Immunostimulant containing lacto-n-tetraose

Abstract

This immunostimulant contains lacto-N-tetraose. The immunostimulant is characterized by being used so as to be ingested or administered in combination with at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I.

Description

Immunostimulant containing lacto-N-tetraose

[0001] The present invention relates to an immunostimulant containing lacto-N-tetraose.

[0002] Human milk oligosaccharides are a general term for oligosaccharides abundantly found in human breast milk and are said to be effective in the healthy development and maturation of infant immune function, intestinal environment, brain function, etc. Regarding immune function, for example, human milk oligosaccharides have been reported to act directly on Toll-like receptors (hereinafter also referred to as "TLRs") involved in innate immunity. Different types of human milk oligosaccharides act on different types of TLRs, and various modes of action have been reported, including activation and inhibition of TLR function.

[0003] There have been many reports on the suppression of immune signaling by human milk oligosaccharides, including one that reports that 3-fucosyllactose, lacto-N-neotetraose, and lactodifucotetraose attenuate TNF-α-induced inflammation in fetal intestinal epithelial cells (Non-Patent Document 1). It has also been reported that 2'-fucosyllactose, 6'-sialyllactose, and 3'-sialyllactose attenuate Toll-like receptor 4 (TLR4) signaling (Patent Document 1, Non-Patent Document 2).

[0004] In contrast, there are reports of human milk oligosaccharides with immunostimulatory effects. For example, lacto-N-triose II has been reported to activate all TLRs in a dose-dependent manner (Non-Patent Document 3), and 3'-sialyllactose, 6'-sialyllactose, and lacto-N-fucopentaose I have been reported to increase the expression level and signaling of TLR4 (Non-Patent Documents 4 and 5).

[0005] International Publication No. 2021 / 195011

[0006] Cheng, Lianghui, et al. "The Human Milk Oligosaccharides 3‐FL, Lacto‐N‐Neotetraose, and LDFT Attenuate Tumor Necrosis Factor‐α Induced Inflammation in Fetal Intestinal Epithelial Cells In Vitro through Shedding or Interacting with Tumor Necrosis Factor Receptor 1." Molecular Nutrition & Food Research 65.7 (2021): 2000425.Sodhi, Chhinder P., et al. "The human milk oligosaccharides 2’-fucosyllactose and 6’-sialyllactose protect against the development of necrotizing enterocolitis by inhibiting toll-like receptor 4 signaling." Pediatric research 89.1 (2021): 91-101.Cheng, Lianghui, et al. "Human milk oligosaccharides and its acid hydrolysate LNT2 show immunomodulatory effects via TLRs in a dose and structure-dependent way." Journal of Functional Foods 59 (2019): 174-184.Asakuma, Sadaki, et al. "Effect of human milk oligosaccharides on messenger ribonucleic acid expression of toll-like receptor 2 and 4, and of MD2 in the intestinal cell line HT-29." Journal of Applied Glycoscience 57.3 (2010): 177-183.Hahn, Won-Ho, et al. "Effect of human breast milk on innate immune response: Up-regulation of bacterial pattern recognition receptors and innate cytokines in THP-1 monocytic cells." European Journal of Inflammation 19 (2021): 20587392211026107.

[0007] Incidentally, human milk oligosaccharides produced by known methods, as well as commonly available commercially available human milk oligosaccharides, often contain high concentrations of endotoxins composed of lipopolysaccharides and the like. Because endotoxins composed of lipopolysaccharides and the like potently activate TLR4 signaling, in order to accurately evaluate the effects of human milk oligosaccharides on the innate immune system, particularly their effects on the innate immune system mediated by TLR4, it is necessary to eliminate the effects of endotoxins from the evaluation system. However, the above-mentioned Patent Document 1 and Non-Patent Documents 1-5 do not adequately examine the removal of endotoxins from each evaluation system. For example, as mentioned above, Patent Document 1 and Non-Patent Document 2 report that 3'-sialyllactose and 6'-sialyllactose attenuate TLR4 signaling, while Non-Patent Document 4 reports that they increase TLR4 expression levels, signaling, etc. Such discrepancies in evaluation results may be due to endotoxin contamination in samples containing 3'-sialyllactose and 6'-sialyllactose. Furthermore, Non-Patent Document 3 was unable to remove high levels of endotoxin in the lacto-N-triose II sample, and merely evaluated the effect of lacto-N-triose II from the difference when an agent that captures LPS was added.

[0008] As described above, it has been difficult to accurately evaluate the effects of human milk oligosaccharides on the innate immune system, particularly their effects mediated by TLR4. However, in our everyday environment, where various infectious diseases such as COVID-19 and influenza are rampant, strengthening one's own immune function through foods or medicines is effective. Therefore, there is a strong demand for the discovery of human milk oligosaccharides with immunostimulatory effects.

[0009] The present invention has been made in consideration of the above circumstances, and aims to provide a new immunostimulant or immunoregulator containing human milk oligosaccharide as an active ingredient.

[0010] The present inventors have discovered a method for sufficiently removing endotoxins from compositions containing human milk oligosaccharides. They have also found that among human milk oligosaccharides, lacto-N-tetraose in particular has immunostimulatory activity. Furthermore, the present inventors have found that certain human milk oligosaccharides suppress the immunostimulatory activity of lacto-N-tetraose.

[0011] The present disclosure provides, for example, the inventions described in the following [1] to

[44] . [1] An immunostimulant containing lacto-N-tetraose. [2] The immunostimulant described in [1], characterized in that it is used to be ingested or administered in combination with at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I. [3] The immunostimulant described in [1] or [2], which is based on enhancing the production of TNF-α. [4] An immunomodulator containing lacto-N-tetraose and at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I. [5] An immunomodulator comprising at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I, characterized in that the immunomodulator is ingested or administered in combination with lacto-N-tetraose. [6] The immunomodulator according to [4] or [5], wherein the lacto-N-tetraose stimulates the immune system, and the at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I suppresses the immune stimulation caused by the lacto-N-tetraose. [7] A composition comprising human milk oligosaccharides, wherein the endotoxin content in the composition is less than 10 endotoxin units per 1 mg of the human milk oligosaccharides. [8] The composition according to [7], wherein the human milk oligosaccharide is lacto-N-tetraose. [9] The composition according to [7] or [8], wherein the composition is in the form of a powder.

[10] Lacto-N-tetraose for use in a therapeutic method for immune stimulation.

[11] Use of lacto-N-tetraose in a non-therapeutic method for stimulating immunity.

[12] The lacto-N-tetraose or use according to

[10] or

[11] , characterized in that the method is used so as to be ingested or administered in combination with at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I.

[13] The lacto-N-tetraose or use according to any of

[10] to

[12] , wherein the method is based on enhancing the production of TNF-α.

[14] Use of lacto-N-tetraose for the manufacture of an immunostimulant.

[15] The use according to

[14] , wherein the immunostimulant is ingested or administered in combination with at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I.

[16] The use according to

[14] or

[15] , wherein the immunostimulant is based on enhancing TNF-α production.

[17] A method for stimulating immunity, comprising administering lacto-N-tetraose to a subject.

[18] The method according to

[17] , wherein the lacto-N-tetraose is administered in combination with at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I.

[19] The method according to

[17] or

[18] , wherein the immune activation is based on enhancing TNF-α production.

[20] Lacto-N-tetraose and at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I for use in a therapeutic method for modulating immunity.

[21] Use of lacto-N-tetraose and at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I in a non-therapeutic method for modulating immunity.

[22] At least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I, administered in combination with lacto-N-tetraose for use in a therapeutic method for modulating immunity.

[23] Use of at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I in a non-therapeutic method for modulating immunity, characterized in that it is ingested in combination with lacto-N-tetraose.

[24] The lacto-N-tetraose and human milk oligosaccharide or use according to any of

[20] to

[23] , wherein the method is based on the immunostimulation of the lacto-N-tetraose and the immunostimulation of the at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I.

[25] Use of lacto-N-tetraose and at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I for the production of an immunomodulator.

[26] Use of at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I for the manufacture of an immunomodulator, characterized in that it is ingested or administered in combination with lacto-N-tetraose.

[27] The use according to

[25] or

[26] , wherein the immunomodulator is based on the immunostimulation of the lacto-N-tetraose and the inhibition of the immunostimulation by the lacto-N-tetraose by at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I.

[28] A method for regulating immunity, comprising administering to a subject lacto-N-tetraose and at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I.

[29] A method for regulating immunity, comprising administering to a subject lacto-N-tetraose in combination with at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I.

[30] The method according to

[28] or

[29] , wherein the immune regulation is based on the immune activation of the lacto-N-tetraose and the suppression of the immune activation by at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I by the lacto-N-tetraose.

[31] An immunostimulant containing lacto-N-fucopentaose I.

[32] The immunostimulant according to

[31] , wherein the endotoxin content is less than 10 endotoxin units per mg of the lacto-N-fucopentaose I.

