Human milk oligosaccharide composition for improving individual adaptability and adult memory capacity and use thereof

Abstract

The present application provides a breast milk oligosaccharide composition for improving adaptability and memory capacity in adulthood of an individual and application thereof. The composition comprises 2'-fucosyllactose (2'-FL), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), and the mass ratio of 2'-fucosyllactose, 3'-sialyllactose and 6'-sialyllactose is (1-12):1:(1-4). The present application also provides food containing the composition, and application of the composition and the food in preparing products for improving and / or improving adaptability of an individual and for improving and / or improving memory capacity of an individual in adulthood. The present application can improve the adaptability of an individual in early life by intervening breast milk oligosaccharides in early life, and has a positive promoting effect on cognitive development in adulthood, especially memory capacity.

Description

Technical Field

[0001] This invention relates to the field of functional nutrient research technology, specifically to a human milk oligosaccharide composition and its application that helps improve an individual's adaptability and adult memory. Background Technology

[0002] Early human development, especially in children aged 0-6, has a profound impact on an individual's long-term health and intellectual development. According to WS / T 580—2017, the "Developmental Behavioral Assessment Scale for Children Aged 0-6," children's developmental behavior mainly includes five attributes: gross motor skills, fine motor skills, language, adaptive abilities, and social behavior. Among these, the adaptive ability attribute refers to a child's capacity to respond to and adapt to their surrounding natural environment and social needs.

[0003] Human milk oligosaccharides (HMOs), the third largest solid substance in breast milk after lactose and fat, play a vital role in the healthy growth and development of infants. Currently, the structures of over 200 HMOs have been determined. Each HMO molecule contains 3-14 monosaccharides linked in a straight-chain or branched manner. Based on their monosaccharide composition, HMOs can be classified into three main categories: fucoidylated human milk oligosaccharides, sialylated human milk oligosaccharides, and neutral human milk oligosaccharides.

[0004] Significant progress has been made in research on the effects of HMOs on infant neural, brain, and cognitive development over the past two decades, but further in-depth research is still needed on the components and specific functions of HMOs. Furthermore, a review revealed a lack of research reports on the long-term effects of HMOs on individual growth. Summary of the Invention

[0005] The inventors in this case discovered in their research that by intervening in specific types of human milk oligosaccharides in early life, they could not only improve an individual's adaptability in early life, but also have a positive effect on cognitive development in adulthood, especially memory ability.

[0006] Specifically, on the one hand, the present invention provides a human milk oligosaccharide composition that helps to enhance and / or improve an individual's adaptability and adult memory, the composition comprising 2'-fucosylated lactose (2'-FL), 3'-sialylated lactose (3'-SL) and 6'-sialylated lactose (6'-SL), wherein the mass ratio of 2'-fucosylated lactose, 3'-sialylated lactose and 6'-sialylated lactose is (1-12):1:(1-4).

[0007] This invention improves an individual's adaptability in early life by intervening in the aforementioned human milk oligosaccharides, and has a positive promoting effect on cognitive development, especially memory ability, in adulthood.

[0008] According to some specific embodiments of the present invention, the methods for enhancing and / or improving memory include methods for assisting in improving memory.

[0009] According to some specific embodiments of the present invention, in the human milk oligosaccharide composition of the present invention which helps to enhance and / or improve individual adaptability and adult memory, the mass ratio of 2'-fucosylated lactose, 3'-sialylated lactose and 6'-sialylated lactose is (1-12):1:(2-3) or (1-12):1:(1.5-2.5).

[0010] According to some specific embodiments of the present invention, in the human milk oligosaccharide composition of the present invention which helps to enhance and / or improve individual adaptability and adult memory, the mass ratio of 2'-fucosylated lactose, 3'-sialylated lactose and 6'-sialylated lactose is (3-11):1:(2-3).

[0011] According to some specific embodiments of the present invention, in the human milk oligosaccharide composition of the present invention which helps to enhance and / or improve individual adaptability and adult memory, the mass ratio of 2'-fucosylated lactose, 3'-sialylated lactose and 6'-sialylated lactose is (4-10):1:(2-3).

[0012] According to some specific embodiments of the present invention, in the human milk oligosaccharide composition of the present invention which helps to enhance and / or improve individual adaptability and adult memory, the mass ratio of 2'-fucosylated lactose, 3'-sialylated lactose and 6'-sialylated lactose is (3-5):1:(2-3), for example (3-5):1:2.

