阅读说明：本技术 一种基于黑枸杞花青素提取物的酸指示剂及其应用 (Acid indicator based on lycium ruthenicum anthocyanin extract and application thereof ) 是由 鄢明辉 游春苹 刘振民 桂敏 扶晓菲 赵磊 于 2019-11-12 设计创作，主要内容包括：本发明公开了一种基于黑枸杞花青素提取物的酸指示剂及其应用,其制备方法为：(1)取黑果枸杞干果25-35g,经粉碎过筛后加入蒸馏水,室温下低速振荡提取2.5-3.5h,取出提取液,再次加入蒸馏水,在45-55℃低速振荡提取0.5-1.5h,取出提取液,将两次提取液合并后浓缩至1.3-1.4g/mL,即得花青素粗提液；(2)超滤、杀菌；(3)在杀菌后的花青素粗提液中加入无菌蒸馏水,洗去强极性组分后,洗脱,收集洗脱液,浓缩、干燥,得花青素提取物粉末,即为所述酸指示剂。本发明提供的酸指示剂能够在不同pH条件下呈现不同颜色,对酸较为敏感,肉眼可辨识度高,能表征出现有酸碱指示剂不能检测的微弱产酸。(The invention discloses an acid indicator based on an anthocyanin extract of lycium ruthenicum and application thereof, wherein the preparation method comprises the following steps: (1) taking 25-35g of lycium ruthenicum dry fruits, crushing and sieving the lycium ruthenicum dry fruits, adding distilled water, performing low-speed oscillation extraction at room temperature for 2.5-3.5h, taking out an extracting solution, adding distilled water again, performing low-speed oscillation extraction at 45-55 ℃ for 0.5-1.5h, taking out the extracting solution, combining the two extracting solutions, and concentrating to 1.3-1.4g/mL to obtain a crude anthocyanin extracting solution; (2) performing ultrafiltration and sterilization; (3) and adding sterile distilled water into the sterilized anthocyanin crude extract, washing away strong polar components, eluting, collecting eluent, concentrating and drying to obtain anthocyanin extract powder, namely the acid indicator. The acid indicator provided by the invention can present different colors under different pH conditions, is sensitive to acid, has high visual identification degree, and can represent weak acid production which cannot be detected by the existing acid-base indicator.)

1. An acid indicator based on lycium ruthenicum anthocyanin extract is characterized in that the preparation method comprises the following steps:

(1) preparing anthocyanin crude extract: taking 25-35g of lycium ruthenicum dry fruits, crushing and sieving the lycium ruthenicum dry fruits, adding distilled water, performing low-speed oscillation extraction at room temperature for 2.5-3.5h, taking out an extracting solution, adding distilled water again, performing low-speed oscillation extraction at 45-55 ℃ for 0.5-1.5h, taking out the extracting solution, combining the extracting solutions obtained in two times, and concentrating the extracting solution at 50-60 ℃ to the concentration of 1.3-1.4g/mL to obtain anthocyanin crude extracting solution;

(2) and (3) sterilization: performing ultrafiltration on the anthocyanin coarse extract by using a filter membrane and then sterilizing;

(3) and (3) purification: adding sterile distilled water into the sterilized anthocyanin crude extract, loading the anthocyanin crude extract to macroporous resin, washing off strong polar components, eluting by using an ethanol water solution with the volume concentration of 60-80%, collecting eluent, concentrating, freezing and drying to obtain anthocyanin extract powder, namely the acid indicator based on the lycium ruthenicum anthocyanin extract.

2. The lycium ruthenicum anthocyanin extract-based acid indicator according to claim 1, wherein the dried lycium ruthenicum in step (1) is produced from Qinghai-Tibet plateau.

3. The acid indicator based on the lycium ruthenicum anthocyanin extract in claim 1, wherein the rotation speed of the pulverizer in the pulverizing in the step (1) is 900-1100r/min, and the mesh number of the screen is 10-30 meshes.

4. The lycium ruthenicum anthocyanin extract-based acid indicator according to claim 1, wherein the rotation speed of the low-speed oscillation in the step (1) is 40-60 rpm;

and/or, the concentration in the step (1) adopts vacuum reduced pressure concentration.

5. The lycium ruthenicum anthocyanin extract-based acid indicator according to claim 1, wherein the pore size of the filter membrane in step (2) is 0.22 μm;

and/or the ozone sterilization conditions in the step (2) are as follows: the ozone amount is 3-5mg/m³The sterilization time is 20-40 min.

6. The lycium ruthenicum anthocyanin extract-based acid indicator according to claim 1, wherein in the step (3), sterile distilled water is added into the sterilized crude extract to 10mL, the mixture is loaded onto AB-8 macroporous resin, strong polar components are washed out by deionized water in 2 column volumes, and then the elution is carried out by using ethanol water solution with the volume concentration of 70%, wherein the elution conditions are as follows: pH 2.0, flow rate 2.0 mL/min.

