CN103897998B - Lactobacillus salivarius and application and functional food composition and preparation method thereof - Google Patents

Abstract

The invention provides a kind of Lactobacillus salivarius (Lactobacillus salivarius), it is characterised in that the deposit number of described Lactobacillus salivarius is CGMCC No.6288.Present invention also offers a kind of functional food composition and preparation method thereof, including: the viable bacteria body of Lactobacillus salivarius as above is inactivated, obtains thalline material.Present invention also offers a kind of functional food composition, described functional food composition contains the viable bacteria body of Lactobacillus salivarius as above.Present invention also offers the Lactobacillus salivarius as above application in the functional food composition preparing slow down aging.Thalline material after the viable bacteria body of Lactobacillus salivarius provided by the invention and inactivation is respectively provided with effect of slow down aging.

Description

Lactobacillus salivarius and its application, functional food composition and preparation method thereof

technical field

The present invention relates to Lactobacillus salivarius and its application, functional food composition and preparation method thereof, in particular to a Lactobacillus salivarius, a functional food composition containing the Lactobacillus salivarius and its preparation method, and the saliva Application of Lactobacillus in the preparation of anti-aging functional food.

Background technique

Aging is a degenerative change in body tissues and organ functions with age (Turnheim, 2003), and is a comprehensive manifestation of various biochemical reactions in the body. The rapid economic development and the advancement of science and technology have made developed countries and some developing countries step into an aging society. Population aging has become a global social problem. By the end of 2009, China's elderly population aged 60 and above reached 167.14 million, accounting for 12.5% of the total population, and it is expected to reach 243 million by 2020. With the growth of age and the aging of the body, diseases related to aging, such as cardiovascular, nervous system diseases, tumors, diabetes, etc., threaten the life span and quality of life of human beings. Therefore, more and more studies are focused on finding substances with anti-aging effects and studying their anti-aging mechanisms. A variety of natural products have been proven to have anti-aging effects. However, at present, the sources of anti-aging substances are relatively single, the cost of separation and purification is high, and the anti-aging drugs have certain side effects, which limits the wide-scale application of these substances. The safety of probiotics is high, and the cost of separation and purification is low. If the anti-aging function of probiotics can be clarified and their anti-aging mechanism can be determined, the prospect of using probiotics as anti-aging products will be very broad.

Lactobacillus salivarius is a lactobacillus widely found in humans and animals. Lactobacillus salivarius can be detected in human oral cavity, breast milk and women's vagina (Li Shaoping, 1991; Olivares et al., 2006). It is the original bacterium of the human body and one of the normal flora existing in the human body; in addition to the human body, Lactobacillus salivarius has also been isolated from the intestines of chickens, pigs, and rabbits (Chen Guiyin, 2006; Tang Xiaoli, 2007; Luo Rui, 2011). Moreover, Lactobacillus salivarius is not a common pathogenic bacteria, and there are few reports of Lactobacillus salivarius causing diseases. Therefore, many studies have conducted functional studies on the screened Lactobacillus salivarius strains, with a view to using them as probiotics for humans and animals.

Contents of the invention

The object of the present invention is to provide a Lactobacillus salivarius with anti-aging function.

In order to achieve the above object, on the one hand, the present invention provides Lactobacillus salivarius, which is characterized in that the preservation number of the Lactobacillus salivarius is CGMCC No.6288.

In a second aspect, the present invention provides a method for preparing a functional food composition, the method comprising: inactivating the living cells of Lactobacillus salivarius as described above to obtain cell substances.

In a third aspect, the present invention provides a functional food composition prepared by the above method.

In a fourth aspect, the present invention provides a functional food composition, which is characterized in that the functional food composition contains the live bacteria of Lactobacillus salivarius as described above.

In the fifth aspect, the present invention provides the application of Lactobacillus salivarius as mentioned above in the preparation of anti-aging functional food composition.

Both the live cells and the inactivated cells of Lactobacillus salivarius provided by the invention have the effect of delaying aging.