[33] Lacto-N-fucopentaose I for use in a therapeutic method of immunostimulating.

[34] Use of lacto-N-fucopentaose I in a non-therapeutic method of immunostimulating.

[35] Use of lacto-N-fucopentaose I for the production of an immunostimulant.

[36] The use according to

[35] , wherein the endotoxin content in the immunostimulant is less than 10 endotoxin units per mg of the lacto-N-fucopentaose I.

[37] A method of immunostimulating immunity, comprising administering lacto-N-fucopentaose I to a subject.

[38] An immunosuppressant containing 6'-sialyllactose.

[39] The immunosuppressant according to

[38] , wherein the endotoxin content is less than 10 endotoxin units per 1 mg of the 6'-sialyllactose.

[40] 6'-sialyllactose for use in a therapeutic method for immunosuppression.

[41] Use of 6'-sialyllactose in a non-therapeutic method for immunosuppression.

[42] Use of 6'-sialyllactose for the manufacture of an immunosuppressant.

[43] The use according to

[42] , wherein the endotoxin content in the immunosuppressant is less than 10 endotoxin units per 1 mg of the 6'-sialyllactose.

[44] A method for immunosuppression, comprising administering 6'-sialyllactose to a subject.

[0012] According to the present invention, it is possible to provide an immunostimulant, immunomodulator, or human milk oligosaccharide-containing composition with a reduced endotoxin content, which contains human milk oligosaccharides.

[0013]

[0033] Figure 1 shows the concentration of TNF-α in the medium when endotoxin-removed lactose or each human milk oligosaccharide was added to a macrophage-derived cell line. Figure 2 shows the concentration of TNF-α in the medium when lacto-N-tetraose and lactose or a specified human milk oligosaccharide were co-added to a macrophage-derived cell line. Figure 3 shows the concentration of TNF-α in the medium when lipopolysaccharide and lactose or a specified human milk oligosaccharide were co-added to a macrophage-derived cell line. Figure 4 shows the concentration of TNF-α in the medium when lactose or a specified human milk oligosaccharide was added to a macrophage-derived cell line followed by the addition of lacto-N-tetraose. Figure 5 shows the concentration of TNF-α in the medium when lactose or a specified human milk oligosaccharide was added to a macrophage-derived cell line followed by the addition of lipopolysaccharide.

[0014] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0015] 1. Immunostimulant containing lacto-N-tetraose An immunostimulant according to one aspect of this embodiment contains lacto-N-tetraose.

[0016] The immunostimulant according to one aspect of this embodiment can improve immune function in a living body. The immunostimulant according to one aspect of this embodiment can, for example, enhance TNF-α production. That is, the immunostimulant according to one aspect of this embodiment may be based on enhancing TNF-α production. Furthermore, with regard to the mechanism of action of the immunostimulant according to one aspect of this embodiment, the inventors believe that one factor in enhancing TNF-α production is that lacto-N-tetraose activates Toll-like receptor 4 (hereinafter also referred to as "TLR4") signaling and the like in macrophages and the like, thereby promoting the secretion of TNF-α from macrophages and the like. Therefore, it is believed that the immunostimulant according to one aspect of this embodiment can activate innate immunity through activation of TLR4 signaling and the like.

[0017] The lacto-N-tetraose may be produced by a known chemical synthesis method, an enzymatic synthesis method, or a microbial synthesis method, or may be a commercially available product. Examples of the microbial synthesis method include those described in WO 2021 / 013708 and WO 2018 / 122225.

[0018] The immunostimulant according to one aspect of this embodiment may contain only lacto-N-tetraose, or may contain components other than lacto-N-tetraose as necessary. That is, the immunostimulant according to one aspect of this embodiment may be an immunostimulating composition containing lacto-N-tetraose, or may be a composition containing an effective amount of lacto-N-tetraose (i.e., an immunostimulating composition containing an effective amount of lacto-N-tetraose). Such compositions may be, for example, food compositions, pharmaceutical compositions, or raw materials thereof.

[0019] The effective amount of lacto-N-tetraose in the immunostimulant according to one aspect of this embodiment is not particularly limited, as long as it is an amount that can achieve an immunostimulatory effect. The effective amount can vary depending on factors such as health status, the method of ingestion or administration, and can be freely determined by those skilled in the art as needed. For example, the effective amount may be 1 mg to 20 g / kg of adult body weight per day, and preferably 5 mg to 500 mg / kg per day. When a subject ingests the immunostimulant according to one aspect of this embodiment, or when the immunostimulant according to one aspect of this embodiment is administered to a subject, the number of ingestions or administrations is not particularly limited, and may be once a day, or multiple times a day, such as twice or three times a day. The timing of ingestion or administration is also not particularly limited, and may be after waking up, before meals, after meals, between meals, or before bedtime. The subject of ingestion or administration may be a mammal, and preferably a human. The route of administration may be, for example, oral, sublingual, intramuscular, enteral, nasal, intravenous, subcutaneous, or intraperitoneal administration, with oral administration being preferred. The preferred route of intake is oral intake.

[0020] The food composition preferably emphasizes the tertiary function (biomodulation function). Examples of foods that emphasize the tertiary function include health foods, functional foods, nutritional compositions, nutritional supplements, supplements, health foods, foods for specified health uses, foods for special dietary uses, foods with nutrient functions, and foods with functional claims. Examples of biomodulation functions include immune care, immune function support, immune function maintenance, and immune function regulation. Examples of foods for specific dietary uses include infant formula and foods for the sick.

[0021] In addition to lacto-N-tetraose, the food composition may contain other ingredients acceptable for use as food, such as human milk oligosaccharides other than lacto-N-tetraose, carbohydrates, proteins, lipids, minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavorings, stabilizers, preservatives, sustained-release regulators, surfactants, solubilizers, and humectants.

[0022] The food composition may be in the form of a supplement (tablet, drink, powder, capsule, etc.), or may be in the form of a beverage, Western confectionery, Japanese confectionery, frozen dessert, cooked food, seasoning, etc. that is commonly consumed as food.

[0023] The method for producing the food composition is not particularly limited and can be any known method. For example, in the case of a supplement, an effective amount of lacto-N-tetraose can be mixed with appropriate additives and then formulated into an appropriate dosage form. In addition, when added to food, an effective amount of lacto-N-tetraose can be mixed with an intermediate product or a final product in the food manufacturing process.

[0024] The pharmaceutical composition may contain, in addition to lacto-N-tetraose, pharmaceutically acceptable additives such as excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers, and suspending agents.

[0025] The pharmaceutical composition may be in any form such as a solid, liquid, or paste, and may be in the form of a tablet (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsule, pill, powder (dispersed medicine), fine granules, granules, liquid, suspension, emulsion, syrup, paste, or injection (including a case where the composition is mixed with distilled water or an infusion such as an amino acid infusion or an electrolyte infusion at the time of use to prepare a liquid).

[0026] The content of lacto-N-tetraose in the immunostimulant according to one aspect of the present embodiment may vary depending on the form of the composition and the method of ingestion or administration, but is not particularly limited as long as the content is such that an immunostimulatory effect can be obtained. The content of lacto-N-tetraose in the immunostimulant according to one aspect of the present embodiment may be, for example, 25% by mass or more, 10% by mass or more, 5% by mass or more, 2% by mass or more, or 1% by mass or more, relative to the total amount of the immunostimulant. The content of lacto-N-tetraose in the immunostimulant according to one aspect of the present embodiment may be, for example, 50% by mass or less, 25% by mass or less, 10% by mass or less, 5% by mass or less, 2% by mass or less, or 1% by mass or less, relative to the total amount of the immunostimulant.

[0027] Furthermore, the immunostimulant containing lacto-N-tetraose according to one aspect of this embodiment may be characterized by being ingested or administered in combination with at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I (hereinafter also referred to as "immunomodulating human milk oligosaccharide" or simply "modulating human milk oligosaccharide"). The modulating human milk oligosaccharide may be ingested or administered as a separate agent from the immunostimulant containing lacto-N-tetraose according to one aspect of this embodiment. In this case, the agent containing the modulating human milk oligosaccharide may be as described below in [2. Immunomodulating agent containing modulating human milk oligosaccharide characterized by being ingested or administered in combination with lacto-N-tetraose]. The human milk oligosaccharides for adjustment suppress the TNF-α production enhancing effect of lacto-N-tetraose contained in the immunostimulant according to one aspect of this embodiment, thereby suppressing the immunostimulation caused by lacto-N-tetraose. Thus, by ingesting or administering the immunostimulant according to one aspect of this embodiment in combination with the human milk oligosaccharides for adjustment, the immunostimulatory effect of lacto-N-tetraose can be weakened as desired.

