Application of three prebiotics to growth and metabolism of lactobacillus plantarum J26

Abstract

The invention relates to application of three prebiotics to growth and metabolism of lactobacillus plantarum J26, in particular to a preparation method for improving growth and metabolism of lactobacillus plantarum J26 by utilizing galactooligosaccharide, fructo-oligosaccharide and isomaltooligosacharide with three different concentration gradients, and belongs to the technical field of microbial production. Compared with the lactobacillus plantarum J26 which is independently used, the prebiotics used in the preparation method can increase the growth rate, the viable count, the acid production capacity and the oxidation resistance of the lactobacillus plantarum J26, and along with the increase of the addition amount of the prebiotics, the increase of various indexes of the lactobacillus plantarum J26 is more and more obvious. The combination of prebiotics and probiotics has the advantage of synergistic interaction, and has certain reference value for research, development and popularization of prebiotics and lactobacillus plantarum J26 related products.

Description

Technical Field

[0001] The invention relates to studying the influence of three prebiotics on the growth metabolism of Lactobacillus plantarum J26 from four aspects of growth curve, acid production capacity, antioxidant capacity and viable bacteria count, and belongs to the technical field of microbial production. Background Art

[0002] Lactobacillus plantarum J26 is an important probiotic isolated from traditional Chinese fermented dairy products. It has the strongest activity on biofilm formation and can improve food industry performance by forming biofilm. In addition, Lactobacillus plantarum J26 can also reduce the content of lipopolysaccharide in the intestine, maintain the intestinal barrier, reduce liver inflammation, restore liver function, and affect the body's cell expression and immune response and many other physiological functions by colonizing, metabolizing and secreting metabolites in the intestinal microenvironment. Prebiotics cannot be directly and completely digested and absorbed by the human body. Instead, they are fermented by microorganisms in the human intestine. The most significant advantage of prebiotics is that they only act on beneficial bacteria to accelerate their growth and metabolism. On the contrary, prebiotics do not have any promoting effect on harmful bacteria with potential pathogenicity or corruption activity. However, prebiotics cannot be directly used by the human body, but use probiotics as a connection with the human body to play a physiological role. Probiotics will produce some substances that are beneficial to human health and play a role by regulating the human intestinal flora. Therefore, the combined use of prebiotics and probiotics has the advantage of synergistic enhancement, and the efficacy is significantly improved compared to the use of probiotics alone.

[0003] In recent years, probiotics and prebiotics have been used in functional foods, and the mode of action of prebiotics and probiotics is closely related to intestinal bacteria, which can promote human health in many ways, improve human health and reduce the incidence of some diseases. In general, prebiotics and probiotics have biological regulatory effects, including increasing appetite, enhancing biological defense, promoting digestion and absorption, and also have obvious control effects on diseases such as hypertension, cancer, diabetes, and anemia. At present, my country is developing, producing and using prebiotics and probiotics on a large scale, and their related products are gradually entering the public eye and are widely known by the public. With the advancement of science and technology and the continuous improvement of living standards, people are paying more and more attention to dietary health, and the popularity of prebiotics and probiotics related products is also continuing to increase. Summary of the invention

[0004] The invention selects three different prebiotics, namely galacto-oligosaccharide, fructo-oligosaccharide and isomalto-oligosaccharide, as research objects, and explores the influence of the three prebiotics on the growth metabolism of Lactobacillus plantarum J26 by measuring the changes in four aspects, namely, the acid production capacity, the antioxidant capacity, the total number of viable bacteria and the growth curve of Lactobacillus plantarum J26 under different concentrations of the three prebiotics, so as to determine a more suitable growth environment for Lactobacillus plantarum J26, and has certain reference value for the research and development and promotion of prebiotics and Lactobacillus plantarum J26 related products, and provides a theoretical basis and data reference for other researches on prebiotics and Lactobacillus plantarum J26.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] The application of three prebiotics to the growth and metabolism of Lactobacillus plantarum J26 includes the following steps:

[0007] Step 1: Activation and preservation of plant lactobacillus J26 strain. Plant lactobacillus J26 is inoculated in MRS liquid medium at an inoculum size of 5% (v / v), cultured at 37°C for 18 hours, and a suitable volume of bacterial suspension is selected and inoculated into MRS solid medium by the three-zone line method to purify the strain. Cultured at 37°C for 18 hours, a single colony is picked and cultured in MRS liquid medium for 18 hours. The purified and activated strain is mixed with 80% sterile glycerol in a 3:1 ratio in a cryopreservation tube and frozen for standby use.

