CN110793963B - Acid indicator based on lycium ruthenicum anthocyanin extract and application thereof - Google Patents
Acid indicator based on lycium ruthenicum anthocyanin extract and application thereof Download PDFInfo
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Abstract
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.
Description
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.
Example 1 preparation of an anthocyanin extract of Lycium ruthenicum Murr
The preparation method of the lycium ruthenicum anthocyanin extract-based acid indicator provided by the embodiment comprises the following steps:
(1) preparing anthocyanin crude extract: taking 30g of dry lycium ruthenicum (purchased from Qinghai Nuo blue wolfberry), crushing by a crusher at 1000r/min, sieving by a 20-mesh sieve, adding 5mL (namely 0.5-time column volume) of distilled water, performing low-speed (50rpm) oscillation extraction at room temperature (25 ℃) for 3h, taking out an extracting solution, adding distilled water again (100 mL each time in two times), performing heat preservation, performing low-speed (50rpm) oscillation extraction at 50 ℃ for 1h, taking out the extracting solution, combining the extracting solutions obtained in two times, and performing vacuum concentration at 50 ℃ until the concentration is 1.3g/mL to obtain a crude anthocyanin extracting solution;
(2) and (3) sterilization: performing ozone sterilization on the anthocyanin crude extract after ultrafiltration by a filter membrane of 0.22 mu m, wherein the sterilization conditions are as follows: the ozone amount was 4mg/m3Sterilizing for 30 min;
(3) and (3) purification: adding sterile distilled water to 10mL of the sterilized anthocyanin crude extract, loading the anthocyanin crude extract to AB-8 macroporous resin (5 multiplied by 30cm), washing out strong polar components (mainly inorganic salt and other impurities) by deionized water with 2 times of column volume, and then eluting the anthocyanin components by ethanol water solution with volume concentration of 70%, wherein the elution conditions are as follows: collecting eluate at pH of 2.0 and flow rate of 2.0mL/min, vacuum concentrating under reduced pressure, and freeze drying to obtain anthocyanin extract powder, i.e. the acid indicator based on Lycium ruthenicum anthocyanin extract.
Example 2
The preparation method of the lycium ruthenicum anthocyanin extract-based acid indicator provided by the embodiment comprises the following steps:
(1) preparing anthocyanin crude extract: taking 25g of dry lycium ruthenicum (purchased from Qinghai Nuo blue wolfberry), crushing by a crusher at 1100r/min, sieving by a 10-mesh sieve, adding 5mL (namely 0.5-time column volume) of distilled water, performing low-speed (40rpm) oscillation extraction at room temperature (25 ℃) for 2.5h, taking out an extracting solution, adding distilled water again (100 mL each time in two times), performing heat preservation at 45 ℃ and low-speed (40rpm) oscillation extraction for 0.5h, taking out the extracting solution, combining the extracting solutions obtained in two times, and performing vacuum concentration at 60 ℃ until the concentration is 1.4g/mL to obtain a crude anthocyanin extracting solution;
(2) and (3) sterilization: performing ozone sterilization on the anthocyanin crude extract after ultrafiltration by a filter membrane of 0.22 mu m, wherein the sterilization conditions are as follows: an amount of ozone of3mg/m3Sterilizing for 40 min;
(3) and (3) purification: adding sterile distilled water to 10mL of the sterilized anthocyanin crude extract, loading the anthocyanin crude extract to AB-8 macroporous resin (5 multiplied by 30cm), washing out strong polar components (mainly inorganic salt and other impurities) by deionized water with 2 times of column volume, and then eluting the anthocyanin components by an ethanol water solution with the volume concentration of 60%, wherein the elution conditions are as follows: collecting eluate at pH of 2.0 and flow rate of 2.0mL/min, vacuum concentrating under reduced pressure, and freeze drying to obtain anthocyanin extract powder, i.e. the acid indicator based on Lycium ruthenicum anthocyanin extract.