[0028] The human milk oligosaccharides for preparation may be produced by known chemical synthesis methods, synthesis methods using enzymes, or synthesis methods using microorganisms, or may be commercially available products. As a synthesis method using a microorganism, for example, for 2'-fucosyllactose, for example, the method described in WO 2021 / 125245 or WO 2022 / 176994, for 3'-sialyllactose and 6'-sialyllactose, for example, WO 2015 / 037698, or the method described in JP 2008-5794 A, lacto-N-neotetraose, for example, WO 2021 / 013708, or the method described in WO 2018 / 122225, for lactodifucotetraose, for example, the method described in WO 2023 / 182527, lactose-N-biose I, for example, the method described in WO 2022 / 034075.

[0029] When the immunostimulant according to one aspect of this embodiment is ingested or administered in combination with a modifying human milk oligosaccharide, the mass ratio of the modifying human milk oligosaccharide to lacto-N-tetraose may vary depending on factors such as health status, the form and ingestion of the composition, and the method of administration, but is not particularly limited as long as it can reduce the immunostimulation caused by lacto-N-tetraose as desired. This mass ratio may be, for example, 20 parts or more, 10 parts or more, 5 parts or more, 2 parts or more, or 1 part or more by mass per part by mass of lacto-N-tetraose. The mass ratio in the immunomodulator according to one aspect of this embodiment may be, for example, 1 part or less by mass, 0.5 parts or less, 0.2 parts or less, 0.1 parts or less, 0.05 parts or less, or 0.01 parts or less by mass. Within the above-mentioned range, a higher mass ratio results in stronger suppression of immunostimulation caused by lacto-N-tetraose, while a lower mass ratio results in weaker suppression of immunostimulation caused by lacto-N-tetraose.

[0030] When the immunostimulant according to one aspect of this embodiment is ingested or administered in combination with human milk oligosaccharides for adjustment, the timing of the ingestion or administration of the immunostimulant according to one aspect of this embodiment is not particularly limited as long as it allows for adjustment of the level of immunostimulatory effect of lacto-N-tetraose as described above, and may be simultaneous with, prior to, or after the ingestion or administration of the human milk oligosaccharides for adjustment. For example, the immunostimulant according to another aspect of this embodiment may be ingested or administered 3 hours to 3 hours, or 1 hour to 1 hour, before or after the ingestion or administration of the human milk oligosaccharides for adjustment.

[0031] 1A. A method for stimulating immunity, comprising administering lacto-N-tetraose to a subject. A method for stimulating immunity according to one aspect of the present embodiment comprises administering lacto-N-tetraose to a subject.

[0032] The administration (ingestion) conditions in the above-mentioned method (e.g., subject to be administered (ingested), amount administered (ingested), number of times administered (ingested), etc.) can be the same as those described above in [1. Immunostimulant containing lacto-N-tetraose] as the administration (ingestion) conditions of the immunostimulant, without any restrictions.

[0033] For example, lacto-N-tetraose may be administered (ingested) to a subject as is, or may be prepared as an immunostimulant as described above in "1. Immunostimulants containing lacto-N-tetraose" and administered (ingested) to a subject. In the former case, lacto-N-tetraose may be administered (ingested) alone or in combination with additional components. The additional components may include human milk oligosaccharides for preparation, components that may be contained in the above-mentioned food compositions or pharmaceutical compositions, etc.

[0034] [1B. Lacto-N-tetraose for use in a therapeutic method for stimulating immunity] One aspect of this embodiment relates to lacto-N-tetraose for use in a therapeutic method for stimulating immunity. Specific embodiments of this lacto-N-tetraose include, without limitation, the embodiments described above in [1. Immunostimulant containing lacto-N-tetraose] and [1A. Method for stimulating immunity, comprising administering lacto-N-tetraose to a subject].

[0035] As used herein, the term "therapeutic method" refers to a method of treating a mammalian body. For example, the method may be a method aimed at or involving medical treatment. More specifically, the method may involve, for example, a medical professional administering a substance to a mammalian body or instructing a medical professional to administer a substance to a mammalian body.

[0036] [1C. Use of lacto-N-tetraose for use in a non-therapeutic method of immunostimulating] One aspect of this embodiment relates to the use of lacto-N-tetraose for use in a non-therapeutic method of immunostimulating. Specific embodiments of this use include, without limitation, the embodiments described above in [1. Immunostimulant containing lacto-N-tetraose] and [1A. Method of immunostimulating immunity comprising administering lacto-N-tetraose to a subject].

[0037] As used herein, the term "non-therapeutic method" refers to a method that does not fall under the category of a therapeutic method. For example, it may be a method that is not intended for and / or does not involve medical treatment. More specifically, it may be a method that does not involve a medical professional administering a substance to a mammal and / or instructing a mammal to administer a substance. Furthermore, a non-therapeutic method may be, for example, a method for the purpose of promoting health.

[0038] [1D. Use of lacto-N-tetraose for producing an immunostimulant] One aspect of this embodiment relates to the use of lacto-N-tetraose for producing an immunostimulant. Specific embodiments of this use include, without limitation, the embodiments described above in [1. Immunostimulant containing lacto-N-tetraose] and [1A. Method for stimulating immunity, comprising administering lacto-N-tetraose to a subject].

[0039] 2. Immunomodulators containing lacto-N-tetraose and specific human milk oligosaccharides (i.e., modulating human milk oligosaccharides); immunomodulators containing modulating human milk oligosaccharides, characterized in that they are ingested or administered in combination with lacto-N-tetraose. The immunomodulators according to one aspect of this embodiment are capable of modulating immune function in the living body. Modulation of immune function includes both immune activation and suppression. The immunomodulators according to one aspect of this embodiment, for example, contain lacto-N-tetraose and modulating human milk oligosaccharides. The immunomodulators according to one aspect of this embodiment, for example, contain modulating human milk oligosaccharides, characterized in that they are ingested or administered in combination with lacto-N-tetraose. The immunomodulators according to one aspect of this embodiment may or may not contain lacto-N-tetraose.

[0040] In the immunomodulator according to one aspect of this embodiment, lacto-N-tetraose can stimulate the immune system by enhancing TNF-α production, while the modulating human milk oligosaccharides can suppress the immune system by suppressing TNF-α production. For example, the modulating human milk oligosaccharides can suppress the immunostimulation caused by lacto-N-tetraose by suppressing the TNF-α production-enhancing effect of lacto-N-tetraose. Thus, the immunomodulator according to one aspect of this embodiment may be an agent in which the immunostimulating effect of lacto-N-tetraose is adjusted, i.e., suppressed, to a degree appropriate for the purpose by the modulating human milk oligosaccharides, or may further exhibit the TNF-α production suppressing effect of the modulating human milk oligosaccharides. From the above, the immunomodulator according to one aspect of this embodiment may be used for the purpose of immune stimulation or immune suppression by appropriately adjusting the content or intake or administration amount of lacto-N-tetraose and the modulating human milk oligosaccharides as described below. When the immunomodulator according to one aspect of this embodiment is used for the purpose of immunosuppression, the modulating human milk oligosaccharide preferably contains 6'-sialyllactose. Furthermore, when the immunomodulator according to one aspect of this embodiment is used for the purpose of immunosuppression, it may contain a compound or composition other than HMO that is known to suppress immunity.

[0041] The human milk oligosaccharides and lacto-N-tetraose for preparation can be the same as those described above in [1. Immunostimulants containing lacto-N-tetraose].

[0042] The immunomodulator according to one aspect of this embodiment may contain only human milk oligosaccharides for modification, or only lacto-N-tetraose and human milk oligosaccharides for modification, or may contain other ingredients as needed. That is, the immunomodulator according to one aspect of this embodiment may be a composition containing an effective amount of human milk oligosaccharides for modification, or effective amounts of lacto-N-tetraose and human milk oligosaccharides for modification (i.e., an immunomodulatory composition containing an effective amount of human milk oligosaccharides for modification; or an immunomodulatory composition containing effective amounts of lacto-N-tetraose and human milk oligosaccharides for modification). Such compositions may be, for example, food compositions, pharmaceutical compositions, or ingredients thereof.

[0043] The effective amount of the human milk oligosaccharide for regulation and the combined effective amount of lacto-N-tetraose and human milk oligosaccharide for regulation in the immunomodulator according to one aspect of this embodiment are not particularly limited, as long as they are sufficient to achieve an immunomodulatory effect. The effective amount may vary depending on factors such as health status and the method of ingestion or administration, and can be determined as needed by those skilled in the art. For example, the effective amount may be 1 mg to 20 g per kg of adult body weight per day, and preferably 5 mg to 500 mg per day. When the immunomodulator according to one aspect of this embodiment is ingested or administered to a subject, the frequency of ingestion or administration is not particularly limited, and may be once a day or multiple times, such as twice or three times a day. The timing of ingestion or administration is also not particularly limited, and may be after waking up, before meals, after meals, between meals, or before bedtime. The subject of ingestion or administration may be a mammal, and preferably a human. The route of administration may be, for example, oral, sublingual, intramuscular, enteral, nasal, intravenous, subcutaneous, or intraperitoneal, and is preferably oral. The route of ingestion is preferably oral ingestion.