7. The lycium ruthenicum anthocyanin extract-based acid indicator according to claim 1, wherein the lycium ruthenicum anthocyanin extract-based acid indicator is used by the method comprising the following steps:

dissolving the anthocyanin extract powder in water solution to make the concentration of the anthocyanin extract be 0.015-0.05% (w/v), and changing the color of the obtained acid indicator under different pH conditions into: red when pH <5.0, purple when 5.0< pH <7.0, blue when pH >7.0, and then a rapid change to yellow.

8. Use of the lycium ruthenicum anthocyanin extract-based acid indicator of claims 1-7 in acid-producing bacterial screening.

9. The use of an acid indicator based on an anthocyanin extract of lycium ruthenicum mill as claimed in claim 8, wherein the acid-producing bacteria species are one or more of lactic acid bacteria, acetic acid bacteria, propionibacterium, butyric acid bacteria.

Technical Field

The invention relates to an acid-base indicator, and particularly relates to an acid indicator based on an anthocyanin extract of lycium ruthenicum and application thereof.

Background

Traditional fermented foods have microorganisms that are health-beneficial to humans and are an important source of leavening agents in the modern food industry. The separation and screening of strains with excellent fermentation performance from sources such as traditional foods and the like is an important direction for the research of modern dairy leavening agents. Strains commonly used in modern food and industrial fermentation, such as lactic acid bacteria, acetic acid bacteria, propionibacterium, clostridium butyricum and the like, have the characteristic of acid production, and the screening and growth characteristic research of the strains by utilizing the characteristic of acid production is a common technical means in the research of food leavening agents at present. The traditional use method is to add calcium carbonate into a culture medium to indicate acid production of microorganisms, and the method has the main problems of low sensitivity to acid, difficult visual differentiation and no food-safe and available acid indicator. Novel acid-base indicators with natural and safe sources are developed by Jiangsu Quezuelian bioengineering technology Limited company and Beijing chemical university, for example, the invention patents with patent numbers ZL201310280878.0 and ZL201610343407.3 realize detection indication of acid, but the sensitivity of the acid indicator to the acid needs to be improved.

Disclosure of Invention

In view of the current situation, and in order to overcome the drawbacks of the prior art, it is an object of the present invention to provide an acid indicator capable of displaying different colors under different pH conditions, which is prepared based on a water-soluble anthocyanin extract from Lycium ruthenicum Murray (Lycium ruthenicum Murray), which is particularly sensitive to acids and which shows a distinct red color when the pH value drops below 4.5. Compared with the traditional calcium carbonate-based acid indicator, the extract is natural and safe, is more sensitive to acid and has high visual identification degree, and can represent weak acid production which cannot be detected by the existing acid-base indicator.

In order to achieve the purpose, the invention adopts the following technical scheme:

an acid indicator based on lycium ruthenicum anthocyanin extract is prepared by the following steps:

(1) preparing anthocyanin crude extract: taking 25-35g of lycium ruthenicum dry fruits, crushing and sieving the lycium ruthenicum dry fruits, adding distilled water, performing low-speed oscillation extraction at room temperature for 2.5-3.5h, taking out an extracting solution, adding distilled water again, performing low-speed oscillation extraction at the temperature of 45-55 ℃ for 0.5-1.5h while keeping the temperature, taking out the extracting solution, combining the extracting solutions obtained in two times, and concentrating the extracting solution at the temperature of 50-60 ℃ until the concentration is 1.3-1.4g/mL to obtain an anthocyanin crude extracting solution;

(2) and (3) sterilization: performing ultrafiltration on the anthocyanin coarse extract by using a filter membrane and then sterilizing;

(3) and (3) purification: adding sterile distilled water into the sterilized anthocyanin crude extract, loading the anthocyanin crude extract to macroporous resin, washing off strong polar components, eluting by using an ethanol water solution with the volume concentration of 60-80%, collecting eluent, concentrating, freezing and drying to obtain anthocyanin extract powder, namely the acid indicator based on the lycium ruthenicum anthocyanin extract.

The petunidin and the derivatives thereof (the main component is the petunidin) in the black medlar account for 95 percent of the total anthocyanin and the derivatives thereof, and the petunidin is more sensitive to acid than other kinds of anthocyanin, so that weak acid production is more easily represented; the dried lycium ruthenicum mill fruits are acidic or weakly acidic after being soaked in water, a large amount of procyanidine in the lycium ruthenicum mill fruits can be decomposed into monomer anthocyanin in the processes of soaking in water and extracting, the anthocyanin extracting efficiency can be effectively improved after two times of extraction, the temperature is properly increased in the second extraction, and experiments prove that the extracting efficiency is further improved.