[0008] Step 2: Cultivation of Lactobacillus plantarum J26. Add different concentration gradients (1%, 2%, 3%) of galacto-oligosaccharide, fructo-oligosaccharide and isomalto-oligosaccharide to the culture medium of Lactobacillus plantarum J26, for a total of 9 groups. Take groups 1-9 as one unit, prepare 4 samples in parallel for each unit, and culture each group of samples at 37°C to determine the acid production capacity, antioxidant capacity, viable count and growth curve.

[0009] Step 3: Determination of the growth curve of Lactobacillus plantarum J26 with prebiotics added. Lactobacillus plantarum J26 with different concentrations of three prebiotics added was inoculated in MRS liquid culture medium at an inoculum size of 5%, and Lactobacillus plantarum J26 without prebiotics added was used as a blank control. The growth curve instrument was used to continuously detect for 48 hours, with each 15 minutes as a time point, and the detection was repeated 3 times at each time point to take the average value.

[0010] Step 4: Determination of the number of viable bacteria of Lactobacillus plantarum J26 with added prebiotics. Lactobacillus plantarum J26 without prebiotics was used as a blank control, and the remaining prebiotic groups were inoculated with 5% Lactobacillus plantarum J26. The prepared samples of each group were placed in a 37°C constant temperature incubator, and diluted after ordinary incubation for 48 hours to determine the number of viable bacteria of Lactobacillus plantarum J26 in each group.

[0011] Step 5: Determination of the acid production capacity of Lactobacillus plantarum J26 with added prebiotics. Take another parallel unit, with the same culture conditions as above, and perform an acidity test on all test groups every two hours in a constant temperature box. Use a pH meter to measure the acidity of each group, and analyze the effects of different concentrations of three prebiotics on the acid production capacity of Lactobacillus plantarum J26 during the growth process. Stop the test when there is no significant difference in the measured data for three consecutive times.

[0012] Step 6: Determination of the antioxidant capacity of Lactobacillus plantarum J26 with prebiotics added. Take another parallel unit, and use the total antioxidant capacity (T-AOC) assay kit and UV spectrophotometer to determine the total antioxidant capacity of Lactobacillus plantarum J26 when three different concentration gradients of galacto-oligosaccharides, fructo-oligosaccharides, and isomaltooligosaccharides are added under the same culture conditions as above.

[0013] The present invention finds that the use of prebiotics can improve the efficacy, growth rate, viable count, acid production capacity and antioxidant capacity of Lactobacillus plantarum J26 when used alone, and as the amount of prebiotics added increases, the effect of prebiotics on the increase of various indicators of Lactobacillus plantarum J26 becomes more and more obvious.

[0014] The beneficial effects achieved by the present invention are:

[0015] 1. Compared with the growth rate of Lactobacillus plantarum J26 in the medium without prebiotics, the three prebiotics all promoted the growth of Lactobacillus plantarum J26. The final total colony count of Lactobacillus plantarum J26 in the growth curve when the addition of galacto-oligosaccharide was 2% and 3% was basically the same, reaching 1.95×10 9 CFU / mL. As the concentration gradient of oligofructose and isomaltose increased, the final total colony count shown in the growth curve of Lactobacillus plantarum J26 also increased. When the addition amount was 3%, it could reach 2.03×10 9 CFU / mL and 2.08×10 9 CFU / mL. When the amount of prebiotics added was 3%, the growth rate of Lactobacillus plantarum J26 added to the galacto-oligosaccharide medium was the highest. Therefore, adding prebiotics can accelerate the growth and reproduction of probiotics.

