CN107217020B - Culture medium suitable for lactobacillus acidophilus and preparation method thereof - Google Patents
Culture medium suitable for lactobacillus acidophilus and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a culture medium suitable for lactobacillus acidophilus and a preparation method thereof, wherein the culture medium comprises the following components in parts by weight: 1000-2000 ml of distilled water, 0.2-1.0 g/L of magnesium sulfate, 0.1-0.5 g/L of manganese sulfate, 10-40 g/L of yeast extract, 1.0-4.0 g/L of dipotassium hydrogen phosphate, 1.0-4.0 g/L of diammonium hydrogen citrate, 5-20 g/L of beef extract, 10-20 g/L of agar, 5-20 g/L of peptone, 800.5-2 g/L of tween, 50-200 g/L of tomato juice, 7.5-30 g/L of sodium acetate, 10-40 g/L of glucose and 0.04-0.2 g/L of dandelion extract. The culture medium is particularly suitable for culturing lactobacillus acidophilus, can obviously improve the viable count of the lactobacillus acidophilus and obviously shorten the time for the lactobacillus acidophilus to enter a stable phase, and the prepared lactobacillus acidophilus also contains a large amount of beneficial metabolites due to the addition of natural factors.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture medium suitable for lactobacillus acidophilus and a preparation method thereof.
Background
Lactobacillus acidophilus (Lactobacillus acidophilus) belongs to the genus Lactobacillus, can produce lactic acid by fermenting carbohydrates, is widely present in the intestinal tracts of humans and animals, has an important effect on the health of humans, especially on maintaining the normal physiological function of the gastrointestinal tract, and is an important beneficial microbial flora in the intestinal tract.
Because the growth conditions of the lactobacillus acidophilus are special, the viable bacteria content of the lactobacillus acidophilus products in the current market is low. Therefore, active research has been conducted on substances that promote the growth of lactobacillus acidophilus to increase the viable count of lactobacillus acidophilus. Meanwhile, the isolation culture of lactobacillus acidophilus and the enzymological research thereof need a good enrichment culture medium, so that the lactobacillus acidophilus can be greatly proliferated in the shortest time, and the lactobacillus acidophilus still has enough viable count after a series of subsequent operations. Therefore, the optimization of the enrichment medium of lactobacillus acidophilus becomes a problem to be solved urgently at present.
Chinese patent application CN103255093B discloses a preparation method of lactobacillus acidophilus, which relates to a liquid culture medium, and the specific components are 2 wt% of glucose, 1 wt% of peptone, 1 wt% of beef extract, 0.5 wt% of yeast extract, 0.2 wt% of dipotassium hydrogen phosphate, 0.5 wt% of NaAc, 0.05 wt% of magnesium sulfate, 0.025 wt% of manganese sulfate, 800.1 vol% of Tween-5, 0.2 wt% of diamine citrate, 1.5 wt% of agar and 5 wt% of tomato juice. The lactobacillus acidophilus prepared from this medium is also accompanied by a large number of other beneficial metabolites: lactein and organic acids such as L-lactic acid, n-butyric acid, acetic acid, formic acid, etc. The products are important energy sources of animal intestinal tracts, can act with probiotics in a synergistic manner, and can remarkably improve the gastrointestinal tract function of animals, improve the immunity and promote the growth of the animals; the product obtained by the invention is prepared into feed, and the digestion and absorption rate of animals on the feed are improved. However, the viable count of the lactobacillus acidophilus obtained by the culture medium is low, and the time for the lactobacillus acidophilus to enter a stable phase is long.
Therefore, it is necessary to provide a culture medium for promoting the growth of lactobacillus acidophilus and significantly reducing the time for lactobacillus acidophilus to enter the stationary phase.
Disclosure of Invention
The invention aims to provide a culture medium suitable for lactobacillus acidophilus and a preparation method thereof. The culture medium is added with natural growth factors, tomato juice and dandelion extract, the natural growth factors, the tomato juice and the dandelion extract can obviously promote the growth and the reproduction of lactobacillus acidophilus, obviously improve the viable count of the lactobacillus acidophilus and obviously shorten the time for the lactobacillus acidophilus to enter a stable period.