[0013] According to some specific embodiments of the present invention, in the human milk oligosaccharide composition of the present invention which helps to enhance and / or improve individual adaptability and adult memory, the mass ratio of 2'-fucosylated lactose, 3'-sialylated lactose and 6'-sialylated lactose is (5-8):1:(2-3), for example (5-7):1:2.

[0014] According to some specific embodiments of the present invention, in the human milk oligosaccharide composition of the present invention which helps to enhance and / or improve individual adaptability and adult memory, the mass ratio of 2'-fucosylated lactose, 3'-sialylated lactose and 6'-sialylated lactose is (9-11):1:(2-3), for example (9-11):1:2.

[0015] According to some specific embodiments of the present invention, the present invention provides a human milk oligosaccharide composition that helps to enhance and / or improve an individual's adaptability and adult memory, which is composed of 2'-fucosylated lactose, 3'-sialylated lactose and 6'-sialylated lactose in any of the aforementioned proportions.

[0016] On the other hand, the present invention also provides an oral product that helps to enhance and / or improve an individual's adaptability and adult memory, the oral product containing a human milk oligosaccharide composition, the human milk oligosaccharide composition being the composition of the present invention described above.

[0017] According to some specific embodiments of the present invention, the oral product of the present invention can be a medicine. The term "medicine" as used herein means any substance that provides a beneficial effect to an individual (e.g., a human or animal). The medicine is not necessarily limited to therapeutic drugs requiring market access, but may also include substances that can be used in nutritional products or supplements, health products or supplements, foods (including functional foods), etc. According to some specific embodiments of the present invention, the beneficial effect provided by the product to an individual is that, compared to similar individuals who have not used the human milk oligosaccharide composition of the present invention or an oral product containing the human milk oligosaccharide composition, use of the human milk oligosaccharide composition of the present invention or an oral product containing the human milk oligosaccharide composition helps to enhance and / or improve an individual's adaptability and adult memory.

[0018] According to some specific embodiments of the present invention, the products of the present invention that help to enhance and / or improve an individual's adaptability and adult memory are snacks, nutritional supplements, dietary supplements, health foods or foods for special medical purposes.

[0019] According to some specific embodiments of the present invention, the products of the present invention that help to enhance and / or improve an individual's adaptability and adult memory are dairy products.

[0020] According to some specific embodiments of the present invention, the products of the present invention that help to enhance and / or improve an individual's adaptability and adult memory are infant formula, toddler formula, milk powder, milk tablets, cheese, liquid dairy products or complementary foods.

[0021] According to some specific embodiments of the present invention, the products of the present invention that help to enhance and / or improve individual adaptability and adult memory contain 10-6000 mg / 100g dry matter of human milk oligosaccharides.

[0022] According to some specific embodiments of the present invention, the product of the present invention that helps to enhance and / or improve an individual's adaptability and adult memory contains 10-2387 mg / 100g dry matter of human milk oligosaccharides.

[0023] According to some specific embodiments of the present invention, the product of the present invention that helps to enhance and / or improve an individual's adaptability and adult memory contains 10-1900 mg / 100g of human milk oligosaccharides.

[0024] According to some specific embodiments of the present invention, the product of the present invention contains 20-1800 mg / 100g dry matter of human milk oligosaccharides.

[0025] According to some specific embodiments of the present invention, the product of the present invention contains 50-1700 mg / 100g dry matter of human milk oligosaccharides.

[0026] According to some specific embodiments of the present invention, the product of the present invention contains 100-1600 mg / 100g dry matter of human milk oligosaccharides.

[0027] According to some specific embodiments of the present invention, the product of the present invention contains 200-1500 mg / 100g dry matter of human milk oligosaccharides.

[0028] According to some specific embodiments of the present invention, the product of the present invention contains 400-1200 mg / 100g dry matter of human milk oligosaccharides.

[0029] According to some specific embodiments of the present invention, the amount of 3'-sialyl lactose in the product of the present invention is usually 10-400 mg / 100g dry matter.

[0030] According to some specific embodiments of the present invention, the amount of 2'-fucosyllactose in the product of the present invention is typically 10-4800 mg / 100g dry matter. For example, the amount of 2'-fucosyllactose in the product can be 10-50 mg / 100g dry matter, 50-100 mg / 100g dry matter, 100-200 mg / 100g dry matter, 200-500 mg / 100g dry matter, 500-1000 mg / 100g dry matter, 1000-2000 mg / 100g dry matter, 2000-3000 mg / 100g dry matter, 3000-4000 mg / 100g dry matter, or 4000-4800 mg / 100g dry matter.