Other features and advantages of the present invention will be described in detail in the following detailed description.

biological deposit

The Lactobacillus salivarius of the present invention was preserved on June 25, 2012 in the General Microbiology Center of the China Microbiological Culture Collection Management Committee (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Microbiological Research, Chinese Academy of Sciences Institute, postal code: 100101) (the abbreviation of the depository unit is CGMCC), and the deposit number is CGMCCNo.6288.

detailed description

Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

On the one hand, the present invention provides a Lactobacillus salivarius, and the preservation number of the Lactobacillus salivarius is CGMCC No.6288.

The Lactobacillus salivarius of the present invention is isolated from the feces of long-lived old people in Bama County, Guangxi.

The Lactobacillus salivarius provided by the present invention can produce a large amount of live cells of Lactobacillus salivarius after being cultivated. The method of cultivation has no special requirements, as long as the Lactobacillus salivarius can proliferate, for example, 10 ⁷ orders of magnitude The bacterial strain is inoculated into a lactobacillus medium, and cultured at a temperature of 15-38° C. for 8-72 hours under anaerobic or aerobic conditions to obtain a culture solution. The lactobacillus culture medium can be various well-known culture mediums suitable for lactobacillus, for example, can be milk and/or "Lactobacillus - Biological Basis and Application" (Yang Jiebin, Light Industry Press, published in 1996) Lactic acid bacteria (MRS) medium described above.

The present invention can further isolate the living cells of Lactobacillus salivarius in the above-mentioned culture medium, and the method of separation is not particularly limited, as long as the cells can be enriched from the culture solution, for example, by centrifugation and/or filtration The method is realized, and the conditions of the centrifugation and the filtration can be known conditions, and the present invention will not repeat them here.

In a second aspect, the present invention provides a method for preparing a functional food composition, the method comprising: inactivating the living cells of Lactobacillus salivarius as described above to obtain cell substances.

The inventors of the present invention unexpectedly found in the research that the bacterial substance obtained after inactivation at 65-85° C. for 0.5-1.5 h has a more significant anti-aging effect. Therefore, the conditions for inactivation preferably include: the temperature is 65-85°C, and the time is 0.5-1.5h.

The method of the present invention also includes adding bacterial substances to food.

According to the present invention, although the purpose of the present invention can be achieved by adding the bacterial substance to the food, that is, the effect of delaying aging, but preferably, based on the total weight of the functional food composition, the total weight of the bacterial substance The added amount is 10-70% by weight, more preferably 30-50% by weight. In the above preferred conditions, the anti-aging effect of the functional food composition is more significant.

In the present invention, the food can be any type of food, such as fruit juice products, dairy products, soybean products and the like. Food can also vary depending on who is eating it. The functional food composition may also contain conventional additives, such as spices, minerals, vitamins, stabilizers, thickeners, preservatives and the like.

In a third aspect, the present invention provides a functional food composition prepared by the above method.

In a fourth aspect, the present invention provides a functional food composition, which contains the above-mentioned live Lactobacillus salivarius.

According to the present invention, although the functional food composition contains the above-mentioned living cells of Lactobacillus salivarius, the purpose of the present invention can be achieved, that is, the effect of delaying aging can be achieved. But preferably, based on the total weight of the functional food composition, the content of live Lactobacillus salivarius is 10 ⁵ -10 ¹⁰ CFU/g, more preferably 10 ⁷ -10 ⁹ CFU/g. In the above preferred conditions, the anti-aging effect of the functional food composition is more significant.

The functional food composition that meets the above requirements may include the culture fluid of Lactobacillus salivarius (for example, fermented milk products produced by fermentation of the Lactobacillus salivarius), isolated live cells of Lactobacillus salivarius, and the like.

In the present invention, the functional food composition also contains food, and the food is as mentioned above, and will not be repeated here.

In the fifth aspect, the present invention provides the application of Lactobacillus salivarius as mentioned above in the preparation of anti-aging functional food composition.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

In addition, various combinations of different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

Example

The following examples will further illustrate the present invention, but do not limit the present invention thereby.

(1) Using nematodes as subjects

C. elegans is the most clearly studied model organism in the fields of development, apoptosis, and aging. Its life cycle is short and it is sensitive to the external environment. Because of the high homology of aging signals, the research on lower organisms is of great significance to the aging of mammals and humans. Direct reference (Greer and Brunet, 2008). Therefore, nematodes were used as test subjects in the following Examples and Comparative Examples.