In order to achieve the purpose, the invention adopts the following technical scheme: a culture medium suitable for lactobacillus acidophilus comprises the following components by weight: 1000-2000 ml of distilled water, 0.2-1.0 g/L of magnesium sulfate, 0.1-0.5 g/L of manganese sulfate, 10-40 g/L of yeast extract, 1.0-4.0 g/L of dipotassium hydrogen phosphate, 1.0-4.0 g/L of diammonium hydrogen citrate, 5-20 g/L of beef extract, 10-20 g/L of agar, 5-20 g/L of peptone, 800.5-2 g/L of tween, 50-200 g/L of tomato juice, 7.5-30 g/L of sodium acetate, 10-40 g/L of glucose and 0.04-0.2 g/L of dandelion extract.
Further, the culture medium consists of the following components in parts by weight: 1000ml of distilled water, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 20g/L of yeast extract, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 10g/L of beef extract, 14g/L of agar, 10g/L of peptone, 801g/L of tween, 150g/L of tomato juice, 15g/L of sodium acetate, 20g/L of glucose and 0.1g/L of dandelion extract.
Further, the tomato juice is prepared by the following steps:
cleaning and slicing fresh tomatoes, adding 1-2 times of purified water and warm water for blanching for 5-15 min, juicing, filtering, adjusting the pH to 6.3, and sterilizing at 110-120 ℃ for 10-15 min to obtain the tomato juice.
Further, the dandelion extract is prepared by the following steps: weighing dandelion, crushing, adding purified water in an amount which is 10-20 times that of the dandelion, decocting for 1-3 times, 0.5-1 h each time, filtering, combining filtrates, concentrating, drying and crushing to obtain the dandelion tea.
The lactobacillus acidophilus has a pH range suitable for growth, and when the hydrogen ion concentration in the culture medium exceeds the adaptation range of the lactobacillus acidophilus, the metabolism of the lactobacillus acidophilus is inhibited. When the environmental pH is in a proper range, the metabolic activity of lactobacillus acidophilus is enhanced, and the growth and the reproduction are accelerated, so that the initial pH of the culture medium is preferably 6.0-6.6, and more preferably the initial pH of the culture medium is 6.2.
The temperature, which is one of the culture conditions, has an important influence on the growth and propagation of lactobacillus acidophilus. Under the condition of proper temperature, the propagation and growth of lactobacillus acidophilus are faster, which is beneficial to the increase of the number of bacteria of lactobacillus acidophilus. Within a certain range, the increase of the temperature can increase the growth rate of the cells, when the optimum temperature is exceeded, the increase of the temperature can affect proteins and nucleic acids in the microorganism, the growth rate is reduced along with the increase of the temperature, and if the temperature is further increased, the cell function is sharply reduced to death. Therefore, the initial temperature of the culture medium is preferably 28-40 ℃, and more preferably 37 ℃.
The size of the inoculation amount in the culture process directly influences the growth and propagation speed of the strains. The inoculation amount is large, the delay period of the thalli is shortened, the proliferation is rapid, but the nutrient substance in the culture medium is easy to lack due to the rapid proliferation. The inoculation amount is small, the growth of the thalli in the adjusting period is slow, the growth period is prolonged, and the possibility of bacterial contamination is easily increased. Therefore, the proper inoculation amount is a key for ensuring the efficient proliferation of the lactic acid bacteria. Preferably, the lactobacillus acidophilus is inoculated in an amount of 2 to 4%, more preferably 3%.
Further, the lactobacillus acidophilus is lactobacillus acidophilus JCM1132, which has a preservation number of: CGMCC1.1878 accordingly, the invention also provides a method for preparing the culture medium for lactobacillus acidophilus, which comprises the following steps:
A) preparing tomato juice: cleaning and slicing fresh tomatoes, adding 1-2 times of purified water and warm water for blanching for 5-15 min, juicing, filtering, adjusting the pH to 6.3, sterilizing at 110-120 ℃ for 10-15 min, and storing in a refrigerator for later use;
B) preparation of dandelion extract: weighing a dandelion herb, crushing, adding purified water in an amount which is 10-20 times that of the dandelion herb, decocting for 1-3 times, 0.5-1 h each time, filtering, combining filtrates, concentrating, drying and crushing to obtain the dandelion herb extract;
C) weighing the components in proportion, mixing, adjusting pH, and sterilizing under high pressure and moist heat.