[0031] According to some specific embodiments of the present invention, the amount of 6'-sialyl lactose in the product of the present invention is typically 5-800 mg / 100g dry matter. For example, the amount of 6'-sialyl lactose in the product can be 5-50 mg / 100g dry matter, 50-100 mg / 100g dry matter, 100-200 mg / 100g dry matter, 200-500 mg / 100g dry matter, or 500-800 mg / 100g dry matter.

[0032] Understandably, the content of each human milk oligosaccharide in the final product should be appropriately adjusted within the range allowed by relevant standards and specifications, depending on the type of product.

[0033] On the other hand, the present invention also provides the use of the composition or the product described herein in the preparation of products that help improve and / or enhance individual adaptability and help improve and / or enhance individual adult memory.

[0034] This invention also provides a method for enhancing and / or improving an individual's adaptability and adult memory, the method comprising: orally administering an effective amount of a human milk oligosaccharide composition to a minor individual in need. The minor individual in need may have normal adaptability, or low or impaired adaptability; and / or, the minor individual in need may have normal memory, or low or impaired memory. By comparing the individual with a similar individual who has not received an effective amount of the human milk oligosaccharide composition of this invention or a product containing the human milk oligosaccharide composition, receiving an effective amount of the human milk oligosaccharide composition of this invention or a product containing the human milk oligosaccharide composition helps to enhance and / or improve an individual's adaptability and adult memory.

[0035] According to some specific embodiments of the present invention, in the application of the present invention, the enhancement and / or improvement of individual adaptability includes: Enhancing an individual's ability to respond to and adapt to their surrounding natural environment; and / or Enhance individuals’ ability to respond to and adapt to the needs of their surrounding society.

[0036] According to some specific embodiments of the present invention, in the application of the present invention, the enhancement and / or improvement of an individual's adult memory ability includes: Enhance and / or improve an individual's object recognition and memory abilities in adulthood; Enhance and / or improve an individual's spatial memory and pattern separation abilities in adulthood; To enhance and / or improve an individual's learning and memory abilities in adulthood; and / or Increase the number of hippocampal neurons in adults.

[0037] According to some specific embodiments of the present invention, in the application of the present invention, the product is an oral product given to an individual in the minor period; Preferably, the product is an oral product given to human individuals aged 0-6 years.

[0038] According to some specific embodiments of the present invention, in the application of the present invention, the effective amount of the breast milk oligomers applied to human individuals is generally 10-200 mg / kg body weight, preferably 40-100 mg / kg body weight.

[0039] According to a specific embodiment of the present invention, the human milk oligosaccharides of the present invention can be derived from commercially available human milk oligosaccharide raw materials with high purity. Unless otherwise specified, the amount or ratio of the human milk oligosaccharides 2'-fucosylated lactose, 3'-sialic acid lactose and 6'-sialic acid lactose in the composition of the present invention is based on the content of human milk oligosaccharides 2'-fucosylated lactose, 3'-sialic acid lactose and 6'-sialic acid lactose in the raw materials.

[0040] In summary, this invention explores the effects of early life intervention with human milk oligosaccharides on early life adaptability and adult cognitive development. Early life intervention with human milk oligosaccharides can enhance early life adaptability and positively promote adult cognitive development, especially memory ability, particularly significantly improving an individual's ability to recognize new objects and new locations in adulthood. Attached Figure Description

[0041] Figure 1 This shows the body weight of rats in each group supplemented with HMO during their juvenile period.

[0042] Figure 2 This shows the eye-opening status of rats in different groups supplemented with HMO during their juvenile period.

[0043] Figure 3 This shows the frequency of entry into the open arm of each group supplemented with HMO during childhood in the elevated cross maze experiment.

[0044] Figure 4 This displays the movement speed of each group supplemented with HMO during childhood.

[0045] Figure 5 This displays the distance traveled by each group during the period of HMO supplementation in infancy.

[0046] Figure 6 This shows the time spent in the central area for each group supplemented with HMO during childhood.

[0047] Figure 7 This shows the results of novel object recognition and memory in rats supplemented with HMO monomers and compositions during juvenile stage.