Although different types of medlar contain anthocyanin, the types of the anthocyanin are different, and the proportion of the anthocyanin in the different types is also different, and experiments prove that the response range and the sensitivity of other common types of medlar extracts to acid are not as good as those of the lycium ruthenicum extract.

In the above acid indicator based on an anthocyanin extract of lycium ruthenicum mill, the dried lycium ruthenicum mill fruits in the step (1) are preferably produced from Qinghai-Tibet plateau, preferably Qinghai.

In the above acid indicator based on the lycium ruthenicum anthocyanin extract, the rotation speed of the pulverizer during pulverization in the step (1) is preferably 900-.

In the above acid indicator based on the lycium ruthenicum anthocyanin extract, the rotation speed of the low-speed oscillation in the step (1) is preferably 40-60rpm, and more preferably 50 rpm.

In the above acid indicator based on lycium ruthenicum anthocyanin extract, preferably, the concentration in step (1) is performed by vacuum reduced pressure concentration.

In the above-mentioned acid indicator based on the lycium ruthenicum anthocyanin extract, the pore size of the filter membrane in the step (2) is preferably 0.22 μm.

In the above acid indicator based on lycium ruthenicum anthocyanin extract, preferably, the ozone sterilization conditions in step (2) are as follows: the ozone amount is 3-5mg/m³Sterilizing for 20-40 min; further preferably, the ozone sterilization conditions are: the ozone amount was 4mg/m³The sterilization time is 30 min.

In the above acid indicator based on lycium ruthenicum anthocyanin extract, preferably, in step (3), sterile distilled water is added to the sterilized crude extract to 10mL, the sample is loaded to an AB-8 macroporous resin, after strong polar components are washed away by deionized water with 2 column volumes, the elution is carried out by using an ethanol aqueous solution, and the elution conditions are as follows: pH 2.0, flow rate 2.0 mL/min.

Preferably, the use method of the lycium ruthenicum anthocyanin extract-based acid indicator is as follows:

dissolving the anthocyanin extract powder in water solution to make the concentration of the anthocyanin extract be 0.015-0.05% (w/v), and changing the color of the obtained acid indicator under different pH conditions into: red when pH <5.0, purple when 5.0< pH <7.0, blue when pH >7.0, and then a rapid change to yellow.

When the concentration of the anthocyanin extract is 0.015-0.05% (w/v), the color change under different pH conditions is obvious and beautiful in sense, and when the concentration is out of the concentration range, the corresponding color can still be presented under different pH conditions, but the state in the sense of the concentration is not as good.

The invention also aims to provide the application of the acid indicator in acid-producing bacteria screening.

Preferably, the acid-producing bacteria is one or more of lactic acid bacteria, acetic acid bacteria, propionibacterium and butyric acid bacteria.

It should be noted that, unless otherwise specified, various reagents, raw materials and equipment used in the present invention can be commercially available.

The separation and screening of fermentation strains from traditional food and other sources is an important research direction in the food field at present, and documents and patent inventions in the prior art screen bacteria from the perspective of specific physiological functions (such as probiotic characteristics of acid resistance, cholesterol reduction, oxidation resistance and the like), and emphasize physiological functions, but are not concerned sufficiently about growth and fermentation characteristics, especially fermentation and acid production characteristics in specific food media, which may bring limitations to the later-stage industrial application of the strains.

Compared with the prior art, the invention has the beneficial effects that:

(1) the acid indicator provided by the invention can simply, conveniently, quickly and intuitively screen out bacteria with acid production characteristics, including weak acid production which cannot be detected by a conventional indicator, such as common fermentation strains in modern food and industrial fermentation, including lactic acid bacteria, acetic acid bacteria, propionibacterium, butyric acid bacteria and the like, and can greatly improve the screening efficiency of the fermentation strains, thereby promoting the development of a novel food leavening agent;

(2) the anthocyanin extract is extracted by pure water, the preparation process is simple, the use is convenient and intuitive, the extract is from Lycium ruthenicum Murray, different colors can be presented under different pH values, the extract is sensitive to acid, and obvious red can appear when the pH value is less than 4.5; compared with the traditional calcium carbonate-based acid indicator, the extract is natural and safe, is more sensitive to acid and has high degree of identification by naked eyes; compared with acid-base indicators developed by Jiangsu resistant sparrow bioengineering technology limited company, Beijing chemical university and the like, the extract is more sensitive to acid, and can represent weak acid production which cannot be detected by the indicator.

Drawings

FIG. 1 is a graph of the color change of an anthocyanin extract under different pH conditions;

FIG. 2 is a schematic diagram showing the change of the molecular structural formula of the anthocyanin extract under different pH conditions;

FIG. 3 is a graph showing color changes of lactic acid bacteria grown on a milk plate when screening lactic acid bacteria with anthocyanin extract.

Detailed Description

The present invention will now be described in more detail, with reference to the following, it being noted that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.