Detection method for fermentation of 2 '-fucosyllactose and extraction of central control sample

Abstract

The invention discloses a detection method for fermentation and extraction of a central control sample of 2 '-fucosyllactose, belongs to the technical field of 2'-fucosyllactose, and aims to solve the problems that the fermentation period of the 2 '-fucosyllactose is basically longer than 50 hours, most detection methods in the fermentation process adopt amino columns and hilic chromatographic columns, the amino columns are short in service life and high in cost, and the detection cost is low. The method solves the problem that the cost is further increased due to the fact that the two chromatographic columns both adopt high-proportion acetonitrile as a mobile phase, and comprises the following steps: S1, weighing glucose, lactose and 2 '-fucosyllactose standard substances in a volumetric flask, and adding pure water to a constant volume to prepare a standard working solution; according to the method, glucose, lactose and 2 '-fucosyllactose can be accurately detected, the mobile phase is an aqueous solution, the cost is very low, meanwhile, the service life of the Aminex HPX-87H chromatographic column is long, the repeatability of hundreds of tests can be kept, and data change and purchase cost caused by frequent replacement of the chromatographic column are avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of 2'-fucosyllactose, and in particular relates to a method for detecting a 2'-fucosyllactose fermentation and extraction intermediate control sample. Background Art

[0002] The 2'-fucosyllactose fermentation process uses lactose and glucose as substrates, and both are in a flow-addition mode, so process control requires real-time monitoring of the content of glucose, lactose and 2'-fucosyllactose. There are many extraction process flows, and each process needs to detect the content of lactose, glucose and 2'-fucosyllactose to determine the next step. In view of the above problems, it is necessary to establish a high-accuracy, easy-to-operate, and low-cost in-process control detection method. At present, the fermentation cycle of 2'-fucosyllactose is basically greater than 50 hours. Most of the detection methods in the fermentation process use amino columns and hilic columns, among which amino columns have a short life and high cost. The above two chromatographic columns use a high proportion of acetonitrile as the mobile phase, which further increases the cost. For this reason, we propose a detection method for 2'-fucosyllactose fermentation and extraction of in-process control samples. Summary of the invention

[0003] The object of the present invention is to provide a method for detecting 2'-fucosyllactose fermentation and extraction of intermediate control samples to solve the problems raised in the above background technology.

[0004] To achieve the above object, the present invention provides the following technical solution: a method for detecting 2'-fucosyllactose fermentation and extraction of intermediate control samples, comprising the following steps:

[0005] Step S1: weigh glucose, lactose and 2'-fucosyllactose standards into a volumetric flask, add pure water to make up to volume, and prepare a standard working solution;

[0006] Step S2: The standard working solution in step S1 is taken, diluted with pure water to form a mass concentration series standard solution, and tested by high performance liquid chromatography, and the peak area is recorded. A standard curve is drawn with the mass concentration of glucose, lactose and 2'-fucosyllactose as the abscissa and the corresponding peak area as the ordinate;

[0007] Step S3: Take a fermentation or extraction control sample, centrifuge it, dilute the supernatant, shake it well, and filter it to obtain a sample solution;

[0008] Step S4: The sample solution obtained in step S3 is tested by liquid chromatography, and the concentrations of glucose, lactose and 2'-fucosyllactose contained in the sample are calculated according to the standard curve in step S2.

[0009] It should be noted in the scheme that the mass concentrations of the glucose, lactose and 2'-fucosyllactose standards in step S1 are standard working solutions of 3-4.5 g / L, 2.5-4 g / L and 5-7 g / L respectively.

[0010] It is further worth noting that the filtration in step S3 is performed using a filter membrane.

[0011] It should be further explained that the pore size of the filter membrane is 0.22 μm.

[0012] As a preferred embodiment, the flow rate in the liquid chromatograph is 0.6 ml / min.

[0013] As a preferred embodiment, the column temperature in the liquid chromatograph is 60°C.

[0014] As a preferred embodiment, the injection volume in the liquid chromatograph is 20 μL.