The invention also aims to provide a lactobacillus acidophilus culture method, which comprises the steps of inoculating the activated lactobacillus acidophilus strain into the culture medium according to the inoculation amount of 2-4%, and culturing in a constant-temperature incubator at 37 ℃ for 20-30 h.
The invention has the following advantages:
the lactobacillus acidophilus culture medium is prepared by adding tomato juice and dandelion extract on the basis of a basic culture medium, is particularly suitable for culturing lactobacillus acidophilus, can obviously improve the viable count of the lactobacillus acidophilus and obviously shorten the time for the lactobacillus acidophilus to enter a stable period, and the prepared lactobacillus acidophilus also contains a large amount of beneficial metabolites due to the addition of natural factors.
Drawings
FIG. 1 shows the effect of different growth factors on the growth of Lactobacillus acidophilus;
FIG. 2 shows the effect of initial pH of the medium on the growth of Lactobacillus acidophilus;
FIG. 3 shows the effect of inoculum size on the growth of Lactobacillus acidophilus;
FIG. 4 shows the growth curves of Lactobacillus acidophilus before and after medium optimization.
Detailed Description
The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.
The invention culture medium prescription part component purchase information
Example 1A culture Medium for Lactobacillus acidophilus
The culture medium comprises the following components in parts by weight: 1000ml of distilled water, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 20g/L of yeast extract, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 10g/L of beef extract, 14g/L of agar, 10g/L of peptone, 801g/L of tween, 150g/L of tomato juice, 15g/L of sodium acetate, 20g/L of glucose and 0.1g/L of dandelion extract.
The preparation method comprises the following steps:
A) preparing tomato juice: cleaning fresh fructus Lycopersici Esculenti, slicing, adding 1 times of purified water, scalding in warm water for 10min, squeezing, filtering, adjusting pH to 6.3, sterilizing at 115 deg.C for 15min, and storing in refrigerator;
B) preparation of dandelion extract: weighing 20g of dandelion, crushing, adding 500mL of water, decocting for 2 times, each time for 0.5h, filtering, combining the filtrates, concentrating, drying and crushing to obtain the dandelion extract;
C) weighing the components in proportion, mixing, adjusting pH to 6.2, and sterilizing under high pressure and moist heat.
Example 2A culture Medium for Lactobacillus acidophilus
The culture medium comprises the following components in parts by weight: 1500ml of distilled water, 0.2g/L of magnesium sulfate, 0.1g/L of manganese sulfate, 10g/L of yeast extract, 1.0g/L of dipotassium phosphate, 1.0g/L of diammonium hydrogen citrate, 5g/L of beef extract, 10g/L of agar, 5g/L of peptone, 800.5g/L of tween, 100g/L of tomato juice, 7.5g/L of sodium acetate, 10g/L of glucose and 0.04g/L of dandelion extract.
The preparation process is referred to example 1.
Example 3A culture Medium for Lactobacillus acidophilus
The culture medium comprises the following components in parts by weight: 2000ml of distilled water, 1.0g/L of magnesium sulfate, 0.5g/L of manganese sulfate, 40g/L of yeast extract, 4.0g/L of dipotassium phosphate, 4.0g/L of diammonium hydrogen citrate, 20g/L of beef extract, 20g/L of agar, 20g/L of peptone, 802g/L of tween, 200g/L of tomato juice, 30g/L of sodium acetate, 40g/L of glucose and 0.2g/L of dandelion extract.
Comparative example 1 culture Medium suitable for Lactobacillus acidophilus
Comparative example 1 differs from example 1 in that the dandelion extract was removed and the weight of the tomato juice was increased to 150.1g/L, with the remaining parameters and operation as in example 1.
Comparative example 2 culture Medium for Lactobacillus acidophilus
Comparative example 2 differs from example 1 in that tomato juice was removed and the weight of the dandelion extract was increased to 150.1g/L, and the remaining parameters and operation were as in example 1.
Comparative example 3 culture Medium for Lactobacillus acidophilus
Comparative example 3 differs from example 1 in that tomato juice and dandelion extract were removed and replaced with 150.1g/L carrot juice, and the remaining parameters and operation were as in example 1.
Preparation of carrot juice:
cleaning fresh carrot 200g with purified water, slicing, adding 200mL of purified water, blanching for 10min, mashing with a juicer, squeezing, filtering with double-layer gauze, adjusting pH to 6.3, sterilizing at 115 deg.C for 15min, and placing in a refrigerator for use.
