DESCRIPTION
การดูดซับแบบ เอ็กซแปนเดดเบด METHODS FOR ISOLATION OF BASIC
MILK PROTEINS ที่รวมถึง แลคโตเฟอร์ริน
TECHNICAL FIELD
[0001] The การเปิดเผยนี้ relates to improved processes for the isolation of basic milk proteins (e.g., proteins that have an isoelectric point @I) value higher than 7.0) or peptides, such as แลคโตเฟอร์ริน, using การดูดซับแบบ เอ็กซแปนเดดเบด
chromatography.
BACKGROUND ART
[0002] Nutritional compositions for infants generally seek to mimic the
composition and function of human breast milk. แลคโตเฟอร์ริน is an 80-kDa member of the transferrin family of iron-binding glycoproteins, and is one of the primary proteins in human milk. แลคโตเฟอร์ริน has the capacity to reversibly bind two iron cations, even at low pH, and can facilitate iron uptake in the human intestine.
Functionally, แลคโตเฟอร์ริน regulates iron absorption and can bind iron based free radicals as well as donate iron for an immunological response. Additionally,
แลคโตเฟอร์ริน exhibits antibacterial activity. Thus, it would useful to include
แลคโตเฟอร์ริน in commercial infant formulas.
[0003] However, the use of แลคโตเฟอร์ริน in infant formulas has been limited by the lack of large-scale, commercially useful sources. Bovine แลคโตเฟอร์ริน, which has about 69% sequence homology to human แลคโตเฟอร์ริน, can be obtained from cow's milk by a variety of purification methods, but such processes are inefficient for
widespread commercial use. Other non-human แลคโตเฟอร์รินs include porcine
แลคโตเฟอร์ริน, equine แลคโตเฟอร์ริน, buffalo แลคโตเฟอร์ริน, goat แลคโตเฟอร์ริน, murine
แลคโตเฟอร์ริน, and camel แลคโตเฟอร์ริน, but similar production inefficiencies exist.
[0004] The concentration of แลคโตเฟอร์ริน in bovine whey, for example, is
generally only about 10 to 100 mg/liter. Stabilized fluid bed adsorption, also called การดูดซับแบบ เอ็กซแปนเดดเบด (EBA) is one process for obtaining proteins from a raw material, such as bovine แลคโตเฟอร์ริน from cow's milk sources. EBA enables the
isolation of biomolecules, such as proteins and plasmids, directly from a crude feed stock using a particulate chromatographic matrix, in which the matrix remains fluidized during loading of the feed stock and optionally during elution. Because
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the matrix is fluidized during loading, solids and undesired materials pass through the matrix, which avoids fouling of the chromatographic material. Thus, EBA can, in certain circumstances, combine the effects of centrifugation, filtration,
concentration and purification into a single process, thereby saving time and
minimizing pre-processing purifying steps. However, current EBA technology has several disadvantages in isolating แลคโตเฟอร์ริน from milk sources. In particular,
current EBA technology still uses a relatively large volume of liquid and also uses a high concentration of sodium hydroxide (e.g. 200 mM) during elution of the
แลคโตเฟอร์ริน, which may cause irreversible structural changes due to denaturing of the protein.
[0005] Additionally, non-human mammalian milk sources contain other
useful proteins, ที่รวมถึง lactoperoxidase, a-lactalbumin, 13-lactoglobulin,
immunoglobulins, glycopeptides, glycomacropeptide, whey protein isolates, and
lysozyme. These proteins and mixtures also may useful as food ingredients or
nutritional supplements. Thus, there is a need for improved processes for isolating and purifying แลคโตเฟอร์ริน and other proteins from milk sources on a commercial scale for use in nutritional products, such as infant formulas.
DISCLOSURE OF THE INVENTION
[0006] It is to be understood that both the foregoing general description and the following detailed description present รูปลักษณะ of the disclosure and are intended to provide an overview or framework for understanding the nature and character of the disclosure as it is claimed. The description serves to explain the principles and operations of the claimed subject matter. Other and further features and advantages of the การเปิดเผยนี้ will be readily apparent to those skilled in the art upon a reading of the following disclosure.