[0015] Compared with the prior art, the detection method of 2'-fucosyllactose fermentation and extraction of intermediate control samples provided by the present invention has at least the following beneficial effects:

[0016] The present invention can accurately detect glucose, lactose and 2'-fucosyllactose, the mobile phase is an aqueous solution, and the cost is very low. At the same time, the Aminex HPX-87H chromatographic column has a long service life and can maintain the repeatability of hundreds of tests, avoiding data changes and procurement costs caused by frequent replacement of chromatographic columns. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the standard solution detection diagram of the present invention;

[0018] Figure 2 It is the detection diagram of the extracted control product of the present invention;

[0019] Figure 3 This is a detection diagram of the fermentation in-process control product of the present invention. DETAILED DESCRIPTION

[0020] The following is a detailed description of a method for detecting 2'-fucosyllactose fermentation and extraction of intermediate control samples provided by the present invention in conjunction with the accompanying drawings and specific embodiments. At the same time, it is explained here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments, and those skilled in the art may also adopt other alternative methods to implement some known technologies; and the accompanying drawings are only for a more specific description of the embodiments, and are not intended to specifically limit the present invention.

[0021] It should be noted that the references to "one embodiment", "an embodiment", "an exemplary embodiment", "some embodiments" and the like in the specification indicate that the embodiments described may include specific features, structures or characteristics, but not every embodiment may include the specific features, structures or characteristics. In addition, when a specific feature, structure or characteristic is described in conjunction with an embodiment, it should be within the knowledge of a person skilled in the art to implement such feature, structure or characteristic in conjunction with other embodiments (whether or not explicitly described).

[0022] In general, a term can be understood, at least in part, from its use in context. For example, depending, at least in part, on the context, the term "one or more" as used herein can be used to describe any feature, structure, or characteristic in the singular sense, or can be used to describe a combination of features, structures, or characteristics in the plural sense. Additionally, the term "based on" can be understood as not necessarily intended to convey an exclusive set of factors, but can instead, depending, at least in part, on the context, allow for the presence of other factors that are not necessarily explicitly described.

[0023] It will be understood that the meanings of “on,” “over,” and “above” in the present invention should be interpreted in the broadest manner, so that “on” not only means “directly on” something, but also includes the meaning of being “on” something with intervening features or layers therebetween, and “on” or “over” not only means “on” or “above” something, but also includes the meaning of being “on” or “above” something with no intervening features or layers therebetween.

[0024] In addition, spatially relative terms such as "under," "beneath," "lower," "above," "upper," etc. may be used herein for descriptive convenience to describe the relationship of one element or feature to another element or features, as shown in the accompanying drawings. The spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the accompanying drawings. The device may be oriented in other ways, and the spatially relative descriptors used herein may be similarly interpreted accordingly.

[0025] The following examples are used to illustrate the present invention, but they cannot be used to limit the scope of protection of the present invention. The conditions in the examples can be further adjusted according to specific conditions. Simple improvements to the method of the present invention under the premise of the concept of the present invention belong to the scope of protection claimed in the present invention.

[0026] See also Figure 1-3 The present invention provides a method for detecting 2'-fucosyllactose fermentation and extraction of intermediate control samples, comprising the following steps:

[0027] Step S1: weigh glucose, lactose and 2'-fucosyllactose standards into a volumetric flask, add pure water to make up to volume, and prepare a standard working solution;

[0028] Step S2: The standard working solution in step S1 is taken, diluted with pure water to form a mass concentration series standard solution, and tested by high performance liquid chromatography, and the peak area is recorded. A standard curve is drawn with the mass concentration of glucose, lactose and 2'-fucosyllactose as the abscissa and the corresponding peak area as the ordinate;

[0029] Step S3: Take a fermentation or extraction control sample, centrifuge it, dilute the supernatant, shake it well, and filter it to obtain a sample solution;

[0030] Step S4: The sample solution obtained in step S3 is tested by high performance liquid chromatography, and the concentrations of glucose, lactose and 2'-fucosyllactose contained in the sample are calculated according to the standard curve in step S2.