Comparative example 4A Medium suitable for Lactobacillus acidophilus
Comparative example 4 differs from example 1 in that tomato juice and dandelion extract were removed and replaced with 150.1g/L beer juice (Qingdao beer, 330 ml/bottle), and the remaining parameters and operation were as in example 1.
Test example I Effect of different growth factors on the growth of Lactobacillus acidophilus
The culture medium described in example 1 and comparative examples 1-4 and a blank control group (compared with example 1, containing no growth factor tomato juice and dandelion extract) were inoculated with 3% lactobacillus acidophilus activating solution, and cultured in an incubator at 37 ℃ for 27h, an enzyme-linked microplate reader was used to measure the OD value of each group at 630nm, and each group was repeated 3 times, and the test results are shown in Table 1 and FIG. 1.
TABLE 1 Effect of different growth factors on the growth of Lactobacillus acidophilus (OD)630)
The results showed that each group promoted the growth of lactobacillus acidophilus to some extent compared to the blank control group; wherein, the growth promoting effect of the carrot juice is least obvious, and the growth promoting effect of the dandelion extract is better; compared with the single action of tomato juice or dandelion extract, the combination of tomato juice and dandelion extract has the most obvious effect of promoting the growth of lactobacillus acidophilus.
Experiment II, influence of initial pH value of culture medium and lactobacillus acidophilus inoculation amount on growth of lactobacillus acidophilus
2.1 Effect of initial pH of Medium on Lactobacillus acidophilus growth
In order to determine the optimal initial pH for the growth of Lactobacillus acidophilus, the OD values of Lactobacillus acidophilus were measured at initial pH values of 6.0, 6.2, 6.4, 6.6, and 6.8 in the medium of example 1 using a microplate reader, respectively, and the results are shown in FIG. 2.
As can be seen from FIG. 2, different initial pH values have an effect on Lactobacillus acidophilus, and both too low and too high initial pH values are detrimental to the growth of Lactobacillus acidophilus. The biological growth of Lactobacillus acidophilus reaches its maximum when the pH is 6.2. Therefore, the initial pH of the culture medium of Lactobacillus acidophilus was selected to be 6.2.
2.2 Effect of inoculum size on Lactobacillus acidophilus growth
The medium of example 1 was inoculated with the respective inoculum sizes of 1%, 2%, 3%, 4%, 5% and 6%, and the effect of the inoculum size of Lactobacillus acidophilus on the growth was examined, and the results are shown in FIG. 3.
As can be seen from FIG. 3, when the inoculation amount is 2% -4%, the lactobacillus rapidly grows to the highest biomass, and the culture period is shortened; when the inoculation amount is 3%, the biomass of lactobacillus acidophilus reaches the maximum; when the inoculation amount is 6%, the number of lactobacillus bacteria is obviously reduced, which indicates that the excessively large inoculation amount causes nutrition deficiency in the later growth period of lactobacillus, so that part of bacteria is dead, and the number of bacteria is reduced, so that the optimal inoculation amount of lactobacillus acidophilus is 3%.
Test example three, Effect of the Medium before and after optimization on the growth Curve of Lactobacillus acidophilus
Respectively inoculating 3% lactobacillus acidophilus into conical flasks containing 200mL of liquid culture medium (compared with the culture medium in example 1, the conical flasks do not contain tomato juice and dandelion extract) and 200mL of the culture medium in example 1, placing the conical flasks in an incubator at 37 ℃ for culturing for 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 48 hours, respectively taking 200 mu L of the culture medium, and measuring by using a microplate reader at 630nm, wherein the culture time is used as a horizontal coordinate and the absorbance is used as a vertical coordinate, so that a growth curve chart 4 of lactobacillus acidophilus before and after the culture medium is optimized is obtained.
