CN114196726B

CN114196726B - Detection method of probiotics in gelatin system

Info

Publication number: CN114196726B
Application number: CN202010979334.3A
Authority: CN
Inventors: 周钡华; 陈琼; 黄盼; 许伟沂
Original assignee: Sirio Pharma Co Ltd
Current assignee: Sirio Pharma Co Ltd
Priority date: 2020-09-17
Filing date: 2020-09-17
Publication date: 2023-10-13
Anticipated expiration: 2040-09-17
Also published as: CN114196726A

Abstract

The invention relates to a detection method of probiotics in a gelatin system. In particular, the invention relates to a method for detecting the content of bacillus coagulans in a bacillus coagulans-containing gelatin soft candy or increasing the detection rate of bacillus coagulans in a bacillus coagulans-containing gelatin soft candy, which comprises the step a) of dissolving the gelatin soft candy with a dissolving solution, characterized in that the dissolving solution comprises 0.01-3% by weight of a nonionic surfactant.

Description

Detection method of probiotics in gelatin system

Technical Field

The invention relates to the field of foods, in particular to a probiotic detection technology. In particular, the invention relates to a method for detecting probiotics in a gelatin system, in particular to a method for detecting the content of bacillus coagulans in gelatin soft sweets containing bacillus coagulans or increasing the detection rate of bacillus coagulans in gelatin soft sweets containing bacillus coagulans.

Background

Gel candies are also known as gummies and soft candies in the daily life of people. In the early stage, people mainly utilize starch biomass with higher amylose content or fruits with higher pectin content to directly produce soft sweets; with the development of technology, attempts have been made to extract substances with gel properties from natural food materials, such as carrageenan, agar, pectin, gelatin, starch, etc., to make soft sweets. At present, the gel candy is produced mainly by taking food gum, starch syrup and granulated sugar as main raw materials, adding functional components and adopting a specific production process. Compared with hard candy, soft candy has a higher water content, generally 15-20% of water content, so that the texture characteristics of soft candy are soft, and meanwhile, different colloid raw materials endow different elasticity and hardness characteristics, and the sugar content of soft candy is lower than that of hard candy, generally 50-80%. The gel soft sweet has the characteristics of elastic taste, long shelf life and the like.

The detection method for the bacillus coagulans content in the prior art generally comprises the steps of dissolving a sample, adjusting pH to be alkaline, heating at a high temperature, and diluting and inoculating. However, the prior art does not mention a method for detecting bacillus coagulans in a gelatin colloid system, and when a dissolution solution adopted in the prior art is applied to a gelatin system containing probiotics, the detection rate of the probiotics is found to be low. Thus, the prior art methods are not suitable for gelatin systems, and in particular for determining the bacillus coagulans content in soft candies.

Thus, there is a need in the art for methods of detecting probiotics in gelatin systems, and in particular for methods of detecting the content of bacillus coagulans in bacillus coagulans-containing gelatin fudge or increasing the detection rate of bacillus coagulans in bacillus coagulans-containing gelatin fudge.

Disclosure of Invention

The invention provides a method for detecting the content of bacillus coagulans in gelatin soft sweets, which is specially used for carrying out homogenization dissolution on the soft sweets by using the dissolving liquid in pretreatment. The invention provides a method for detecting gelatin soft sweets containing bacillus coagulans, which is easier to dissolve and has higher detection rate.

The first aspect of the present invention relates to a method for detecting the content of bacillus coagulans in a bacillus coagulans-containing gelatin gum or increasing the detection rate of bacillus coagulans in a bacillus coagulans-containing gelatin gum, said method comprising the step of a) dissolving the gelatin gum with a dissolving solution, characterized in that the dissolving solution comprises 0.01% -3% by weight of a nonionic surfactant.

In some embodiments, the nonionic surfactant is selected from one or more of emetic and span, optionally the tween is tween 20 or tween 80, and optionally the span is span 80. In some embodiments, the dissolution solution further comprises one or more selected from the group consisting of a sodium chloride solution, a phosphate buffer solution, and an aqueous peptone solution, optionally the sodium chloride solution is 0.6% -1.0% by weight sodium chloride solution, for example 0.85% by weight sodium chloride solution or 0.9% by weight sodium chloride solution; optionally the phosphate buffer solution is a phosphate buffer solution having a pH of 6.0-8.5, e.g. pH 7; and optionally the peptone aqueous solution is 0.01% -1% by weight peptone aqueous solution, e.g. 0.1% by weight peptone aqueous solution. In some embodiments, the dissolution solution comprises 0.05% to 0.35% by weight of a nonionic surfactant, optionally selected from tween 20, tween 80, span 80, and combinations thereof. In some embodiments, the dissolution solution comprises 0.05% to 0.35% by weight, e.g., 0.1% by weight, tween 80. In some embodiments, the dissolution liquid does not comprise an anionic surfactant.