[0016] 2. Compared with the viable bacterial count of Lactobacillus plantarum J26 when no prebiotics were added to the culture medium, the three prebiotics all had a proliferation effect on Lactobacillus plantarum J26, and with the increase of prebiotic concentration, the effect of the three prebiotics on increasing the viable bacterial count became more obvious. When the amount of oligosaccharide added in the culture medium was increased from 2% to 3%, the viable bacterial count of Lactobacillus plantarum J26 increased the most, and the viable bacterial count at this time was 4.77×10 9 CFU / mL. Therefore, prebiotics promote the growth and reproduction of probiotics.

[0017] 3. The acid production capacity of Lactobacillus plantarum J26 promoted by prebiotics was positively correlated with the amount of prebiotics added, and the pH of Lactobacillus plantarum J26 decreased most significantly with the increase of oligofructose added in the culture medium. When 3% isomaltooligosaccharide was added to the culture medium, the final pH value of Lactobacillus plantarum J26 was the lowest, which was 3.23.

[0018] 4. With the increase of the amount of prebiotics added, the total antioxidant capacity of Lactobacillus plantarum J26 also increased. When the amount of oligomaltosaccharide added in the culture medium was 3%, the total antioxidant capacity of Lactobacillus plantarum J26 was the highest, which was 43.16U / mL. Then the total antioxidant capacity of Lactobacillus plantarum J26 in the culture medium with 3% oligofructose added was 35.02U / mL, and the total antioxidant capacity of Lactobacillus plantarum J26 in the culture medium with 3% galacto-oligosaccharide added was 26.27U / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the growth curve of Lactobacillus plantarum J26 when galacto-oligosaccharide is added to the culture medium;

[0020] Figure 2 This is the growth curve of Lactobacillus plantarum J26 when oligofructose is added to the culture medium;

[0021] Figure 3 This is the growth curve of Lactobacillus plantarum J26 when isomaltooligosaccharide was added to the culture medium;

[0022] Figure 4 This is the growth curve of Lactobacillus plantarum J26 when the prebiotics addition amount is 3%;

[0023] Figure 5 is the total colony count of Lactobacillus plantarum J26 when prebiotics were added;

[0024] Figure 6 The acid production capacity of Lactobacillus plantarum J26 when different concentrations of galacto-oligosaccharide were added to the culture medium;

[0025] Figure 7 The acid production capacity of Lactobacillus plantarum J26 when different concentrations of oligofructose were added to the culture medium;

[0026] Figure 8 The acid production capacity of Lactobacillus plantarum J26 when different concentrations of isomaltooligosaccharide were added to the culture medium;

[0027] Fig. 9 is the acid production capacity of Lactobacillus plantarum J26 when the prebiotics addition amount is 3%;

[0028] Fig.10 is the total antioxidant capacity of Lactobacillus plantarum J26 when prebiotics were added. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0030] The application of the three prebiotics to the growth and metabolism of Lactobacillus plantarum J26 comprises the following steps:

[0031] Step 1: Activation and preservation of plant lactobacillus J26 strain. Plant lactobacillus J26 is inoculated in MRS liquid medium at an inoculum size of 5% (v / v), cultured at 37°C for 18 hours, and a suitable volume of bacterial suspension is selected and inoculated into MRS solid medium by the three-zone line method to purify the strain. Cultured at 37°C for 18 hours, a single colony is picked and cultured in MRS liquid medium for 18 hours. The purified and activated strain is mixed with 80% sterile glycerol in a 3:1 ratio in a cryopreservation tube and frozen for standby use.

[0032] Step 2: Cultivation of Lactobacillus plantarum J26. Add different concentration gradients (1%, 2%, 3%) of galacto-oligosaccharide, fructo-oligosaccharide and isomalto-oligosaccharide to the culture medium of Lactobacillus plantarum J26, for a total of 9 groups. Take groups 1-9 as one unit, prepare 4 samples in parallel for each unit, and culture each group of samples at 37°C to determine the acid production capacity, antioxidant capacity, viable count and growth curve.

[0033] Step 3: Determination of the growth curve of Lactobacillus plantarum J26 with prebiotics added. Lactobacillus plantarum J26 with different concentrations of three prebiotics added was inoculated in MRS liquid culture medium at an inoculum size of 5%, and Lactobacillus plantarum J26 without prebiotics added was used as a blank control. The growth curve instrument was used to continuously detect for 48 hours, with each 15 minutes as a time point, and the detection was repeated 3 times at each time point to take the average value.