[0044] The immunomodulator according to one aspect of this embodiment may take the form of a food composition, pharmaceutical composition, or raw material thereof as described above in [1. Immunostimulant containing lacto-N-tetraose].

[0045] When the immunomodulator according to one aspect of this embodiment contains lacto-N-tetraose, the content of lacto-N-tetraose in the immunomodulator can be the same as that in [1. Immunostimulant containing lacto-N-tetraose] above.

[0046] The content of the modulating human milk oligosaccharide in the immunoregulator according to one aspect of this embodiment may vary depending on the form of the composition and the method of ingestion or administration, but is not particularly limited as long as it is a content that provides an immunomodulatory effect. The content of the modulating human milk oligosaccharide in the immunoregulator according to one aspect of this embodiment may be, for example, 25% by mass or more, 10% by mass or more, 5% by mass or more, 2% by mass or more, or 1% by mass or more, based on the total amount of the immunoregulator. The content of the modulating human milk oligosaccharide in the immunoregulator according to one aspect of this embodiment may be, for example, 50% by mass or less, 25% by mass or less, 10% by mass or less, 5% by mass or less, 2% by mass or less, or 1% by mass or less, based on the total amount of the immunoregulator.

[0047] The mass ratio of the modulating human milk oligosaccharides to lacto-N-tetraose in the immunomodulator according to one aspect of this embodiment, or the mass ratio administered or ingested (hereinafter collectively referred to as "mass ratio"), may vary depending on factors such as health status, the form or ingestion of the composition, and the method of administration, but is not particularly limited as long as it is a mass ratio that provides an immunomodulatory effect. The mass ratio in the immunomodulator according to one aspect of this embodiment may be, for example, 20 parts by mass or more, 10 parts by mass or more, 5 parts by mass or more, 2 parts by mass or more, or 1 part by mass or more, relative to 1 part by mass of lacto-N-tetraose. The mass ratio in the immunomodulator according to one aspect of this embodiment may be, for example, 1 part by mass or less, 0.5 parts by mass or less, 0.2 parts by mass or less, 0.1 parts by mass or less, 0.05 parts by mass or less, or 0.01 parts by mass or less. By lowering the mass ratio within the above-mentioned range, the immunostimulatory effect of the immunomodulator according to one aspect of this embodiment can be improved. Thus, by lowering the mass ratio within the above-mentioned range, the immunomodulator according to one aspect of the present embodiment can be used for the purpose of immunostimulating immunity. By increasing the mass ratio within the above-mentioned range, the immunostimulating effect of the immunomodulator according to one aspect of the present embodiment can be reduced, or the immunosuppressive effect of the immunomodulator according to one aspect of the present embodiment can be improved. Thus, by increasing the mass ratio within the above-mentioned range, the immunomodulator according to one aspect of the present embodiment can be used for the purpose of immunosuppressing immunity.

[0048] In the immunomodulator according to another aspect of this embodiment, when the modulating human milk oligosaccharide is ingested or administered in combination with lacto-N-tetraose, the timing of ingestion or administration of the modulating human milk oligosaccharide is not particularly limited as long as it is capable of regulating immune function, and may be simultaneous with, before, or after the ingestion or administration of lacto-N-tetraose. For example, the immunostimulant according to another aspect of this embodiment may be ingested or administered 3 hours to 3 hours, or 1 hour to 1 hour, before or after the ingestion or administration of lacto-N-tetraose.

[0049] 2A. Methods for modulating immunity comprising administering lacto-N-tetraose and modulating human milk oligosaccharides to a subject; methods for modulating immunity comprising administering modulating human milk oligosaccharides in combination with lacto-N-tetraose to a subject. A method for modulating immunity according to one aspect of this embodiment comprises administering lacto-N-tetraose and modulating human milk oligosaccharides to a subject, or administering modulating human milk oligosaccharides in combination with lacto-N-tetraose to a subject.

[0050] The administration (ingestion) conditions in the above-mentioned method (e.g., the subject to be administered (ingested), the amount administered (ingested), the number of times administered (ingested), etc.) can be, without limitation, the conditions described above as the administration (ingestion) conditions for the immunomodulator in [2. Immunomodulator containing lacto-N-tetraose and a specific human milk oligosaccharide (i.e., modulating human milk oligosaccharide); Immunomodulator containing modulating human milk oligosaccharide, characterized in that it is used so as to be ingested or administered in combination with lacto-N-tetraose].

[0051] For example, lacto-N-tetraose and human milk oligosaccharides for modification may be administered (ingested) to a subject as is, or may be prepared as an immunomodulator as described above in [2. Immunomodulators containing lacto-N-tetraose and specific human milk oligosaccharides (i.e., human milk oligosaccharides for modification); Immunomodulators containing human milk oligosaccharides for modification, characterized in that they are used to be ingested or administered in combination with lacto-N-tetraose] and then administered (ingested) to a subject. In the former case, lacto-N-tetraose and human milk oligosaccharides for modification may be administered (ingested) without being combined with other ingredients, or may be administered (ingested) in combination with additional ingredients. The additional ingredients may be ingredients that may be contained in the food compositions or pharmaceutical compositions described above.

[0052] [2B. Lacto-N-tetraose and Modulatory Human Milk Oligosaccharides for Use in Therapeutic Methods for Modulating Immunity; Modulatory Human Milk Oligosaccharides Administered in Combination with Lacto-N-tetraose for Use in Therapeutic Methods for Modulating Immunity] One aspect of this embodiment relates to lacto-N-tetraose and modulatory human milk oligosaccharides for use in therapeutic methods for modulating immunity, and modulatory human milk oligosaccharides administered in combination with lacto-N-tetraose for use in therapeutic methods for modulating immunity. Specific embodiments of the lacto-N-tetraose and the modulatory human milk oligosaccharides, and the modulatory human milk oligosaccharides administered in combination with lacto-N-tetraose, include the immunomodulators described above in [2. An immunomodulator comprising lacto-N-tetraose and a specific human milk oligosaccharide (i.e., a modulatory human milk oligosaccharide); an immunomodulator comprising a modulatory human milk oligosaccharide, which is used to be ingested or administered in combination with lacto-N-tetraose] and the immunomodulators described above in [2A. The embodiments described above can be applied without limitation: a method for modulating immunity, comprising administering lacto-N-tetraose and modulating human milk oligosaccharides to a subject; a method for modulating immunity, comprising administering modulating human milk oligosaccharides in combination with lacto-N-tetraose to a subject.

[0053] 2C. Use of lacto-N-tetraose and modulating human milk oligosaccharides for use in non-therapeutic immune modulation methods; Use of modulating human milk oligosaccharides administered in combination with lacto-N-tetraose for use in non-therapeutic immune modulation methods. One aspect of this embodiment relates to the use of lacto-N-tetraose and modulating human milk oligosaccharides for use in non-therapeutic immune modulation methods, and the use of modulating human milk oligosaccharides administered in combination with lacto-N-tetraose for use in non-therapeutic immune modulation methods. Specific embodiments of these uses include the above-mentioned 2. Immunomodulators containing lacto-N-tetraose and specific human milk oligosaccharides (i.e., modulating human milk oligosaccharides); Immunomodulators containing modulating human milk oligosaccharides, which are ingested or administered in combination with lacto-N-tetraose, and the above-mentioned 2A. The embodiments described above can be applied without limitation: a method for modulating immunity, comprising administering lacto-N-tetraose and modulating human milk oligosaccharides to a subject; a method for modulating immunity, comprising administering modulating human milk oligosaccharides in combination with lacto-N-tetraose to a subject.

[0054] 2D. Use of lacto-N-tetraose and modulating human milk oligosaccharides for the production of an immunomodulator; Use of modulating human milk oligosaccharides for the production of an immunomodulator, characterized in that the immunomodulator is ingested or administered in combination with lacto-N-tetraose. One aspect of this embodiment relates to the use of lacto-N-tetraose and modulating human milk oligosaccharides for the production of an immunomodulator, and the use of modulating human milk oligosaccharides for the production of an immunomodulator, characterized in that the immunomodulator is ingested or administered in combination with lacto-N-tetraose. Specific embodiments of these uses include those described above in 2. An immunomodulator comprising lacto-N-tetraose and a specific human milk oligosaccharide (i.e., modulating human milk oligosaccharide); an immunomodulator comprising modulating human milk oligosaccharide, characterized in that the immunomodulator is ingested or administered in combination with lacto-N-tetraose, and those described above in 2A. The embodiments described above can be applied without limitation: a method for modulating immunity, comprising administering lacto-N-tetraose and modulating human milk oligosaccharides to a subject; a method for modulating immunity, comprising administering modulating human milk oligosaccharides in combination with lacto-N-tetraose to a subject.

[0055] 3. Immunostimulant Containing Lacto-N-Fucopentaose I An immunostimulant according to another aspect of this embodiment contains lacto-N-fucopentaose I.