In some embodiments, the method further comprises the steps of: b) Adjusting the pH of the solution obtained in step a) to alkaline; c) Incubating the solution obtained in step b) at an elevated temperature and then immediately cooling; d) Diluting the cooled solution and inoculating; and e) incubating the diluted solution and then counting. In some embodiments, the pH of the solution obtained in step a) is adjusted to 8.5±0.2 in step b). In some embodiments, the solution obtained in step b) is incubated for 30 minutes in a water bath at 75 ℃ in step c), then removed and immediately placed in ice water to cool to below 45 ℃. In some embodiments, the cooled solution is diluted in a 10-fold gradient in step d) and inoculated into a dish, the medium is poured in and shaken up. In some embodiments, in step e), after the medium has solidified, it is incubated upside down at 40.+ -. 2 ℃ for 48 hours, and then counted. In some embodiments, the medium is a glucose yeast extract medium.

A second aspect of the invention relates to the use of a dissolving solution as described in the first aspect for dissolving a gelatin gum containing bacillus coagulans to increase the detection rate of bacillus coagulans.

In a second aspect the invention relates to a kit for detecting bacillus coagulans in bacillus coagulans-containing gelatin soft candy, characterized in that the kit comprises a dissolution liquid as described in the first aspect.

Detailed Description

Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One of ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods.

The dissolving solution in the prior art is not suitable for dissolving gelatin soft sweets, and soft sweets cannot be completely dissolved in the dissolving solution in the prior art, so that bacillus coagulans cannot be completely dispersed, and the problems of lower detection rate, poor repeatability of detection results and the like are caused. The invention adopts the dissolving solution added with the nonionic surfactant such as Tween to promote the dissolution and dispersion of the gelatin soft candy in the solution, and provides a simple and effective detection method for the quality control of bacillus coagulans in the gelatin soft candy.

Definition of the definition

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the open-ended expressions "comprising" and "comprising" are to be interpreted to mean that structural components or method steps which are not mentioned can also be included, but it is to be noted that the open-ended expression also covers the case of only being composed of the components and method steps mentioned (i.e. the case of the closed-ended expression "composed of … …" is to be covered).

As used throughout, ranges are used as shorthand for describing each and every value that is within the range. Any numerical value, particularly integer values, within a range may be selected as the end of the range. For example, a range of 0.01-3% is used to describe all values within the range, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07% … … 2.5.5%, 2.6%, 2.7%, 2.8%, 2.9% and 3%, and includes all subranges, such as 0.01% -1%, 0.05% -0.15%, 1% -2%, 2% -3%, etc.

All parts and percentages used herein are by weight unless otherwise indicated.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Bacillus coagulans @Bacillus coagulans) Is a gram positive bacterium belonging to the genus Bacillus belonging to the phylum of the genus hard (or thick) wall fungus. In the present invention, the bacillus coagulans content in the gelatin gum tested is not limited and may be present in a reasonable additive amount of probiotics or in an amount allowed by regulatory authorities, for example in a therapeutically effective amount or in an amount effective to promote health. In one implementationIn embodiments, the gelatin gum may contain 0.00001% to 30% by weight, for example 0.01% to 20% by weight, 0.1% to 10% by weight, 1% to 8% by weight, and 3% to 8% by weight bacillus coagulans. In one embodiment, the gelatin gum may contain 1X 10 ⁶ -1×10 ¹² CFU/g Bacillus coagulans.

The gel soft candy takes one or more hydrophilic colloids as a main material and is added with food raw materials such as sucrose, starch and the like, and the gel soft candy is formed by heating and dissolving the gel soft candy to a certain concentration. The most common of the jelly is gelatin jelly. Compared with soft sweets such as pectin, carrageenan and the like, the gelatin soft sweets have soft and elastic matrix and unique taste; and gelatin is a hydrolysate of collagen, contains rich amino acids essential to human body, so gelatin matrix soft sweets are deeply favored by consumers. Gelatin fondants generally comprise gelatin, sugar alcohol and water, and may optionally comprise agar, acid, essence, pigment, fermentation broth, coating, probiotics, and the like. Those skilled in the art can routinely determine their amounts as desired.