[0034] Step 4: Determination of the number of viable bacteria of Lactobacillus plantarum J26 with added prebiotics. Lactobacillus plantarum J26 without prebiotics was used as a blank control, and the remaining prebiotic groups were inoculated with 5% Lactobacillus plantarum J26. The prepared samples of each group were placed in a 37°C constant temperature incubator, and diluted after ordinary incubation for 48 hours to determine the number of viable bacteria of Lactobacillus plantarum J26 in each group.

[0035] Step 5: Determination of the acid production capacity of Lactobacillus plantarum J26 with added prebiotics. Take another parallel unit, with the same culture conditions as above, and perform an acidity test on all test groups every two hours in a constant temperature box. Use a pH meter to measure the acidity of each group, and analyze the effects of different concentrations of three prebiotics on the acid production capacity of Lactobacillus plantarum J26 during the growth process. Stop the test when there is no significant difference in the measured data for three consecutive times.

[0036] Step 6: Determination of the antioxidant capacity of Lactobacillus plantarum J26 with prebiotics added. Take another parallel unit, and use the total antioxidant capacity (T-AOC) assay kit and UV spectrophotometer to determine the total antioxidant capacity of Lactobacillus plantarum J26 when three different concentration gradients of galacto-oligosaccharides, fructo-oligosaccharides, and isomaltooligosaccharides are added under the same culture conditions as above.

[0037] Table 1 Total antioxidant capacity (T-AOC) assay kit operation

[0038]

[0039]

[0040] Note: Preparation of reagent 2: Use 120mL of double distilled water to fully dissolve each powder (the powder is difficult to dissolve at room temperature, so it can be placed in a 37℃ water bath to accelerate dissolution). The prepared powder can be stored at room temperature for at least 1 month. Reagent 3 application solution: Dilute the stock solution with diluent at a ratio of 1:19. This reagent should be prepared before use.

[0041] One total antioxidant capacity unit (U) means that at 37°C, the absorbance (OD) value of the reaction system increases by 0.01 per milliliter of plant lactobacillus J26 per minute. The calculation formula is as follows:

[0042]

[0043] Where, T: reaction time, 30min; V 反总 : Total volume of reaction system, mL; V 样 : Sampling volume, mL; N: dilution factor of the sample before testing.

[0044] In summary, the use of prebiotics can improve the growth rate, viable count, acid production capacity and antioxidant capacity of Lactobacillus plantarum J26 alone, and with the increase in the amount of prebiotics added, the effect of prebiotics on the various indicators of Lactobacillus plantarum J26 becomes more and more obvious. Galacto-oligosaccharides can significantly accelerate the growth rate of Lactobacillus plantarum J26. When the addition amount is 3%, its growth rate is the fastest compared with other addition amounts. When 3% isomaltooligosaccharide is added to the culture medium, the viable count is the highest (5.7×10 9 CFU / mL), the acid production capacity and antioxidant capacity were also significantly improved (pH was 3.23, total antioxidant capacity was 43.16U / mL).

[0045] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of three prebiotics on the growth metabolism of Lactobacillus plantarum J26, characterized in that: Three different concentration gradients (1%, 2%, 3%) of galacto-oligosaccharide, fructo-oligosaccharide or isomaltooligosaccharide were added to the culture medium. Among them, the growth rate of J26 in the culture medium with 3% galacto-oligosaccharide was the fastest; the other indicators of J26 in the culture medium with 3% isomaltooligosaccharide were relatively good, and the number of viable bacteria could reach 5.7×10 9 CFU / mL, the lowest pH was 3.23, and the highest total antioxidant capacity was 43.16U / mL.

2. The application of the three prebiotics to the growth metabolism of Lactobacillus plantarum J26 according to claim 1, characterized in that: The use of prebiotics can improve the efficacy, growth rate, viable count, acid production capacity and antioxidant capacity of Lactobacillus plantarum J26 when used alone, and as the amount of prebiotics added increases, its effect on the various indicators of Lactobacillus plantarum J26 becomes more and more obvious.

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