[0048] Figure 8This shows the results of novel location recognition memory in rats supplemented with HMO during juvenile stage (easy mode).

[0049] Figure 9 This shows the results of novel location recognition memory (difficult mode) in rats supplemented with HMO during juvenile period.

[0050] Figure 10 The results of staining show the number of newly generated neurons in the hippocampus of rats supplemented with HMO in their early years. Detailed Implementation

[0051] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0052] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints may be selected.

[0053] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0054] As used herein, the term "enhanced adaptability" means an improvement in an individual's adaptability, including but not limited to the ability to respond to and adapt to the surrounding natural and social environment, compared to the same response in the absence of the human milk oligosaccharides of this invention. In relation to any beneficial effects on memory capacity, the term "enhanced" is synonymous with "strengthened," "improved," or "promoted." In some embodiments of this invention, particularly in cases where an individual's adaptability is impaired, deteriorated, or threatened, the term "enhanced adaptability" means an effect that reduces, mitigates, or alleviates the degree of impairment or deterioration of adaptability, or reduces the risk of impairment or deterioration of adaptability, including but not limited to: alleviating adverse symptoms associated with impairment or deterioration of adaptability; slowing or preventing the onset of symptoms before they occur; slowing or halting the progression of impairment or deterioration of adaptability; slowing or halting the worsening of impairment or deterioration of adaptability; slowing or halting irreversible damage in the progressive (or chronic) phase of impairment or deterioration of adaptability; delaying the onset of (progressive) impairment or deterioration of adaptability; reducing the severity of impairment or deterioration of adaptability; curing impairment or deterioration of adaptability; and preventing impairment or deterioration of adaptability from occurring. The term "adaptive impairment" as used in this article refers to damage, decline, or deterioration of adaptive capacity, to the point that it is difficult to maintain or achieve normal function without intervention.

[0055] As used herein, the term "enhanced memory" means an improvement in an individual's memory capacity (including short-term memory, long-term memory, memory recall, and / or memory recognition, etc.) compared to the same response in the absence of the human milk oligosaccharides of this invention. This includes, but is not limited to, enhanced object recognition memory, enhanced spatial and pattern separation memory, enhanced learning memory, and increased hippocampal neuron count. In relation to any beneficial effects on memory capacity, the term "enhanced" is synonymous with "strengthened," "improved," or "promoted." In some embodiments of the present invention, particularly in cases where an individual's memory capacity is impaired, declining, or threatened, the term "improving memory capacity" refers to actions that reduce, mitigate, or alleviate the degree of memory impairment or decline, or reduce the risk of memory impairment or decline, including but not limited to: alleviating adverse symptoms associated with memory impairment or decline; slowing or preventing the onset of symptoms before they occur; slowing or halting the progression of memory impairment or decline; slowing or halting the worsening of memory impairment or decline; slowing or halting irreversible damage in the progressive (or chronic) phase of memory impairment or decline; delaying the onset of (progressive) memory impairment or decline; reducing the severity of memory impairment or decline; curing memory impairment or decline; and preventing the occurrence of memory impairment or decline. As used herein, the term "memory impairment" refers to impaired, diminished, or declining learning, memory, and / or cognitive functions, to the point that normal functioning is difficult to maintain or achieve without intervention. Some common signs of memory decline include confusion, short-term or long-term memory loss, and decreased object recognition and / or spatial judgment.

[0056] As used herein, the term "individual" means any animal or human who may benefit from the products of this invention that contribute to improved learning, memory, and / or cognitive function. Generally, when an individual is a human, it can be an infant or a child. The term "individual" refers to a human aged 0-3 years from birth, and the term "child" refers to a human aged 3-6 years from birth. For ease of description, the terms 0-6 years or human are used interchangeably throughout this document.

[0057] In addition to the specific methods, equipment, and materials used in the embodiments, based on the knowledge of those skilled in the art and the description of this invention, any prior art methods, equipment, and materials similar to or equivalent to those described, equipment, and materials in the embodiments of this invention can be used to implement this invention.

[0058] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0059] In all embodiments and experiments, all human milk oligosaccharides used were commercially available food-grade raw materials. Unless otherwise specified, the amount of human milk oligosaccharides mentioned in this invention is based on the effective content.