In some embodiments, the gelatin gum may contain 30% -90% by weight, e.g., 55% -85% by weight, sweetener, 1% -20% by weight, e.g., 1% -10% by weight and 5% -8% by weight gelatin, 0.00001% -30% by weight, e.g., 1-10% by weight and 3-8% by weight probiotics, 0.1% -10% by weight, e.g., 2.5% -5% by weight acidulant, 0-3% by weight, e.g., 0.2% -1.5% by weight flavor, 0-3% by weight, e.g., 0-1% by weight color, 0-3% by weight, e.g., 0.2-1% by weight film coating, and/or 0-15% by weight, e.g., 0-6% by weight water. In some embodiments, the gelatin gum may also contain 0.1% to 1% by weight, e.g., 0.2% to 0.7% by weight, of other gelling agents such as pectin, agar, starch, modified starch, acacia, carrageenan, gellan, xanthan, sodium alginate and locust bean gum. In some embodiments, the sweetener is selected from one or more of the following: glucose syrup, fructose syrup, lactose, fructose, sorbitol, mannitol, maltose syrup, maltitol, isomalt, erythritol, xylitol, trehalose, fructo-oligosaccharides, polydextrose, inulin, white granulated sugar, glucose, and combinations thereof. In some embodiments, the gelatin gum may contain 30% -50% by weight glucose syrup, 25% -50% by weight white granulated sugar, and 1% -12% by weight erythritol. In some embodiments, the gelatin gum may contain 30% -50% by weight glucose syrup, 25% -50% by weight white granulated sugar, and 1% -12% by weight sorbitol. In some embodiments, the gelatin gum may contain 30% -50% by weight glucose syrup, 25% -50% by weight white granulated sugar, and 1% -12% by weight maltitol. In some embodiments, the acidulant is selected from one or more of the following: citric acid, malic acid, lactic acid, tartaric acid, fumaric acid, and edible salts thereof, and any combination thereof. In some embodiments, the gelatin soft candy of the present invention may optionally further comprise vitamins, minerals, antioxidants, amino acids, essential oils, herbs, and polyphenols, among other nutritional ingredients, as well as other additives such as candy flavors, processing aids, additional flavors, seasonings, colorants, preservatives, excipients, and the like, and combinations thereof.

The dissolution of the gelatin gum of the present invention comprises 0.01-3% by weight, for example 0.01% -1% by weight, 0.05% -0.35% by weight, 0.08% -0.15% by weight, 0.2% -0.4% by weight, 0.6% -0.8% by weight, 1% -2% by weight, 2% -3% by weight and any subrange therebetween of nonionic surfactant. In some embodiments, the dissolution solution comprises 0.01 wt%, 0.03 wt%, 0.05 wt%, 0.08 wt%, 0.1 wt%, 0.15 wt%, 0.2 wt%, 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt%, 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, or 3 wt% nonionic surfactant. In some embodiments, the nonionic surfactant is selected from one or more of emetic and span, optionally the tween is tween 20 or tween 80, and optionally the span is span 80. In some embodiments, the dissolution solution further comprises one or more selected from the group consisting of a sodium chloride solution, a phosphate buffer solution, and an aqueous peptone solution, optionally the sodium chloride solution is 0.6% -1.0% by weight sodium chloride solution, for example 0.85% by weight sodium chloride solution or 0.9% by weight sodium chloride solution; optionally the phosphate buffer solution is a phosphate buffer solution having a pH of 6.0-8.5, e.g. pH 6.5, 7, 7.5, 8 or 8.5; and optionally the peptone aqueous solution is 0.01-1% by weight peptone aqueous solution, e.g. 0.1% by weight peptone aqueous solution. In some embodiments, the dissolution liquid does not comprise an anionic surfactant. In some embodiments, the dissolution solution consists of a nonionic surfactant and one or more solutions selected from the group consisting of sodium chloride solution, phosphate buffer solution, and peptone aqueous solution.

Detection method

The detection method of the invention comprises the following basic steps:

a) Using the dissolving solution of the invention to dissolve gelatin soft sweets;

b) Adjusting the pH of the solution obtained in step a) to 8.5+/-0.2;

c) Incubating the solution obtained in step b) for 30 minutes in a water bath at 75 ℃, then taking out and immediately putting into ice water to cool to below 45 ℃;

d) Diluting the cooled solution in a gradient of 10 times, inoculating the diluted solution into a plate, pouring a culture medium, and shaking uniformly; and

e) After the solidification of the medium, the culture was inverted at 40.+ -. 2 ℃ for 48 hours, and then counting was performed.