[0056] An immunostimulant according to another aspect of this embodiment can improve immune function in a living body. The immunostimulant according to another aspect of this embodiment can, for example, enhance TNF-α production. That is, the immunostimulant according to another aspect of this embodiment may be based on enhancing TNF-α production. Furthermore, the present inventors believe that one of the mechanisms by which the immunostimulant according to another aspect of this embodiment enhances TNF-α production is that lacto-N-fucopentaose I activates TLR4 signaling and the like in macrophages and the like, thereby promoting the secretion of TNF-α from macrophages and the like. Therefore, the immunostimulant according to another aspect of this embodiment is thought to be able to activate innate immunity via activation of TLR4 signaling and the like.

[0057] Furthermore, the immunostimulant according to another aspect of this embodiment may be based on promoting immunostimulation by endotoxin, or may be based on promoting enhanced TNF-α production by endotoxin.

[0058] The lacto-N-fucopentaose I may be produced by a known chemical synthesis method, an enzymatic synthesis method, or a microbial synthesis method, or may be a commercially available product. Examples of the microbial synthesis method include the method described in WO 2023 / 182528.

[0059] An immunostimulant according to another aspect of this embodiment may contain only lacto-N-fucopentaose I, or may contain, as necessary, components other than lacto-N-fucopentaose I. That is, an immunostimulant according to another aspect of this embodiment may be an immunostimulating composition containing lacto-N-fucopentaose I, or may be a composition containing an effective amount of lacto-N-fucopentaose I (i.e., an immunostimulating composition containing an effective amount of lacto-N-fucopentaose I). Such compositions may be, for example, food compositions, pharmaceutical compositions, or raw materials thereof.

[0060] The effective amount of lacto-N-fucopentaose I in the immunostimulant according to another aspect of this embodiment is not particularly limited, as long as it is an amount that can achieve an immunostimulatory effect. The effective amount may vary depending on factors such as health status and the method of ingestion or administration, and can be freely determined by those skilled in the art as needed. For example, the effective amount may be 1 mg to 20 g / kg of adult body weight per day, and preferably 5 mg to 500 mg / kg per day. When a subject ingests the immunostimulant according to another aspect of this embodiment, or when the immunostimulant according to another aspect of this embodiment is administered to a subject, the number of ingestions or administrations is not particularly limited, and may be once a day, or may be divided into multiple doses, such as twice or three times a day. Furthermore, the timing of ingestion or administration is not particularly limited, and may be after waking up, before meals, after meals, between meals, or before bedtime. The subject of ingestion or administration may be a mammal, and preferably a human. The route of administration may be, for example, oral, sublingual, intramuscular, enteral, nasal, intravenous, subcutaneous, or intraperitoneal, and is preferably oral. The route of ingestion is preferably oral ingestion.

[0061] The immunomodulator according to another aspect of this embodiment may take the form of a food composition, pharmaceutical composition, or raw material thereof as described in [1. Immunostimulant containing lacto-N-tetraose] above.

[0062] The content of lacto-N-fucopentaose I in the immunostimulant according to another aspect of the present embodiment may vary depending on the form of the composition and the method of ingestion or administration, but is not particularly limited as long as it is a content that provides an immunostimulatory effect. The content of lacto-N-fucopentaose I in the immunostimulant according to another aspect of the present embodiment may be, for example, 25% by mass or more, 10% by mass or more, 5% by mass or more, 2% by mass or more, or 1% by mass or more, relative to the total amount of the immunostimulant. The content of lacto-N-fucopentaose I in the immunostimulant according to another aspect of the present embodiment may be, for example, 50% by mass or less, 25% by mass or less, 10% by mass or less, 5% by mass or less, 2% by mass or less, or 1% by mass or less, relative to the total amount of the immunostimulant.

[0063] 3A. A method for stimulating immunity, comprising administering lacto-N-fucopentaose I to a subject. A method for stimulating immunity according to one aspect of this embodiment comprises administering lacto-N-fucopentaose I to a subject.

[0064] The administration (ingestion) conditions in the above-mentioned method (e.g., subject to be administered (ingested), amount administered (ingested), number of times administered (ingested), etc.) can be the same as those described above in [3. Immunostimulant containing lacto-N-fucopentaose I] as the administration (ingestion) conditions of the immunostimulant, without any restrictions.

[0065] For example, lacto-N-fucopentaose I may be administered (ingested) directly to a subject, or may be prepared as an immunostimulant as described above in [3. Immunostimulant containing lacto-N-fucopentaose I] and administered (ingested) to a subject. In the former case, lacto-N-fucopentaose I may be administered (ingested) alone or in combination with an additional component. The additional component may be a human milk oligosaccharide for preparation, a component that may be contained in the above-mentioned food composition or pharmaceutical composition, or the like.

[0066] [3B. Lacto-N-fucopentaose I for use in a therapeutic method for stimulating immunity] One aspect of this embodiment relates to lacto-N-fucopentaose I for use in a therapeutic method for stimulating immunity. Specific embodiments of the lacto-N-fucopentaose I include, without limitation, the embodiments described above in [3. Immunostimulants containing lacto-N-fucopentaose I] and [3A. Methods for stimulating immunity, comprising administering lacto-N-fucopentaose I to a subject].

[0067] [3C. Use of lacto-N-fucopentaose I for use in a non-therapeutic method of stimulating immunity] One aspect of this embodiment relates to use of lacto-N-fucopentaose I for use in a non-therapeutic method of stimulating immunity. Specific embodiments of this use include, without limitation, the embodiments described above in [3. Immunostimulants containing lacto-N-fucopentaose I] and [3A. Methods of stimulating immunity comprising administering lacto-N-fucopentaose I to a subject].

[0068] [3D. Use of lacto-N-fucopentaose I for producing an immunostimulant] One aspect of this embodiment relates to the use of lacto-N-fucopentaose I for producing an immunostimulant. Specific embodiments of this use include, without limitation, the embodiments described above in [3. Immunostimulant containing lacto-N-fucopentaose I] and [3A. Method for stimulating immunity, comprising administering lacto-N-fucopentaose I to a subject].

[0069] 4. Immunosuppressant containing 6'-sialyllactose An immunosuppressant according to one aspect of this embodiment contains 6'-sialyllactose.

[0070] The immunosuppressant according to one aspect of this embodiment is capable of suppressing immune function in a living body. The immunosuppressant according to one aspect of this embodiment is capable of suppressing, for example, the production of TNF-α. That is, the immunosuppressant according to one aspect of this embodiment may be based on the suppression of TNF-α production. Furthermore, with regard to the mechanism of action of the immunosuppressant according to one aspect of this embodiment for suppressing TNF-α production, the inventors believe that one factor is that 6'-sialyllactose suppresses TLR4 signaling and the like in macrophages and the like, thereby suppressing TNF-α secretion from macrophages and the like. Therefore, it is believed that the immunosuppressant according to one aspect of this embodiment is capable of suppressing immune function via the suppression of TLR4 signaling and the like.

[0071] Furthermore, the immunosuppressant according to one aspect of this embodiment may be based on suppressing immunostimulation by lacto-N-tetraose or endotoxin, or may be based on suppressing the enhancement of TNF-α production by lacto-N-tetraose or endotoxin.

[0072] The 6'-sialyllactose that can be used is the same as that described above in [1. Immunostimulant containing lacto-N-tetraose].

[0073] The immunosuppressant according to one aspect of this embodiment may contain only 6'-sialyllactose, or may contain components other than 6'-sialyllactose as necessary. That is, the immunostimulant according to another aspect of this embodiment may be a composition containing an effective amount of 6'-sialyllactose (i.e., an immunostimulatory composition containing an effective amount of 6'-sialyllactose). Such compositions may be, for example, food compositions, pharmaceutical compositions, or raw materials thereof.

[0074] The effective amount of 6'-sialyllactose in the immunosuppressant according to one aspect of this embodiment is not particularly limited, as long as it is an amount that can achieve an immunosuppressive effect. The effective amount may vary depending on factors such as health status, the method of ingestion or administration, and can be freely determined by those skilled in the art as needed. For example, the amount may be 1 mg to 20 g / day, and preferably 5 mg to 500 mg / day, per kg of adult body weight. When the immunosuppressant according to one aspect of this embodiment is ingested by a subject or administered to a subject, the number of ingestions or administrations is not particularly limited, and may be once a day or multiple times, such as twice or three times a day. Furthermore, the timing of ingestion or administration is not particularly limited, and may be after waking up, before meals, after meals, between meals, or before bedtime. The subject of ingestion or administration may be a mammal, and preferably a human. The route of administration may be, for example, oral, sublingual, intramuscular, enteral, nasal, intravenous, subcutaneous, or intraperitoneal administration, with oral administration being preferred. The preferred route of intake is oral intake.

[0075] The immunosuppressant according to one aspect of this embodiment may take the form of a food composition, pharmaceutical composition, or raw material thereof as described above in [1. Immunostimulant containing lacto-N-tetraose].