[0031] In step S1, the mass concentrations of the glucose, lactose and 2'-fucosyllactose standards are 3-4.5 g / L, 2.5-4 g / L and 5-7 g / L standard working solutions, respectively.

[0032] In step S2, pure water is used to dilute the solution into a mass concentration series standard solution. The specific concentrations are shown in Table 1

[0033] STD1 STD2 STD3 STD4 STD5 STD6 STD7 glucose 3.5750 2.9792 2.3833 1.7875 1.1917 0.5958 0.2979 lactose 3.2154 2.6795 2.1436 1.6077 1.0718 0.5359 0.2679 2'-fucosyllactose 6.1030 5.0858 4.0732 3.0515 2.0343 1.0172 0.5086

[0034] Table 1

[0035] The conditions for HPLC were:

[0036] Column: Bio-Rad Aminex HPX–87H;

[0037] Mobile phase: 0.005 M aqueous sulfuric acid;

[0038] Flow rate: 0.6ml / min;

[0039] Column temperature: 60°C;

[0040] Detector: differential refractometer (40°C);

[0041] Injection volume: 20 μL

[0042] In step S3, the filtration is performed using a filter membrane, wherein the pore size of the filter membrane is 0.22 μm.

[0043] Unless otherwise defined, the technical or scientific terms used in the present invention shall have the usual meanings understood by persons with ordinary skills in the field to which the present invention belongs. The words "including" or "comprising" and the like used in the present invention mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, but do not exclude other elements or objects. The words "connect" or "connected" and the like are not limited to physical or mechanical connections, but may also include electrical connections, whether direct or indirect. "Up", "down", "left", "right", etc. are only used to indicate relative position relationships. When the absolute position of the object being described changes, the relative position relationship may also change accordingly.

[0044] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting 2'-fucosyllactose fermentation and extraction of intermediate control samples, characterized in that: The following steps are involved: Step S1: Weigh glucose, lactose and 2'-fucosyllactose standards into a volumetric flask, add pure water to make up to volume, and prepare a standard working solution; Step S2: The standard working solution in step S1 is taken, diluted with pure water to form a mass concentration series standard solution, and tested by high performance liquid chromatography, and the peak area is recorded. A standard curve is drawn with the mass concentration of glucose, lactose and 2'-fucosyllactose as the abscissa and the corresponding peak area as the ordinate; Step S3: Take a fermentation or extraction control sample, centrifuge it, dilute the supernatant, shake it well, and filter it to obtain a sample solution; Step S4: The sample solution obtained in step S3 is tested by liquid chromatography, and the concentrations of glucose, lactose and 2'-fucosyllactose contained in the sample are calculated according to the standard curve in step S2.

2. A method for detecting 2'-fucosyllactose fermentation and extraction of intermediate control samples according to claim 1, characterized in that: In the step S1, the mass concentrations of the glucose, lactose and 2'-fucosyllactose standards are 3-4.5 g / L, 2.5-4 g / L and 5-7 g / L, respectively, in the standard working solutions.

3. A method for detecting 2'-fucosyllactose fermentation and extraction of intermediate control samples according to claim 1, characterized in that: In the step S3, the filtration is performed using a filter membrane.

4. A method for detecting 2'-fucosyllactose fermentation and extraction of intermediate control samples according to claim 3, characterized in that: The pore size of the filter membrane is 0.22 μm.

5. A method for detecting 2'-fucosyllactose fermentation and extraction of intermediate control samples according to claim 1, characterized in that: The flow rate in the liquid chromatograph was 0.6 ml / min.

6. A method for detecting 2'-fucosyllactose fermentation and extraction of intermediate control samples according to claim 1, characterized in that: The column temperature in the liquid chromatograph is 60°C.

7. A method for detecting 2'-fucosyllactose fermentation and extraction of intermediate control samples according to claim 1, characterized in that: The injection volume in the liquid chromatograph was 20 μL.

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