In some embodiments, the gelatin gum may be pre-treated, e.g., divided into small pieces or homogenized, etc., prior to dissolution using the dissolution fluids of the present invention, to enhance dissolution.

Kit for detecting a substance in a sample

Kits of the invention may be prepared by methods conventional in the art. The kit may comprise materials or reagents (including the solubilizate of the invention) used in carrying out the method of the invention. The kit may include storage reagents (e.g., a solution in a suitable container, etc.) and/or support materials (e.g., buffers, instructions for performing the assay, etc.). For example, the kit may include one or more containers (e.g., test tubes) containing the respective reagents and/or support materials. The kit may also contain internal standards for quality control, positive and negative controls, and the like. The above examples are not to be construed as limiting the kits and their contents suitable for use in the present invention.

While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Many modifications to the disclosed embodiments may be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described embodiments.

Examples

Unless otherwise indicated, the materials used in the examples herein are commercially available, and the various specific experimental methods used to conduct the experiments are either routine in the art or are in accordance with the procedures and conditions suggested by the manufacturer and can be routinely determined by one of skill in the art as desired. Some materials and methods are detailed below.

EXAMPLE 1 improvement of Bacillus coagulans detection Rate in gelatin fondants containing Bacillus coagulans by the dissolution solution of the present invention

First, a gelatin soft candy containing 2.0 million CFU/g bacillus coagulans was taken, crushed into small pieces with a garlic press, then a sample was sampled with a sterile sampling spoon, and then dissolved by a solution having the composition shown in table 1, and after homogenization, completely dispersed in the solution. The samples were divided into 4 parts, 30g each, the first 3 parts were used for trial adjustment, adjusted to 8.5.+ -. 0.2, and the last 1 part were used for formal sample testing. After the preparation, the sample is subjected to a high-temperature water bath at 75 ℃ for 30 minutes, taken out and immediately placed into ice water for cooling to below 45 ℃. The cooled sample was diluted 10-fold in gradient and inoculated in a dish, the glucose yeast extract medium was poured in and shaken well. After the solidification of the medium, the culture was inverted at 40.+ -. 2 ℃ for 48 hours and counted. The detection rate of Bacillus coagulans by each solution is shown in Table 1.

TABLE 1

Annotation: the gelatin soft candy comprises: 3% of bacillus coagulans; gelatin 7%; agar 0.7%; 47.5% of glucose syrup; 25% of white granulated sugar; erythritol 12%; 3.8% of citric acid; and 1% of a coating agent.

As can be seen from table 1, compared with the dissolution solution without the nonionic surfactant, the dissolution solutions with the two nonionic surfactants (tween and span) added respectively have different degrees of significant improvement on the detection rate of bacillus coagulans. In addition, the addition of anionic surfactant (SDS) to the dissolution results in a detection rate of only 19%, indicating that this type of surfactant is not suitable for gelatin systems.

It can also be seen from table 1 that the detection rate of bacillus coagulans in the gelatin system is improved to different degrees by the nonionic surfactant in the concentration range of 0.01% -3%, which indicates that the nonionic surfactant is suitable for detecting bacillus coagulans in the gelatin system in the concentration range.

EXAMPLE 2 detection Rate of Bacillus coagulans in different dosage forms by the dissolution solution of the present invention

The dosage forms described in table 2 below were tested in the same procedure as in example 1, but using the dissolution solutions shown in table 2 below. The results are shown in Table 2.

TABLE 2

As can be seen from Table 2, in the gelatin soft candy containing Bacillus coagulans, the detection rate of the gelatin soft candy is unexpectedly found to be remarkably improved by 96% by adopting the solution added with Tween 80. In the same other dosage form containing Bacillus coagulans, however, the detection rate of Bacillus coagulans was found to be low when different dosage form products were dissolved separately using the same dissolution liquid. It is explained that the dissolution rate of the invention can not be significantly improved when the dissolution liquid is applied to all dosage forms, and the dissolution liquid of the invention is only applicable to gelatin systems.

Example 3 Effect of adding nonionic surfactant at different stages on detection Rate

In the same manner as in example 1, but with the addition of the nonionic surfactant to the solution or the medium, respectively, gelatin fondants containing 2.0 hundred million CFU/g Bacillus coagulans were examined. The results are shown in Table 3 below.