[0060] Example 1: Early intervention with human milk oligosaccharides improves individual adaptability

[0061] 1. Animal models

[0062] The Long Evans rat breeding pairs used in this embodiment were purchased from Envigo, Inc., USA, and were used for the internal breeding of rats required for the experiment. To ensure that the mother rats could provide adequate care for each pup, litters with more than 10 pups were artificially adjusted to have fewer than 10 pups within 0 to 1 day after birth. The pups were weaned on day 21 after birth. The rats were housed in an environment with reversed diurnal cycles, i.e., 12 hours of darkness and 12 hours of light (lights off at 9:00 AM and on at 9:00 PM), to adapt to the rats' active cycles.

[0063] 2. HMO intervention

[0064] The project included three different human lactose oligosaccharides (HMOs): 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), and 2'-fucosylated lactose (2'-FL). These HMOs were administered as single HMOs or in different combinations to juvenile rats (from day 8 to day 35 after birth), resulting in the following group settings: control group (receiving drinking water), single HMO group, and HMO triple combination group (2'-FL + 3'-SL + 6'-SL) in ratios of 10:1:2, 6:1:2, and 4:1:2. The control group received no HMOs but was given plain drinking water in the same manner as other animals receiving HMOs. HMOs were administered orally by gavage at a dose of 400 mg / kg body weight daily.

[0065] 3. Adaptive Behavioral Research

[0066] Eye opening and weight gain are common physiological parameters for tracking the development of newborn rodents. Weight is considered a general indicator of health and development, while eye opening is considered an indicator of brain maturity. Behavioral studies were subsequently conducted in elevated cruciform maze and open-field tests.

[0067] The elevated plus maze (EPM) is an experimental method for evaluating anxiety responses in rodents. The EPM consists of two open arms and two closed arms, intersecting in a cross shape, with the central area at the intersection. Throughout the project, the EPM was placed in the same room with identical environmental settings: a temperature of 20°C, humidity of 40%, and a light intensity of 10 lux for the open arms. Each animal was placed in the center of the maze, facing one of the open arms, and its behavior was recorded for 5 minutes. After each animal's test, the apparatus was thoroughly cleaned with 40% ethanol and allowed to dry. If an animal jumped off the maze, it was retested on the maze after 2-3 days.

[0068] The open field test consists of four white square fields, each measuring 50 cm × 50 cm × 50 cm (length × width × height), arranged in a 2 × 2 grid (see...). Figure 2 The system can simultaneously track four animals in each trial. Open-field testing was conducted in a room at 20°C with 40% humidity and a light intensity of 10 lux. A camera was mounted above the open-field area to record behavior for subsequent offline behavioral scoring using Noldus Ethovision (version 15.00) software. The edge areas were defined as 15 cm wide zones extending from each wall, surrounding the central area, which was a 20 cm x 20 cm (length x width) square in the center of the field. The time animals spent at the edges or in the center, and the frequency of their movement between different areas, were typically used as indicators of anxiety, motor activity, or exploratory drive. Generally, anxious rats avoided open spaces and tended to stay close to the walls, while curious rats were more willing to explore the entire field. Each animal was placed in the center of the open field, and its behavior was recorded for 15 minutes. After 15 minutes, the rats were returned to their cages, and the experimental area was thoroughly cleaned with 40% ethanol and allowed to dry. The open field was white to maximize contrast and facilitate camera detection.

[0069] 4. Statistical Analysis

[0070] The study used IBM SPSS software version 20.0 for data analysis. A difference was considered statistically significant when the reported p-value was less than 0.05, and statistical trend was defined as a p-value less than or equal to 0.1.

[0071] In this embodiment, the group information is shown in Table 1.

[0072] Table 1

[0073] 5. Experimental Results: (1) Weight gain of rats in each group The changes in rat body weight were tracked, and there were no significant differences between the groups. The results are as follows: Figure 1 As shown, HMO intervention has no effect on rat body weight.

[0074] (2) Eye opening status of rats in each group

[0075] Opening the eyes is considered one of the indicators of brain maturity, as experimental results show... Figure 2 As shown, there was a strong correlation between eye opening in newborn rats and the HMO intervention they received. The results indicated that, compared to the HMO monomer, the HMO composition increased the number of eyes open on days 13 and 15, suggesting that the HMO composition promoted brain development in newborn rats. On day 16, all newborn rats in the HMO intervention group, except for the control group, had both eyes open.