FIG. 4 shows that 0-6 h after inoculating Lactobacillus acidophilus with a pre-optimized culture medium (not containing tomato juice and dandelion extract), the Lactobacillus acidophilus is in a growth retardation phase in this period, the Lactobacillus acidophilus is in a logarithmic phase for 6-24 h, the Lactobacillus acidophilus enters a stationary phase for 24-33 h, and the thallus begins to enter a decline and death phase; however, the optimized culture medium (added with tomato juice and dandelion extract) is more beneficial to the growth of lactobacillus acidophilus, after lactobacillus acidophilus begins to enter a logarithmic phase for about 12 hours after about 3 hours of a lag phase, the thallus enters a stationary phase, the absorbance is maximum at 21 hours, and the time for entering the stationary phase is obviously shortened.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (7)
1. A culture medium suitable for Lactobacillus acidophilus is characterized by comprising the following components in parts by weight: 1000-2000 ml of distilled water, 0.2-1.0 g/L of magnesium sulfate, 0.1-0.5 g/L of manganese sulfate, 10-40 g/L of yeast extract, 1.0-4.0 g/L of dipotassium hydrogen phosphate, 1.0-4.0 g/L of diammonium hydrogen citrate, 5-20 g/L of beef extract, 10-20 g/L of agar, 5-20 g/L of peptone, 800.5-2 g/L of tween, 50-200 g/L of tomato juice, 7.5-30 g/L of sodium acetate, 10-40 g/L of glucose and 0.04-0.2 g/L of dandelion extract; the dandelion extract is prepared by the following steps:
weighing dandelion, crushing, adding purified water in an amount which is 10-20 times that of the dandelion, decocting for 1-3 times, 0.5-1 h each time, filtering, combining filtrates, concentrating, drying and crushing to obtain the dandelion tea.
2. The culture medium suitable for lactobacillus acidophilus according to claim 1, characterized by consisting of the following components in weight: 1000ml of distilled water, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 20g/L of yeast extract, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 10g/L of beef extract, 14g/L of agar, 10g/L of peptone, 801g/L of tween, 150g/L of tomato juice, 15g/L of sodium acetate, 20g/L of glucose and 0.1g/L of dandelion extract.
3. A culture medium suitable for lactobacillus acidophilus according to claim 1 or 2, characterized in that the tomato juice is obtained by the following steps:
cleaning and slicing fresh tomatoes, adding 1-2 times of purified water and warm water for blanching for 5-15 min, juicing, filtering, adjusting the pH to 6.3, and sterilizing at 110-120 ℃ for 10-15 min to obtain the tomato juice.
4. The culture medium suitable for lactobacillus acidophilus according to claim 1 or 2, characterized in that it has an initial pH comprised between 6.0 and 6.6.
5. The culture medium suitable for lactobacillus acidophilus according to claim 1 or 2, characterized in that it has an initial temperature of between 28 and 40 ℃.
6. A method for preparing a culture medium for Lactobacillus acidophilus according to any of claims 1 to 5, characterized by comprising the following steps:
A) preparing tomato juice: cleaning and slicing fresh tomatoes, adding 1-2 times of purified water and warm water for blanching for 5-15 min, juicing, filtering, adjusting the pH to 6.3, sterilizing at 110-120 ℃ for 10-15 min, and storing in a refrigerator for later use;
B) preparation of dandelion extract: weighing a dandelion herb, crushing, adding purified water in an amount which is 10-20 times that of the dandelion herb, decocting for 1-3 times, 0.5-1 h each time, filtering, combining filtrates, concentrating, drying and crushing to obtain the dandelion herb extract;
C) weighing the components in proportion, mixing, adjusting pH, and sterilizing under high pressure and moist heat.
7. A method for culturing Lactobacillus acidophilus, characterized in that activated Lactobacillus acidophilus strain is inoculated into the culture medium according to any of claims 1 to 5 or the culture medium prepared according to claim 6 in an inoculum size of 2 to 4%, and cultured in a constant temperature incubator at 37 ℃ for 20 to 30 hours.
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| CN110106133A (en) * | 2019-05-23 | 2019-08-09 | 北京大学 | Adjust the culture solution and adjusting method of growth of lactobacillus speed and bacterium interior element content |
| CN111926053A (en) * | 2020-08-07 | 2020-11-13 | 辽宁傲农饲料有限公司 | Culture medium for counting lactic acid bacteria and saccharomycetes in compound bacteria fermented feed and preparation method and application thereof |
| CN112961797A (en) * | 2021-02-01 | 2021-06-15 | 武汉微康益生菌研究院有限公司 | Lactobacillus acidophilus high-density fermentation medium and application thereof |
| CN115820515A (en) * | 2023-01-03 | 2023-03-21 | 东北农业大学 | Preparation of cheap lactobacillus PTG medium and application of cheap lactobacillus PTG medium in pickled vegetable fermentation |
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