TABLE 3 Table 3

As can be seen from table 3, when tween 80 was applied to the culture medium, no nonionic surfactant was added to the pretreated solution and diluent, and no significant improvement in the detection rate was found; when the surfactant is applied to the pretreated solution (dissolution solution and dilution solution), the detection rate of bacillus coagulans is greatly improved, which indicates that the onset stage of the nonionic surfactant is in the pretreatment process.

Example 4 detection Rate of the solution of the present invention for different species

The gelatin gum was tested in the same manner as in example 1, except that the gelatin gum contained 2.0 hundred million CFU/g of Bacillus coagulans or 2.0 hundred million CFU/g of Bacillus subtilis. The results are shown in Table 4 below.

TABLE 4 Table 4

As can be seen from Table 4, the 0.1% peptone aqueous solution added with 0.1% Tween 80 was applied as a dissolution solution to the gelatin soft candy containing Bacillus subtilis, and the detection rate was found to be not improved, but the detection rate of Bacillus coagulans in the gelatin soft candy containing Bacillus coagulans was significantly improved, which indicates that the dissolution solution of the present invention was suitable for the gelatin soft candy containing Bacillus coagulans, and the detection rate of the gelatin soft candy containing Bacillus subtilis was not improved.

Example 5 detection Rate of different dissolution Components

A gelatin soft candy containing 2.0 hundred million CFU/g Bacillus coagulans was examined in the same manner as in example 1 except that the dissolution liquid shown in Table 5 below was used. The results are shown in Table 5.

TABLE 5

As can be seen from table 5, the addition of the nonionic surfactant (tween 80) to the dissolution solution commonly used in the art still resulted in a very high detection rate of bacillus coagulans in gelatin soft candy, indicating that the nonionic surfactant has a significant promoting effect and is very suitable for detection of bacillus coagulans in gelatin soft candy containing bacillus coagulans.

Claims

1. A method for detecting the content of bacillus coagulans in a bacillus coagulans-containing gelatin gum or increasing the detection rate of bacillus coagulans in a bacillus coagulans-containing gelatin gum, the method comprising the steps of a) dissolving the gelatin gum with a dissolving solution, characterized by: the dissolution liquid comprises 0.01% -3% by weight of nonionic surfactant; the nonionic surfactant is at least one selected from Tween 20, tween 80 and span 80; the dissolution solution further comprises at least one of a sodium chloride solution, a phosphate buffer solution and an aqueous peptone solution.

2. The method of claim 1, wherein: the sodium chloride solution is 0.6-1.0% of sodium chloride solution by weight; the peptone water solution is 0.01% -1% of peptone water solution by weight.

3. The method of claim 2, wherein: the sodium chloride solution is 0.85% sodium chloride solution or 0.9% sodium chloride solution by weight; the peptone aqueous solution was 0.1% by weight peptone aqueous solution.

4. The method of claim 1, wherein: the dissolution liquid contains 0.05-0.35% by weight of nonionic surfactant.

5. The method of claim 1, wherein: the dissolution solution contains 0.05% -0.35% by weight of tween 80.

6. The method of claim 5, wherein: the solution contained 0.1% by weight of tween 80.

7. The method of claim 1, wherein: the dissolution liquid does not contain an anionic surfactant.

8. The method of claim 1, wherein: the method further comprises the steps of: b) Adjusting the pH of the solution obtained in step a) to alkaline; c) Incubating the solution obtained in step b) at high temperature and then immediately cooling; d) Diluting the cooled solution and inoculating; and e) incubating the diluted solution; and then counting.

9. The method as recited in claim 8, wherein: the method further comprises the steps of: b) Adjusting the pH of the solution obtained in step a) to 8.5+/-0.2; c) Incubating the solution obtained in step b) for 30 minutes in a water bath at 75 ℃, then taking out and immediately putting into ice water to cool to below 45 ℃; d) Diluting the cooled solution in a gradient of 10 times, inoculating the diluted solution into a plate, pouring a culture medium, and shaking uniformly; and e) after the culture medium is solidified, reversely culturing for 48 hours at 40+/-2 ℃; and then counting.

10. The method of claim 9, wherein: the culture medium is a glucose yeast extract culture medium.

11. Use of the dissolution solution of any one of claims 1-7 to dissolve a gelatin gum containing bacillus coagulans to increase the detection rate of bacillus coagulans.