Example 3
The preparation method of the lycium ruthenicum anthocyanin extract-based acid indicator provided by the embodiment comprises the following steps:
(1) preparing anthocyanin crude extract: taking 35g of dry lycium ruthenicum (purchased from Qinghai Nuo blue wolfberry), crushing by a crusher at 900r/min, sieving by a 30-mesh sieve, adding 5mL (namely 0.5-time column volume) of distilled water, performing low-speed (60rpm) oscillation extraction at room temperature (25 ℃) for 3.5h, taking out an extracting solution, adding distilled water again (100 mL each time in two times), performing heat preservation at 55 ℃ and low-speed (60rpm) oscillation extraction for 1.5h, taking out the extracting solution, combining the extracting solutions obtained in two times, and performing vacuum concentration at 55 ℃ until the concentration is 1.35g/mL to obtain a crude anthocyanin extracting solution;
(2) and (3) sterilization: performing ozone sterilization on the anthocyanin crude extract after ultrafiltration by a filter membrane of 0.22 mu m, wherein the sterilization conditions are as follows: the ozone amount was 5mg/m3Sterilizing for 20 min;
(3) and (3) purification: adding sterile distilled water to 10mL of the sterilized anthocyanin crude extract, loading the anthocyanin crude extract to AB-8 macroporous resin (5 multiplied by 30cm), washing out strong polar components (mainly inorganic salt and other impurities) by deionized water with 2 times of column volume, and then eluting the anthocyanin components by an ethanol water solution with the volume concentration of 80%, wherein the elution conditions are as follows: collecting eluate at pH of 2.0 and flow rate of 2.0mL/min, vacuum concentrating under reduced pressure, and freeze drying to obtain anthocyanin extract powder, i.e. the acid indicator based on Lycium ruthenicum anthocyanin extract.
To illustrate the effect of the anthocyanidin extract of lycium ruthenicum provided by the invention, the applicant carried out the following experiments:
experimental example 1 correspondence of Lycium ruthenicum anthocyanin extract to acid (color change)
The anthocyanin extract powder in example 1 is dissolved in aqueous solution with different pH conditions at a final concentration of 0.015% w/v, and the color change of the anthocyanin extract powder under different pH conditions is observed, and the result is shown in figure 1, wherein the color of the anthocyanin extract in the invention is shown according to the pH conditions, wherein, the thick short line represents the pH range of milk under normal conditions (6.0-7.0), and the horizontal line with an arrow indicates the indicated pH range of the anthocyanin extract to acid; the molecular structure of the anthocyanin extract and the transformation between different forms thereof are shown in figure 2; as can be seen from fig. 1-2, the anthocyanin extract can show different colors under different pH conditions, specifically, a red color when the pH is less than 5.0, a prominent red color when the pH is less than 4.5, a purple color when the pH is less than 5.0 and 7.0, and a blue color when the pH is greater than 7.0, but the anthocyanin extract is unstable and quickly oxidized to become yellow, and the experiment shows that the anthocyanin extract is sensitive to acid and is expected to be used for the separation and screening of microorganisms which weakly produce acid.
As can be seen from fig. 2, the molecular structural formula of the anthocyanin extract in the aqueous solution with pH <5.0 is:
the molecular structural formula of the anthocyanin extract in an aqueous solution with a pH of 5.0< 7.0 is as follows:
the molecular structural formula of the anthocyanin extract in an aqueous solution with the pH value of more than 7.0 is as follows:
the acid indicator powders prepared in example 2 and example 3 were dissolved in an aqueous solution to make the concentration of the anthocyanin extract be 0.015-0.05% (w/v), such as 0.015% (w/v), 0.01% (w/v), 0.02% (w/v), 0.03% (w/v), 0.04% (w/v), 0.05% (w/v), and the color change under different pH conditions is also obvious and pleasant, so that the color change is easy to be distinguished by the naked eye of the skilled person, and the color development rule is the same as above, and is not repeated.