[0076] (3) Elevated cross maze behavioral experiment

[0077] The elevated cruciate maze is a classic behavioral test used to assess anxiety behavior in rats. The frequency of entering the open arms is a key indicator in this test. Because the open arms lack protection, animals typically feel exposed and uneasy, and tend to remain in the closed arms. A higher frequency of entering the open arms generally indicates lower anxiety levels and greater adaptability to new environments; conversely, a lower frequency indicates higher anxiety levels and less adaptability to new environments.

[0078] The results are as follows Figure 3 As shown, compared to the control group, HMO intervention increased the frequency of rats entering the open arm, indicating improved adaptability to new environments and exploratory behavior, with lower anxiety levels. HMO compositions 1 and 2, in particular, performed best among all groups.

[0079] (4) Results of the open field behavioral experiment

[0080] In behavioral experiments, locomotion refers to spontaneous movement in animals within a specific environment. It is commonly used to assess an animal's activity level, exploratory behavior, and adaptability to new environments. Locomotion typically includes data such as movement speed and distance. Results are as follows... Figure 4 As shown, HMO intervention had a positive effect on the movement speed and distance of rats. The HMO intervention groups, especially the HMO composition intervention groups, increased the movement speed and distance of rats in the open field, indicating that HMO intervention can increase the exploratory behavior, activity level, and adaptability to new environments of rats. The results showed that HMO intervention improved movement speed compared to the control group; compared to HMO monomers, HMO composition 1 and HMO composition 3 both showed higher movement speeds.

[0081] Figure 5 The distance traveled in the open field was measured for each group. Compared to the control group, HMO intervention increased the distance traveled by rats, indicating that HMO intervention enhanced the exploratory behavior and activity levels of rats. Compared to the HMO monomer groups, both HMO composition 1 and HMO composition 3 significantly increased the distance traveled by rats in the open field.

[0082] Figure 6 The time spent in the central area of ​​the open field for each group is recorded. This is a commonly used indicator in open field behavioral experiments, referring to the time rats spend in the central area while freely moving around in the open field. Generally, the longer an animal spends in the central area, the lower its anxiety level may be, and the more active its exploratory behavior, because the central area is relatively more exposed and vulnerable to threats. Compared to the control group, HMO intervention increased the time rats spent in the central area, indicating that HMO intervention enhanced the rats' exploratory behavior. Compared to the HMO monotherapy groups, HMO compositions 1-3, especially HMO composition 2, significantly increased the time rats spent in the center of the open field, indicating that HMO intervention enhanced the rats' exploratory behavior.

[0083] Example 2: Experimental Protocol for Early Intervention with Human Milk Oligosaccharides to Improve Adult Memory

[0084] 1. Animal models

[0085] The Long Evans rat breeding pairs used in this experiment were purchased from Envigo, USA, for internal breeding of the rats required for the experiment. To ensure that the mother rats could provide adequate care for each pup, litters with more than 10 pups were artificially adjusted to fewer than 10 pups within the first 0 to 1 day after birth. The pups were weaned on day 21 after birth. The rats were housed in an environment with reversed diurnal cycles, i.e., 12 hours of darkness and 12 hours of light (lights off at 9:00 AM and on at 9:00 PM), to adapt to the rats' active cycles.

[0086] 2. HMO intervention

[0087] This study included three different human lactose oligosaccharides (HMOs): 2'-fucosylated lactose (2'-FL), 3'-sialyl lactose (3'-SL), and 6'-sialyl lactose (6'-SL), alone or in combinations of two or three. These HMOs were administered as individual HMOs or in different combinations to juvenile rats (from day 8 to day 35 after birth), forming different group settings with 27 rats in each group. The control group received no HMOs but was given plain drinking water in the same manner as other animals receiving HMOs. The HMOs, either individual or combined, were administered orally by gavage at a daily dose of 400 mg / kg body weight.

[0088] 3. Behavioral research

[0089] After the intervention period ended, all rats in each group were housed in a uniform environment with reversed day and night cycles, fed standard rat diet (without HMOs), and allowed free access to food.

[0090] Rats were assessed behaviorally using memory tests during weeks 32-34, as detailed below: The Novel Object Recognition Test (NORT) is a behavioral task used to assess recognition memory. It measures the time an animal takes to explore a newly introduced object after becoming familiar with a pair of identical objects. In this task, two identical objects are initially presented in a test box, and in the retest phase, one of the objects is replaced with a new object. The time an animal spends exploring the replaced object reflects its object recognition memory ability. NORT is based on an animal's preference for exploring new objects. In the experiment, animals typically spend more time exploring new objects than familiar ones. This preference reflects the animal's ability to recognize and remember new objects.

