test mice was observed.

[0054] The experimental results are as follows Figure 1-7 Shown

[0055] from Figure 1-2 It can be seen that after continuous gavage of the experimental mice for 5 weeks, the edible oligosaccharide composition of the present invention and the HMO group samples can significantly increase the time and distance of the mice exploring the new arm. This shows that edible oligosaccharide composition can significantly improve the exploration and memory ability of mice, and the effect is similar to that of the HMO group. In addition, edible GOS and FOS did not significantly increase the time and distance of mice exploring the new arm, indicating that GOS and FOS are ineffective in improving the exploration and memory ability of edible mice.

[0056] from Figure 3-4 It can be seen that after continuous gavage of the experimental mice for 5 weeks, the edible oligosaccharide composition of the present invention and the HMO group samples can significantly increase the time and number of explorations of the mice for new objects. This shows that edible oligosaccharide composition can significantly improve the cognitive memory ability of mice, and the effect is similar to that of the HMO group. In addition, edible GOS and FOS did not significantly increase the time and number of explorations of mice for new objects, indicating that GOS and FOS are ineffective in improving the cognitive memory ability of edible mice.

[0057] from Figure 5-7It can be seen that after continuous gavage of the experimental mice for 5 weeks, the proportion of the time spent exercising in the quadrant where the platform is located in the mice that consumed the oligosaccharide composition and the HMO group samples of the present invention was significantly higher than that of the control group mice. The number of times that the mice that consumed the oligosaccharide composition and the HMO group samples crossed the platform was also significantly higher than that of the control group mice. The proportion of the distance traveled in the quadrant where the platform is located in the total distance traveled in the mice that consumed the oligosaccharide composition and the HMO group samples was also significantly higher than that of the control group mice. These data show that consuming the oligosaccharide composition samples can significantly improve the learning and memory ability of mice for spatial position and sense of direction (spatial positioning), and the effect is similar to that of the HMO group. In addition, consuming GOS and FOS did not significantly improve the proportion of the time spent exercising in the quadrant where the platform is located in the total time, the number of times the platform is crossed, and the proportion of the distance traveled in the quadrant where the platform is located in the total distance traveled, indicating that GOS and FOS are ineffective in improving the learning and memory ability of mice for spatial position and sense of direction (spatial positioning).

[0058] Those skilled in the art will easily understand that the above descriptions are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A composition simulating whole breast milk oligosaccharides, characterized in that: The composition simulating whole breast milk oligosaccharide comprises 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyl lactose.

2. The composition of simulated whole breast milk oligosaccharide according to claim 1, characterized in that The mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyl lactose in the composition simulating whole breast milk oligosaccharides is (3.9-8.1): (0.8-1.2): (0.8-1.2).

3. The composition of simulated whole breast milk oligosaccharide according to claim 1, characterized in that The mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyl lactose in the composition simulating whole breast milk oligosaccharide is (3.95-8.05): (0.95-1.05): (0.95-1.05).

4. The composition of simulated whole breast milk oligosaccharide according to claim 1, characterized in that The mass ratio of 2'-fucosyllactose, lactose-N-neotetraose and 3'-sialyl lactose in the composition simulating whole breast milk oligosaccharides is (4-8):1:

1.

5. The use of the composition simulating whole human milk oligosaccharide according to any one of claims 1 to 4, characterized in that: The application is to use the composition simulating whole breast milk oligosaccharides in food to improve the cognitive behavioral ability of the consumer.

6. The use of the composition simulating whole breast milk oligosaccharides according to claim 5, characterized in that: The edible objects are infants, children over 3 years old, teenagers or the elderly.

7. The use of the composition simulating whole breast milk oligosaccharides according to claim 5, characterized in that: The food is infant formula powder, complementary food or nutritional supplement.

8. The use of the composition simulating whole breast milk oligosaccharides according to claim 5, characterized in that: The improvement of the cognitive behavior ability of the edible object is specifically manifested in that the exploration index of the edible object in the Y-maze test can be significantly increased.

9. The use of the composition simulating whole breast milk oligosaccharides according to claim 5, characterized in that: The improvement of the cognitive behavior ability of the edible object is specifically manifested in that the cognitive index of the edible object in a new object recognition test can be significantly increased.

10. The use of the composition simulating whole breast milk oligosaccharides according to claim 5, characterized in that: The improvement of cognitive behavioral ability of the edible subject is specifically manifested in significantly increasing the memory ability of the edible subject in a water maze test; preferably, the edible subject consumes the composition simulating whole human milk oligosaccharides at an amount of 300 to 600 mg / kg / d.

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