[0076] The content of 6'-sialyllactose in the immunosuppressant according to one aspect of the present embodiment may vary depending on the form of the composition and the method of ingestion or administration, but is not particularly limited as long as it is a content that provides an immunosuppressive effect. The content of 6'-sialyllactose in the immunosuppressant according to one aspect of the present embodiment may be, for example, 25% by mass or more, 10% by mass or more, 5% by mass or more, 2% by mass or more, or 1% by mass or more, relative to the total amount of the immunosuppressant. The content of 6'-sialyllactose in the immunosuppressant according to one aspect of the present embodiment may be, for example, 50% by mass or less, 25% by mass or less, 10% by mass or less, 5% by mass or less, 2% by mass or less, or 1% by mass or less, relative to the total amount of the immunosuppressant.

[0077] 4A. A method for suppressing immunity comprising administering 6'-sialyllactose to a subject A method for suppressing immunity according to one aspect of this embodiment comprises administering 6'-sialyllactose to a subject.

[0078] The administration (ingestion) conditions in the above method (e.g., the subject to be administered (ingested), the administration (ingestion) amount, the number of administrations (ingestions), etc.) can be the same as those described above as the administration (ingestion) conditions for immunosuppressants in [Immunosuppressant containing 4,6'-sialyllactose], without any restrictions.

[0079] For example, 6'-sialyllactose may be administered (ingested) to a subject as is, or may be prepared as an immunosuppressant as described above in [4. Immunosuppressant containing 6'-sialyllactose] and administered (ingested) to a subject. In the former case, 6'-sialyllactose may be administered (ingested) alone, or may be administered (ingested) in combination with an additional component. The additional component may be a component that may be contained in the food composition or pharmaceutical composition described above.

[0080] [4B. 6'-Sialyllactose for use in a therapeutic method for immunosuppression] One aspect of this embodiment relates to 6'-sialyllactose for use in a therapeutic method for immunosuppression. Specific embodiments of the 6'-sialyllactose include, without limitation, the embodiments described above in [4. Immunosuppressant containing 6'-sialyllactose] and [4A. Method for immunosuppression comprising administering 6'-sialyllactose to a subject].

[0081] [4C. Use of 6'-sialyllactose for use in a non-therapeutic method of immunosuppression] One aspect of this embodiment relates to the use of 6'-sialyllactose for use in a non-therapeutic method of immunosuppression. Specific embodiments of this use include, without limitation, the embodiments described above in [4. Immunosuppressant containing 6'-sialyllactose] and [4A. Method of immunosuppression comprising administering 6'-sialyllactose to a subject].

[0082] [4D. Use of 6'-sialyllactose for producing an immunosuppressant] One aspect of this embodiment relates to the use of 6'-sialyllactose for producing an immunosuppressant. Specific embodiments of this use include, without limitation, the embodiments described above in [4. Immunosuppressant containing 6'-sialyllactose] and [4A. Method for suppressing immunity comprising administering 6'-sialyllactose to a subject].

[0083] [5. Composition containing human milk oligosaccharides] The composition of this embodiment is a composition containing human milk oligosaccharides, and the endotoxin content of the composition is less than 10 endotoxin units (hereinafter also referred to as "EU") per 1 mg of the human milk oligosaccharides.

[0084] The composition according to this embodiment contains less than 10 EU of endotoxin per 1 mg of human milk oligosaccharides, and therefore has the effects of human milk oligosaccharides, such as immunostimulating activity and intestinal environment regulation, without substantially exhibiting the effects of endotoxin, such as endotoxin-induced TLR4 signaling activation, etc. This effect is more pronounced when the endotoxin content is less than 0.5 EU, less than 0.05 EU, less than 0.005 EU, less than 0.0005 EU, or less than 0.00005 EU per 1 mg of human milk oligosaccharides.

[0085] The endotoxin content in the composition according to this embodiment refers to the endotoxin content determined by endpoint colorimetry, which involves reacting the endotoxin with Limulus amebocyte lysate. Specifically, the measurement of the endotoxin content by endpoint colorimetry may be performed by the method described in the Examples below.

[0086] Human milk oligosaccharides are not particularly limited and include, for example, 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lactodifucotetraose, lactose-N-biose I, lacto-N-neotetraose, lacto-N-tetraose, lacto-N-triose II, N-acetyllactosamine, 3-fucosyllactose, and lacto-N-fucopentaose I. The composition according to this embodiment may contain one type of human milk oligosaccharide, or may contain a combination of two or more types of human milk oligosaccharides.

[0087] Human milk oligosaccharides may be those produced by known chemical synthesis methods, synthesis methods using enzymes, or synthesis methods using microorganisms, or commercially available products may be used. Examples of synthesis methods using microorganisms include those described in WO 2023 / 120615 for lacto-N-triose II, WO 2022 / 034075 for N-acetyllactosamine, and WO 2022 / 168991 or WO 2022 / 168992 for 3-fucosyllactose. For lacto-N-tetraose, 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I, similar to those described above in [1. Immunostimulants containing lacto-N-tetraose], and those described above in [3. Immunostimulants containing lacto-N-tetraose] can be used. For lacto-N-fucopentaose I, those described above in [3. The same as the immunostimulant containing lacto-N-fucopentaose I can be used.

[0088] The content of human milk oligosaccharides in the composition of this embodiment is not particularly limited, but may be, for example, 5 to 50% by mass, or 10 to 25% by mass, based on the total amount of the composition of this embodiment.

[0089] In the composition of this embodiment, when the human milk oligosaccharide is lacto-N-tetraose or lacto-N-fucopentaose I, the composition may be an immunostimulant. In addition, in the composition of this embodiment, when the human milk oligosaccharide is 6'-sialyllactose, the composition may be an immunosuppressant.

[0090] In the composition of this embodiment, when the human milk oligosaccharide comprises lacto-N-tetraose and the above-mentioned human milk oligosaccharide for modification, or when the human milk oligosaccharide comprises the above-mentioned human milk oligosaccharide for modification, the composition may be an immunomodulator.

[0091] The composition according to this embodiment may take the form of a food composition, pharmaceutical composition, or raw material thereof described in the above section [1. Immunostimulant containing lacto-N-tetraose]. Among these, powder is preferred. Examples of powder include spray-dried powder and freeze-dried powder.

[0092] The method for producing the composition according to this embodiment is not particularly limited and may include, for example, a step of removing endotoxins from the composition by ultrafiltration of a solution containing the composition containing human milk oligosaccharides. The composition containing human milk oligosaccharides used in the above production may be, for example, a composition produced by a known chemical synthesis method or a synthesis method using a microorganism, or a composition generally available commercially as human milk oligosaccharides. The endotoxin content of these compositions is typically greater than 0.002 EU, and often greater than 0.22 EU, per 0.1 mmol of human milk oligosaccharides.

[0093] The solvent for the solution is not particularly limited and may be, for example, ultrapure water. The content of human milk oligosaccharides in the solution is not particularly limited and may be, for example, 1 to 1000 mmol / L based on the solution.

[0094] The ultrafiltration in the above step is not particularly limited, and may be carried out by, for example, centrifugal ultrafiltration.

[0095] The filter used for ultrafiltration in the above process is not particularly limited as long as it can filter human milk oligosaccharides and retain endotoxins, but for example, an ultrafiltration membrane with a molecular weight cutoff of 1 to 4 kDa may be used.

[0096] When the filter used for ultrafiltration in the above step contains glycerol as a preservative, the filter may be used after removing the glycerol. Glycerol may be removed from the filter by dissolving the glycerol in ultrapure water, for example, by repeating the procedure of passing ultrapure water through the filter 3 to 5 times.

[0097] In the above process, ultrafiltration may be performed one or more times. The number of times ultrafiltration may be appropriately set so that the endotoxin content in the filtrate is less than 10 EU per 1 mg of human milk oligosaccharides, preferably less than 0.5 EU, more preferably less than 0.005 EU, even more preferably less than 0.0005 EU, and most preferably less than 0.00005 EU. The number of times ultrafiltration is not particularly limited, but if the endotoxin concentration in the human milk oligosaccharide-containing composition subjected to the above process exceeds 0.22 EU / mL, ultrafiltration may be performed five or more times.

[0098] The present invention will be specifically described below based on examples, but the present invention is not limited to these examples.

[0099] Example 1: Removal of Endogenous Endotoxin from Sugar Samples To prevent activation of Toll-like receptor 4 (hereinafter also referred to as "TLR4") signaling due to endotoxin contamination, endotoxin was removed from the lactose and human milk oligosaccharide (hereinafter also referred to as "HMO") samples used for evaluation using the following method. First, on a clean bench, each component of a centrifugal ultrafiltration filter, Amicon Ultra -0.5 3 kD (model number UFC500396, manufactured by Merck) was arranged on aluminum foil and sterilized for 15 minutes under an ultraviolet germicidal lamp. The centrifugal ultrafiltration filter was then assembled, 0.5 mL of ultrapure water was added, and the filter was centrifuged at 14,500 g and 4°C for 10 minutes to remove the filter residue and the flow-through liquid. Similarly, ultrapure water was added to the above centrifugal ultrafiltration filter, centrifuged, and the filter residue and the flow-through liquid were removed. This procedure was repeated three times, and then all water adhering to the filter was removed. By the above procedure, glycerol used as a preservative in the centrifugal ultrafiltration filter was removed from the filter. In the following procedures, the centrifugal ultrafiltration filter from which glycerol had been removed by the above method was used.