[0091] In addition to novel object recognition, this experimental study also included a novel object location task (NOLT) to measure the animals' learning and memory abilities. The NOLT utilizes the rats' natural curiosity: when an animal recognizes an object has been moved, it is more inclined to explore the moved object. The variation in the time taken to explore the object at different possible locations was used as a measure of pattern separation. NOLT is a behavioral task used to measure spatial memory and pattern separation (van Goethem, van Hagen & Prickaerts, 2018). The time an animal takes to explore a moved object reflects its spatial memory and pattern separation abilities. The object locations were labeled P1 (location 1), P3 (location 3), and P5 (location 5). Larger numbers indicate greater displacement of the object from the center of the test box, with P1 being the intermediate location (the same as the object location in the acquisition phase). The NOLT study used in this project included two experimental conditions: 1) Easy condition (location 5, P5): In the easy condition, the object was displaced a greater distance from its original location during the test phase. This is considered a low-mode separation condition because displacement of an object over a larger distance is considered easier for the animal to detect; 2) Difficult condition (position 3, P3): Under this condition, the object is displaced a small distance from its original position during the testing phase. This is considered a high-mode separation condition because small-distance displacements are considered harder for the animal to detect.

[0092] 4. Fluorescent staining experiment

[0093] After behavioral studies confirmed that the HMO composition had activity promoting adult cognitive development, the effects of the HMO composition on the rat brain were further investigated. The number of newly generated neurons in the hippocampus was determined using a 5-bromodeoxyuridine staining assay. 5-bromodeoxyuridine is an analog of thymine nucleoside, which can be incorporated into newly synthesized DNA during the DNA synthesis phase of the cell cycle and is commonly used to detect proliferating cells. Female rats in the HMO intervention group underwent the staining experiment 3 days before sacrifice. Rats received intraperitoneal (IP) injections of 5-bromodeoxyuridine at the same time for 3 consecutive days at a dose of 100 mg / kg body weight to ensure consistent labeling of newly generated neurons. Brain samples were collected after rat sacrifice, and hippocampal sections were stained. 5-bromodeoxyuridine-positive cells were manually counted using Cell-Counter in ImageJ.

[0094] 5. Statistical Analysis

[0095] The study used IBM SPSS software version 20.0 for data analysis. A difference was considered statistically significant when the reported p-value was less than 0.05, and statistical trend was defined as a p-value less than or equal to 0.1.

[0096] In this embodiment, following the aforementioned "Experimental Protocol for Early Intervention of Test Substances to Improve Adult Memory," three different human lactose oligosaccharides (HMOs) were tested: 2'-fucosylated lactose (2'-FL), 3'-sialylated lactose (3'-SL), and 6'-sialylated lactose (6'-SL). These HMOs were administered as individual HMOs or in different combinations to juvenile rats (from day 8 to day 35 after birth), resulting in the following group settings: control group (receiving drinking water), HMO monomer groups (2'-FL, 3'-SL, and 6'-SL groups), HMO composition 1, HMO composition 2, and HMO composition 3. Each control and HMO monomer group consisted of 27 rats, and each HMO combination group consisted of 27 rats. The control group received no HMOs but was given ordinary drinking water in the same manner as other animals receiving HMOs. The daily dose of either the HMO monomer or the combination administered orally via gavage was 400 mg / kg body weight (the gavage dose for the HMO combination group was the total HMO dose of 400 mg / kg body weight).

[0097] In this embodiment, the experimental group information is shown in Table 2.

[0098] Table 2. Experimental group settings

[0099] Figure 7This study shows the results of novel object recognition and memory in rats supplemented with HMOs during juvenile stage. The NORT experiment was conducted at 32-34 weeks of rat life, and data are expressed as the mean and standard error of the discrimination index. The results showed that the novel object discrimination index was increased in the HMO combination intervention groups compared to the control group. In particular, HMO combination 1 group had the highest object recognition index, followed by HMO combination 3 group and the 6'-SL group, indicating that intervention with human milk oligosaccharides during juvenile stage significantly improves memory ability in adulthood, and the three HMO combinations showed a certain synergistic effect.