In the following examples and comparative examples:

The nematode was Caenorhabditis elegans strain, wild-type Caenorhabditis legans N2, purchased from the American Nematode Genetics Center (Caenorhabditis Genetics Center (CGC)).

Strains: nematode standard food Escherichia coli (E.coli) OP50, purchased from American Center for Nematode Genetics; Lactobacillus salivarius A is the Lactobacillus salivarius of the present invention (the bacterial strain was preserved in China Microorganisms on June 25, 2012 General Microbiology Center of Species Preservation Management Committee (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101) (the abbreviation of the depository unit is CGMCC), and the deposit number is CGMCCNo.6288) .

The basic nematode operation method refers to the Wormbook standard nematode culture method.

1M Potassium Phosphate Buffer (pH 6.0): 108.3gKH ₂ PO ₄ , 46.8gK ₂ HPO ₄ ·3H ₂ O, distilled water to 1L, sterilized at 121°C for 15min for later use.

NGM medium: 3.0g NaCl, 20g agar powder, 2.5g bacto-peptone, add 970mL of distilled water, sterilize at 121°C for 15min, place in a 60°C water bath, add the following ingredients under sterile conditions: _1mL1MMgSO4 , _1mL1MCaCl2 , 25mL1M potassium phosphate buffer solution, 3.2mL of 5mg/mL cholesterol in ethanol solution; after mixing, pour the plate. After the plate is solidified, place it at room temperature for 2 days, volatilize the water and check that the plate is free from bacterial contamination, and then store it in a sealed box at 4°C for future use.

mNGM medium: before pouring the NGM medium into the plate, add filter-sterilized FUdR and carbenicillin under sterile conditions to a final concentration of 50 μM and 0.5 mM respectively, mix well and pour into the plate, and place it at room temperature for 2 days to evaporate the water after solidification Afterwards, the plate can be smeared with bacteria or stored in a sealed box at 4°C, and used within 1 month.

M9 buffer: 3gKH ₂ PO ₄ , 15.12gNa ₂ HPO ₄ ·12H ₂ O, 5gNaCl, add distilled water to make up to 1L, sterilize at 121°C for 15min, cool to room temperature, add 1mL 1MMgSO ₄ aseptically, mix well and set aside.

Nematode lysate: 1mL 8M NaOH, 0.6mL 9% sodium hypochlorite solution, 3.4mL sterile ddH ₂ O, mixed well before use, ready to use.

LB medium: tryptone 10g, yeast extract powder 5g, NaCl 10g (add 15g agar to the solid medium), add 950mL distilled water, adjust the pH to 7.0, and sterilize at 121°C for 15 minutes for later use.

MRS medium: purchased from Beijing Land Bridge Company.

Example 1.1

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

(1) Inoculate Escherichia coli OP50 into LB liquid medium with 1% inoculum amount, shake and culture in a constant temperature shaker at 37°C and 220rpm for 8 hours to the end of the logarithm, and obtain Escherichia coli OP50 culture fluid; inoculate Lactobacillus salivarius A with 1% The inoculation amount of the inoculum was inoculated into MRS liquid medium, and cultured in nitrogen-filled anaerobic bottle at 37°C for 12 hours to the end of logarithm to obtain Lactobacillus salivarius A culture solution.

Centrifuge the above-mentioned Escherichia coli OP50 and Lactobacillus salivarius A culture solution at 4000×g for 15 minutes to collect the bacteria, repeat centrifugation and washing twice with M9 buffer, then centrifuge at 16000×g at 4°C for 15 minutes, remove all supernatant, Weigh the wet weight of the bacteria. Resuspend the bacteria with a certain amount of M9, so that the final concentration of the bacteria is 400mg/mL, and distribute them in 1.5mL centrifuge tubes as concentrated bacteria.