[0100] Next, lactose (lactose monohydrate, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter also referred to as "Lac") and 11 types of HMO samples, namely, 2'-fucosyllactose (hereinafter also referred to as "2'-FL"), 3'-sialyllactose (hereinafter also referred to as "3'-SL"), 6'-sialyllactose (hereinafter also referred to as "6'-SL"), lactodifucotetraose (hereinafter also referred to as "LDFT"), lactose-N-biose I (hereinafter also referred to as "LNB"), lacto-N-neotetraose (hereinafter also referred to as "LNnT"), lacto-N-tetraose (hereinafter also referred to as "LNT"), lacto-N-triose II (hereinafter also referred to as "LNT"). II). N-acetyllactosamine (hereinafter also referred to as "LacNAc"), 3-fucosyllactose (hereinafter also referred to as "3-FL"), and lacto-N-fucopentaose I (hereinafter also referred to as "LNFP I") were each dissolved in ultrapure water to prepare 100 mM solutions (hereinafter also referred to as "untreated solutions"). Each of the 11 HMO samples was produced and purified by genetically modified microorganisms. 0.5 mL of each 100 mM untreated solution was added to a centrifugal ultrafiltration filter and centrifuged at 14,500 g and 4°C for 40 minutes, and the flow-through A was collected. Then, 0.4 mL of each new 100 mM untreated solution was added to the same centrifugal ultrafiltration filter that had been used to filter the same solution, and the flow-through B was collected. The flow-through fractions A and B were combined to form the first flow-through fraction. Each of the first flow-through fractions was added to a new centrifugal ultrafiltration filter and centrifuged at 14,500 g and 4°C for 40 minutes, and the resulting flow-through fraction was used as the second flow-through fraction. The same procedures as those used to obtain the first and second flow-through fractions were repeated three more times for each second flow-through fraction, and approximately 0.5 mL of the fifth flow-through fraction was obtained.

[0101] Subsequently, the endotoxin concentrations of each of the fifth pass-through fractions and each of the untreated solutions were measured by endpoint colorimetry using an Endospecy ES-24s (Seikagaku Corporation) according to the manufacturer's protocol. Specifically, standard endotoxin, ultrapure water, 100 mM of each of the fifth pass-through fractions, or 100 mM of each of the untreated solutions were added to Limulus Amebocyte Lysate (LAL) reagent, and after 30 minutes of incubation at 37°C, the absorbance at 405 nm was measured. Table 1 shows the endotoxin concentrations of each of the untreated solutions and each of the fifth pass-through fractions. A calibration curve was prepared using endotoxin standard samples diluted to 0.22-0.0017 EU / mL. The measurement results were analyzed using a four-parameter logistic (4-PL) curve. In Table 1, ">0.22" indicates that the endotoxin concentration exceeds the upper detection limit of 0.22 EU / ml. In Table 1, "<0.002" indicates that the endotoxin concentration is below the lower detection limit of 0.002 EU / ml.

[0102]

[0103] As shown in Table 1, the endotoxin concentrations of each untreated solution not subjected to ultrafiltration were 0.0139 EU / mL and 0.2050 EU / mL for 3'-SL and 6'-SL, respectively, less than 0.002 EU / mL for lactose, and more than 0.22 EU / mL for other HMOs. In contrast, the absorbance at 405 nm for all of the fifth pass-through fractions was 0.0310, the lower limit of detection, and the endotoxin concentrations of all of the fifth pass-through fractions were less than 0.002 EU / mL.

[0104] Example 2: Measurement of Changes in TNF-α Production Amounts Due to Addition of Lactose or HMO Using RAW264.7 Cells A frozen vial containing RAW264.7 cells (ECACC, model number 91062702), a mouse macrophage-derived cell line, was partially thawed in a 37°C water bath and suspended in DMEM medium (10% by volume FBS, 1% by volume penicillin-streptomycin solution), followed by centrifugation at 800 g for 3 minutes. The supernatant was discarded, and fresh DMEM medium was added and the cells were seeded into a 100 mm dish. After culturing until 90% confluent, the medium was removed and 10 mL of DMEM medium was added. The cells were all detached using a cell scraper, and 1 mL of cells from the DMEM medium was added to 10 mL of fresh DMEM medium, seeded into a 100 mm dish, and subcultured. After several passages, the RAW264.7 cells, whose viability was 96-99%, were used in the following procedure.

[0105] The fifth pass-through fractions obtained by the method described in Example 1 were diluted with sterile water to prepare 10 mM and 20 mM lactose solutions and each HMO solution. These were then diluted 2-fold with DMEM medium (2-fold concentrated) to prepare lactose-containing DMEM medium and each HMO-containing DMEM medium (hereinafter collectively referred to as "test medium") with final concentrations of 5 mM and 10 mM, respectively. ５ The above RAW264.7 cells were seeded at 0.1 mL per well in a 96-well plate at 1000 ng / ml and cultured overnight. The medium was removed, and 0.1 mL of each of the above test media, negative control DMEM medium, or positive control lipopolysaccharide (lipopolysaccharide from Escherichia coli O111:B4, Sigma-Aldrich; hereafter also referred to as "LPS")-containing DMEM medium was added to the 96-well plate and cultured for 24 hours. LPS was prepared at 2.5 mg / mL with sterile water and then diluted to 0.1 μg / mL with DMEM medium to prepare LPS-containing DMEM medium.

[0106] The media from each 96-well plate was then collected and transferred to a 96-well PCR plate, followed by centrifugation at 4000 g for 5 minutes. After centrifugation, the TNF-α concentration in the supernatant was measured using a TNF-α Mouse DuoSet Kit (model number DY410, manufactured by R&D Systems). Specifically, 800 ng / mL of Capture antibody (100 μL / well) was added to each well of the 96-well ELISA plate and incubated overnight at room temperature. The Capture antibody was then removed from each well, and the wells were washed three times with PBS-T (300 μL / well). Then, 100 μL of each of the supernatants obtained by the centrifugation procedure described above was added and incubated for 2 hours at room temperature. After washing each well three times with PBS-T (300 μL / well), 37.5 ng / mL detection antibody (100 μL / well) was added and incubated for 2 hours. After washing each well three times with PBS-T (300 μL / well), Streptavidin-HRP (a 40-fold dilution of the stock solution was added per well) was added and incubated for 20 minutes. After washing each well three times with PBS-T (300 μL / well), 50 μL of TMB (3,3',5,5'-tetramethylbenzidine) substrate was added and incubated for 30 minutes. Finally, 50 μL / well of 2N sulfuric acid was added to each well, and the absorbance was measured using a plate reader (450 nm, reference 600 nm). The TNF-α standard was diluted with dilution buffer to 2000 to 15.6 pg / mL before use. The measurement results of TNF-α concentration are shown in Tables 2 and 3 below and in Figure 1. The TNF-α concentrations listed in Tables 2 and 3 represent the average values ​​of n=3. In Table 3 and Figure 1, "vehicle" represents the negative control. Comparison of the TNF-α concentration of the negative control with other TNF-α concentrations was performed by Dunnett's test using GraphPad Prism 8.

[0107]

[0108]

[0109] As shown in Tables 2 and 3 and Figure 1, HMOs other than LNT did not promote or barely promoted TNF-α production, whereas 5 mM or 10 mM LNT significantly promoted TNF-α production. Therefore, it was demonstrated that LNT has the effect of stimulating immunity through the production of TNF-α. The promotion of TNF-α production by LNT is thought to be due to the activation of TLR4 signaling and the like by LNT. Incidentally, it has been reported that all LNT IIs activate Toll-like receptors (Non-Patent Document 3), and that 3'-SL, 6'-SL, and LNFP I activate TLR4 signaling (Non-Patent Documents 4 and 5). However, Tables 2 and 3 and FIG. 1 show that LNT has a superior effect of promoting TNF-α production compared to LNT II, ​​3'-SL, 6'-SL, and LNFP I, and therefore has a superior immunostimulatory effect.

[0110] Example 3: Measurement of Changes in TNF-α Production Amounts by Simultaneous Addition of LNT or LPS and Lac or HMO in Combination A cryovial containing mouse RAW264.7 cells (ECACC, model number 91062702) was partially thawed in a 37°C water bath and suspended in DMEM medium (10% by volume FBS, 1% by volume penicillin-streptomycin solution), followed by centrifugation at 800 g for 3 minutes. The supernatant was discarded, and fresh DMEM medium was added and the cells were seeded into a 100 mm dish. After culturing until 90% confluent, the medium was removed and 10 mL of DMEM medium was added. The cells were completely detached with a cell scraper, and 1 mL of cells was added to 10 mL of fresh DMEM medium, seeded into a 100 mm dish, and subcultured. After several passages, cells with a viability of 96-99% were used for the following procedure.