[0100] Figure 8 The results show the novel location recognition memory outcomes (easy mode) of rats supplemented with HMOs during juvenile stage. The NOLT experiment was conducted at 32-34 weeks of age, and data are expressed as the mean and standard error of the number of correct answers. The results showed that the novel location discrimination index was increased in HMO combination 1 and HMO combination 3 groups compared with the control group. This indicates that intervention with human milk oligosaccharides during juvenile stage, especially the combination of 2'-FL, 3'-SL, and 6'-SL, has a significant effect on improving learning and memory abilities in adulthood.

[0101] Figure 9 The results show the novel location recognition and memory outcomes (difficult mode) of rats supplemented with HMOs during juvenile stage. The closer the novel location is to the center, the greater the recognition difficulty. Therefore, the performance of rats in the difficult mode can indicate their performance in handling complex tasks. In this experiment, no meaningful statistical data were obtained for the 2'-FL group and the HMO composition 3 group due to operational abnormalities, and they are not shown in the figure. The experimental results show that, compared with the control group, the novel location discrimination index of the HMO composition 1 group and the HMO composition 2 group intervention group was increased. This indicates that HMO composition intervention during juvenile stage, especially the combination of 2'-FL, 3'-SL, and 6'-SL, has a significant effect on improving learning and memory abilities in adulthood, especially the ability to handle complex situations.

[0102] After behavioral experiments confirmed the positive impact of early intervention with HMO combinations on cognitive development in adulthood, the number of newly generated neurons in the brains of rats in the HMO combination groups was measured. Fluorescent staining was used to analyze the differences in newly generated neurons in the brains of rats in the control group and the HMO combination groups 1, 2, and 3. Figure 10 The results show the staining results of the number of newly generated neurons in the hippocampus of rats supplemented with HMOs during childhood. The results showed that, compared with the control group, the intervention groups (HMO composition 1 and HMO composition 3) had an increased number of fluorescently positive cells, indicating an increase in the number of newly generated neurons in the hippocampus. This suggests that intervention with HMO compositions during childhood, especially the combinations of 2'-FL, 3'-SL, and 6'-SL, may enhance cognitive development in adulthood by increasing the number of newly generated neurons in the hippocampus.

[0103] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A human milk oligosaccharide composition that helps enhance and / or improve an individual's adaptability and adult memory, wherein, The composition comprises 2'-fucosyllactose (2'-FL), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), wherein the mass ratio of 2'-fucosyllactose, 3'-sialyllactose and 6'-sialyllactose is (1-12):1:(1-4).

2. The human milk oligosaccharide composition according to claim 1, which helps to enhance and / or improve individual adaptability and adult memory, wherein, The mass ratio of 2'-fucosylated lactose, 3'-sialylated lactose and 6'-sialylated lactose is (1-12):1:(2-3) or (1-12):1:(1.5-2.5).

3. The human milk oligosaccharide composition according to claim 1, which helps to enhance and / or improve individual adaptability and adult memory, wherein, The mass ratio of 2'-fucosylated lactose, 3'-sialylated lactose and 6'-sialylated lactose is (4-10):1:(2-3).

4. An oral product that helps enhance and / or improve an individual's adaptability and adult memory, wherein, The oral product contains a human milk oligosaccharide composition, which is the composition described in any one of claims 1-3.

5. The product according to claim 4, wherein, The products are snacks, nutritional supplements, dietary supplements, health foods, or foods for special medical purposes.

6. The product according to claim 4, wherein, The product contains 10-6000 mg of human milk oligosaccharides per 100g of dry matter.

7. The use of the composition of any one of claims 1-3 or the product of any one of claims 4-6 in the preparation of products that help improve and / or enhance individual adaptability and help improve and / or enhance individual adult memory.

8. The application according to claim 7, wherein, The enhancement and / or improvement of individual adaptability includes: Enhancing an individual's ability to respond to and adapt to their surrounding natural environment; and / or Enhance individuals’ ability to respond to and adapt to the needs of their surrounding society.

9. The application according to claim 7, wherein, The enhancement and / or improvement of an individual's adult memory ability includes: Enhance and / or improve an individual's object recognition and memory abilities in adulthood; Enhance and / or improve an individual's spatial memory and pattern separation abilities in adulthood; To enhance and / or improve an individual's learning and memory abilities in adulthood; and / or Increase the number of hippocampal neurons in adults.

10. The application according to any one of claims 7-9, wherein, The product in question is an oral product intended for use by minors. Optionally, the product is an oral product given to human individuals aged 0-6 years.

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