Experimental example 2 response of Lycium ruthenicum anthocyanin extract to lactic acid
Lactic acid and lactic acid bacteria, and the anthocyanin extract powder in example 1 are taken as examples to illustrate the potential application of the extract in the separation and screening of acid-producing microorganisms. When the plate screening is carried out, lactic acid bacteria secrete lactic acid to the periphery of colonies, so that the pH value of a local area of the colonies is reduced. A color change occurs when the pH falls within the acid indicator response range.
The pH values corresponding to lactic acid with different concentrations are shown in table 1, and the anthocyanin extract can be used for detecting the lactic acid with the concentration as low as 0.0001mol/L by combining with the analysis of the corresponding characteristics of the anthocyanin extract on acidity, wherein the pH value corresponding to the lactic acid is less than 4.0, and the anthocyanin extract is bright red.
TABLE 1 pH values for different concentrations of lactic acid
Experimental example 3 screening of lactic acid bacteria required for fermentation of milk products
Taking the special pickles in the south of the Yangtze river as an example, lactic acid bacteria which can grow in milk and produce acid are screened from the characteristic pickles so as to be applied to milk fermentation, and the specific screening process is as follows:
(1) preparation of growth-indicating screening plates used: weighing 10g of skimmed milk powder, adding 100mL of distilled water, dissolving completely, and sterilizing at 100 deg.C for 15 min; weighing 1.5g of agar powder, adding 100mL of distilled water, mixing well, and sterilizing at 100 deg.C for 15 min; and cooling to 60 ℃ after sterilization, adding 10mL of the sterilized degreasing emulsion into the agar powder solution, slightly shaking and uniformly mixing, adding the anthocyanin extract with the final concentration of 0.025-0.05% (slowly adding by using a liquid transfer gun while slightly shaking), fully mixing, pouring the mixture into a flat plate, naturally solidifying the flat plate, and cooling for later use.
(2) Taking 1mL of pickle juice of Jiangnan special pickle, diluting by 100 times with sterile distilled water, uniformly coating the pickle juice on the milk flat plate in the step (1), and placing the milk flat plate in an anaerobic incubator (35 ℃) for overnight culture, wherein the color change condition of the milk flat plate is noticed during the culture.
(3) After 24h of incubation, a colony on the milk plate showed a color change, which was presumed to be a lactic acid bacterium capable of growing in milk, as shown in FIG. 3.
The experiments show that the anthocyanin extract obtained in the invention can be used as an indicator for separating and screening lactic acid bacteria, the indicator is expected to bring convenience to screening of dairy fermentation strains, and research and development of novel fermented dairy products are promoted.
The anthocyanin extract obtained by the invention can also be used for screening acid-producing strains such as acetic acid bacteria, propionibacterium, butyric acid bacteria and the like, and the concentration of the anthocyanin extract is 0.015-0.05% (w/v).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and simplifications made in the spirit of the present invention are intended to be included in the scope of the present invention.
Claims (9)
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 according to any one of claims 1 to 7 in acid-producing bacteria species 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.
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| CN101912480A (en) * | 2010-07-23 | 2010-12-15 | 中国科学院西北高原生物研究所 | Method for preparing procyanidin extract of lycium ruthenicum murr |
| CN105816413A (en) * | 2016-05-30 | 2016-08-03 | 新疆源森康乐生物科技有限公司 | Black barbary wolfberry fruit procyanidine mask powder and preparation method thereof |
| CN108991471A (en) * | 2018-06-27 | 2018-12-14 | 浙江农林大学暨阳学院 | A kind of natural additive for foodstuff with potent antioxidant activity |
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| CN101912480A (en) * | 2010-07-23 | 2010-12-15 | 中国科学院西北高原生物研究所 | Method for preparing procyanidin extract of lycium ruthenicum murr |
| CN105816413A (en) * | 2016-05-30 | 2016-08-03 | 新疆源森康乐生物科技有限公司 | Black barbary wolfberry fruit procyanidine mask powder and preparation method thereof |
| CN108991471A (en) * | 2018-06-27 | 2018-12-14 | 浙江农林大学暨阳学院 | A kind of natural additive for foodstuff with potent antioxidant activity |
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