[0111] DMEM media containing LNT at final concentrations of 5 mM and 10 mM (hereinafter referred to as "5 mM LNT treatment medium" and "10 mM LNT treatment medium," respectively) and 5 mM lactose-containing DMEM media, as well as 10 types of 5 mM HMO-containing DMEM media other than LNT, were prepared in the same manner as in Example 2. A 2.5 mg / mL aqueous solution of LPS (lipopolysaccharide from Escherichia coli O111:B4, Sigma-Aldrich) was diluted with sterile water to 1.896 ng / mL, and then diluted to 0.948 ng / mL with DMEM medium (2x concentrated). This was used as the LPS-treated medium. Then, the 20 mM LNT treatment medium or the 1.896 ng / mL LPS treatment medium was mixed with 10 mM lactose-containing DMEM medium or DMEM medium containing 10 mM of each of the 10 types of HMOs other than LNT at a volume ratio of 1:1 to obtain a 10 mM LNT treatment medium and a 0.948 ng / mL LPS treatment medium containing 10 mM lactose or 10 mM of each HMO, respectively. ５ RAW264.7 cells (0.1 mL / well) were seeded into a 96-well plate at 0.1 mL per well and cultured overnight. The medium was removed, and 0.1 mL of 10 mM LNT-treated medium, LPS-treated medium, and 5 mM LNT-treated medium, each containing or not containing 10 mM lactose or 10 types of 10 mM HMOs other than LNT, were added and cultured for 24 hours. The above media without 10 mM lactose or 10 types of 10 mM HMOs other than LNT served as the control group. Additionally, 0.1 mL of DMEM medium alone was added as a vehicle for the control group and cultured for 24 hours.

[0112] The media for each treatment section was then collected from the 96-well plate, transferred to a 96-well PCR plate, and centrifuged at 4000 g for 5 minutes. After centrifugation, the TNF-α concentration in the supernatant was measured using a TNF-α Mouse DuoSet Kit (model number DY410, manufactured by R&D Systems). Specifically, 800 ng / mL of Capture antibody (100 μL / well) was added to each well of the 96-well ELISA plate and incubated overnight at room temperature. The Capture antibody was then removed from each well, and the wells were washed three times with PBS-T (300 μL / well). Then, 100 μL of each of the supernatants obtained by the centrifugation procedure described above was added and incubated for 2 hours at room temperature. Each well was then washed three times with PBS-T (300 μL / well), after which 37.5 ng / mL detection antibody (100 μL / well) was added and incubated for 2 hours. Each well was then washed three times with PBS-T (300 μL / well), and Streptavidin-HRP (a 1000-fold dilution of the stock solution was added per well) was added and incubated for 20 minutes. Each well was then washed three times with PBS-T (300 μL / well), and 100 μL of Immunostar LD (Fujifilm Wako Pure Chemical Industries, Ltd., 296-69901) was added, incubated for 5 minutes, and the absorbance was immediately measured using a plate reader (450 nm, reference 600 nm). The TNF-α standard was diluted with dilution buffer to 240-10 pg / mL before use. The measurement results of TNF-α concentration are shown in Tables 4 and 5 below, as well as Figures 2 and 3. The TNF-α concentrations listed in Tables 4 and 5 represent the average values ​​of n=3. In Table 5, Figures 2 and 3, "vehicle" represents the negative control. Comparison of the TNF-α concentration of the negative control with other TNF-α concentrations was performed by Dunnett's test using GraphPad Prism 8.

[0113]

[0114]

[0115] As shown in Tables 4 and 5 and Figures 2 and 3, the addition of LNT in combination with any of 2'-FL, 3'-SL, 6'-SL, LDFT, LNB, and LNnT significantly suppressed TNF-α production compared to the addition of LNT alone. This suggests that 2'-FL, 3'-SL, 6'-SL, LDFT, LNB, and LNnT can suppress the immunostimulatory activity of other oligosaccharides, particularly LNT. Furthermore, the addition of LPS in combination with LNFP I increased TNF-α production compared to the addition of LPS alone, and the addition of LPS in combination with 6'-SL significantly suppressed TNF-α production compared to the addition of LPS alone. The inhibitory or enhancing effects of various HMOs on the promoting effect (immunostimulation) of TNF-α production by LPS and LNT were different, suggesting that the mechanism of action of LNT to promote TNF-α production may be different from that of LPS.

[0116] Example 4 Measurement of changes in TNF-α production levels due to administration of LNT or LPS after administration of Lac or HMO Next, we investigated whether the immunostimulatory activity of LNT or LPS could be suppressed by each HMO by administering LNT or LPS after administration of Lac or each HMO. Similar to the method described in Example 3, a medium for an LNT treatment group, a medium for an LPS treatment group, and a DMEM medium containing 5 mM lactose or 10 types of DMEM medium containing 5 mM of each HMO other than LNT were prepared. 2 × 10 ５RAW264.7 cells (0.1 mL / well) were seeded into a 96-well plate and cultured overnight. The medium was removed, and 80 μL of 5 mM lactose-containing DMEM medium or 5 mM each of 10 HMOs other than LNT was added. After 10 minutes, 20 μL of 50 mM LNT-treated medium or 4.74 ng / mL LPS-treated medium containing 5 mM lactose or 10 HMOs other than LNT was added. The cells were then cultured for 24 hours. For the control group, 80 μL of 5 mM or 10 mM LNT-treated medium or 0.948 ng / mL LPS-treated medium was added after the medium was removed. After 10 minutes, 20 μL of 5 mM or 10 mM LNT-treated medium or 0.948 ng / mL LPS-treated medium was added and cultured for 24 hours. As a control vehicle, after removing the medium, 80 μL of DMEM medium alone was added, and after 10 minutes, 20 μL of DMEM medium alone was added again, followed by culturing for 24 hours.

[0117] The media from each 96-well plate was then collected and transferred to a 96-well PCR plate, followed by centrifugation at 4000 g for 5 minutes. After centrifugation, the TNF-α concentration in the supernatant was measured using a TNF-α Mouse DuoSet Kit (model number DY410, manufactured by R&D Systems) in the same manner as in Example 3. The results of the TNF-α concentration measurements are shown in Tables 6 and 7, as well as Figures 4 and 5. The TNF-α concentrations listed in Tables 6 and 7 represent the average values ​​of n=3. In Table 7, Figures 4 and 5, "vehicle" refers to the negative control. Comparison of the TNF-α concentration of the negative control with other TNF-α concentrations was performed using Dunnett's test using GraphPad Prism 8.

[0118]

[0119]

[0120] As shown in Tables 6 and 7 and Figures 4 and 5, when LNT was added after the addition of any of 2'-FL, 3'-SL, 6'-SL, LDFT, LNB, and LNnT, TNF-α production was significantly suppressed compared to when LNT was added alone. Taking into account the results of Example 3, it was shown that 2'-FL, 3'-SL, 6'-SL, LDFT, LNB, and LNnT have an inhibitory effect on the promotion of TNF-α production (immunostimulatory effect) by LNT, regardless of the timing of addition. Furthermore, when LPS was added after the addition of LNFP I, TNF-α production increased compared to when LPS was added alone, and when LPS was added after the addition of 6'-SL, TNF-α production was significantly suppressed compared to when LPS was added alone. Taking into account the results of Example 3, it was shown that LNFP I has an enhancing effect on the promotion of TNF-α production by LPS (immunostimulatory effect) regardless of the timing of addition, and 6'-SL has an inhibitory effect on the promotion of TNF-α production by LPS (immunostimulatory effect) regardless of the timing of addition.

Claims

1. An immunostimulant containing lacto-N-tetraose.

2. The immunostimulant described in claim 1, characterized in that it is used by being ingested or administered in combination with at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I.

3. The immunostimulant according to claim 1 or 2, which is based on enhancing the production of TNF-α.

4. An immunomodulator comprising lacto-N-tetraose and at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose and lactose-N-biose I.

5. An immunomodulator containing at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I, characterized in that it is ingested or administered in combination with lacto-N-tetraose.

6. The immunomodulator according to claim 4 or 5, wherein the lacto-N-tetraose stimulates the immune system, and the at least one human milk oligosaccharide selected from the group consisting of 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-neotetraose, lactodifucotetraose, and lactose-N-biose I suppresses the immune system stimulation caused by the lacto-N-tetraose.

7. A composition containing human milk oligosaccharides, wherein the composition contains less than 10 endotoxin units per mg of the human milk oligosaccharides.

8. The composition of claim 7, wherein the human milk oligosaccharide is lacto-N-tetraose.

9. The composition of claim 7 or 8, wherein the composition is a powder.

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