[0007] The การเปิดเผยนี้ provides improved EBA processes for isolating proteins from milk sources. In particular รูปลักษณะ, the การเปิดเผยนี้
provides a process for isolating a milk protein, such as แลคโตเฟอร์ริน, from a milk
source ซึ่งประกอบรวมด้วย establishing an การดูดซับแบบ เอ็กซแปนเดดเบด column ซึ่งประกอบรวมด้วย a particulate matrix, applying a milk source to the matrix, and eluting the แลคโตเฟอร์ริน from the matrix with an elution buffer ซึ่งประกอบรวมด้วย about 0.3 to about 2.0 M sodium chloride. Any mammalian milk source may be used in the present processes,
although in particular รูปลักษณะ, the milk source is a bovine milk source. The
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milk source ประกอบรวมด้วย, in some รูปลักษณะ, whole milk, reduced fat milk, skim milk, whey, casein, or mixtures thereof.
BEST MODE FOR CARRYING OUT THE INVENTION
[0008] Reference now will be made in detail to the รูปลักษณะ of the
การเปิดเผยนี้, one or more examples of which are set forth herein below. Each example is provided by way of explanation of the nutritional composition of the
การเปิดเผยนี้ and is not a limitation. In fact, it will be apparent to those
skilled in the art that various modifications and variations can be made to the
teachings of the การเปิดเผยนี้ without departing from the scope or spirit of the disclosure. For instance, features illustrated or described as part of one
รูปลักษณะ, can be used with another รูปลักษณะ to yield a still further
รูปลักษณะ.
[0009] Thus, it is intended that the การเปิดเผยนี้ covers such
modifications and variations as come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the การเปิดเผยนี้ are disclosed in or are apparent from the following detailed description. It is to be
understood by one of ordinary skill in the art that the present discussion is a
description of exemplary รูปลักษณะ only and is not intended as limiting the
broader aspects of the การเปิดเผยนี้.
[0010] All references to singular characteristics or limitations of the การเปิดเผยนี้ shall include the corresponding plural characteristic or limitation, and vice versa, unless otherwise specified or clearly implied to the contrary by the
context in which the reference is made.
[0011] All combinations of method or process steps as used herein can be
performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.
[0012] The methods of the การเปิดเผยนี้, ที่รวมถึง components thereof, can ประกอบรวมด้วย, consist of, or consist essentially of the essential elements and
limitations of the รูปลักษณะ described herein, as well as any additional or
optional steps.
[0013] As used herein, the term "about" should be construed to refer to both of the numbers specified in any range. Any reference to a range should be
considered as providing support for any subset within that range.
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(0014] The การเปิดเผยนี้ relates to improved processes for purifying
proteins from a raw material, such as a mammalian milk source, using EBA. The milk source can be, in certain รูปลักษณะ, whole milk, skim milk, reduced fat milk, or a product derived from milk, such as whey or casein. While the milk
source can be any mammalian milk source, in particular รูปลักษณะ, it is a
bovine milk source, and more particularly, bovine whey. In particular
รูปลักษณะ, the target protein is แลคโตเฟอร์ริน, though other milk proteins, such as lactoperoxidases or lactalbumins, also may be isolated. EBA is particularly
suitable for handling large volumes of crude feed material and is therefore useful for producing commercially useful amounts of purified milk proteins. In some
รูปลักษณะ, the process ประกอบรวมด้วย the steps of establishing an การดูดซับแบบ เอ็กซแปนเดดเบด column ซึ่งประกอบรวมด้วย a particulate matrix, applying a milk source to the matrix, and eluting the แลคโตเฟอร์ริน from the matrix with about 0.3 to about 2.0M sodium chloride. In other รูปลักษณะ, the แลคโตเฟอร์ริน is eluted with about 0.5 to about 1.0 M sodium chloride, while in further รูปลักษณะ, the แลคโตเฟอร์ริน is
eluted with about 0.7 to about 0.9 M sodium chloride.
[0015] The การดูดซับแบบ เอ็กซแปนเดดเบด column can be any known in the art,
such as those described in U.S. Patent Nos. 7,812,138, 6,620,326, and 6,977,046. In some รูปลักษณะ, the column is be equipped with at least one inlet and at least one outlet, and can be operated in both an expanded mode and a packed mode.
More particularly, in certain รูปลักษณะ, a milk source is applied to the column in an expanded mode, and the elution is performed in either expanded or packed mode. In particular รูปลักษณะ, the elution is performed in an expanded mode. For example, the expansion ratio in the expanded mode may be about 1 to about 3, or about 1.3 to about 1.7. EBA technology is further described in international
published applicatio