Heat inactivate the above two kinds of concentrated bacteria at 75°C for 1 hour respectively. After cooling, mix Escherichia coli OP50 and Lactobacillus salivarius A in a weight ratio of 3:2 (that is, Lactobacillus salivarius is 40% by weight) to obtain a mixture, and mix the mixture according to Suspend in M9 buffer at a ratio of 400 mg/mL, spread on the surface of mNGM plates with a diameter of 60 mm, so that each plate has a bacterial cell volume of 10 mg, a total of 6 plates, and seal and store at 4 °C for later use.

(2) On the surface of NGM medium with a diameter of 90 mm, evenly spread 200 μL of Escherichia coli OP50 culture solution, incubate at 37°C for 10 hours, use it as a plate for nematode subculture, and store in a sealed container at 4°C for later use.

Under sterile conditions, use a scalpel to cut a small piece of culture medium about 1cm×1cm from the NGM plate with nematodes growing on it, buckle the front side down on the plate for nematode passage, and culture it at 25°C, and the 60mm plate usually lasts for 7 days Passage once to maintain nematode activity.

The plate on which most of the adult worms are in the egg-laying stage after subculture is used for nematode synchronization:

Add 2mL of LM9 buffer solution to the plate, pipette with a sterile pipette, wash down the worms on the plate as much as possible, transfer the worms to a 1.5mL centrifuge tube, centrifuge at 800×g for 1min, discard the supernatant to collect the worms, add 1mL of LM9 and centrifuge again Wash the worms and suck up the supernatant as much as possible.

Add 1 mL of fresh nematode lysate, immediately vortex for 10 s, centrifuge at 800×g for 1 min, and pipette away the supernatant.

Immediately add 1mL LM9 to wash the parasites, and centrifuge at 800×g for 1min twice in a row to remove residual lysate. Finally, about 100 μL of the M9 suspension containing eggs remained in the tube.

Using a sterile pipette, the eggs were dropped onto the OP50-coated NGM plate, cultured at 25° C. for 48 hours, and nematodes synchronized for 48 hours were obtained.

(3) Place the nematodes synchronized for 48 hours under a stereomicroscope, and use a platinum shovel to pick L4 stage hermaphrodites with transparent half-moon-shaped genital openings on the mNGM plates obtained in step (1). Pick 10 per plate. Only, the plates were cultured at 25°C. Observe the living conditions of nematodes every day. If the nematodes have no active response to the light touch of the platinum shovel, the nematodes are considered dead. Exclude missing, internal hatching and unnatural death nematodes. When all nematodes died, the experiment ended. The average lifespan of nematodes is calculated in Table 1.

Example 1.2

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

(1) Inoculate Escherichia coli OP50 into LB liquid medium with 1% inoculum amount, shake and culture in a constant temperature shaker at 37°C and 220rpm for 8 hours to the end of the logarithm, and obtain Escherichia coli OP50 culture fluid; inoculate Lactobacillus salivarius A with 1% The inoculation amount of the inoculum was inoculated into MRS liquid medium, and cultured in nitrogen-filled anaerobic bottle at 37°C for 12 hours to the end of logarithm to obtain Lactobacillus salivarius A culture solution.

Centrifuge the above-mentioned Escherichia coli OP50 and Lactobacillus salivarius A culture solution at 4000×g for 15 minutes to collect the bacteria, repeat centrifugation and washing twice with M9 buffer, then centrifuge at 16000×g at 4°C for 15 minutes, remove all supernatant, Weigh the wet weight of the bacteria. Resuspend the bacteria with a certain amount of M9, so that the final concentration of the bacteria is 400mg/mL, and distribute them in 1.5mL centrifuge tubes as concentrated bacteria.

Heat inactivate the above two kinds of concentrated bacteria at 65°C for 1.5 hours respectively, and after cooling, mix Escherichia coli OP50 and Lactobacillus salivarius A in a weight ratio of 7:3 (that is, Lactobacillus salivarius is 30% by weight) to obtain a mixture, and the mixture Suspend in M9 buffer at a ratio of 400 mg/mL, spread on the surface of mNGM plates with a diameter of 60 mm, so that the amount of bacteria per plate is 10 mg, a total of 6 plates, and seal and store at 4 °C for later use.

(2) On the surface of NGM medium with a diameter of 90 mm, evenly spread 200 μL of Escherichia coli OP50 culture solution, incubate at 37°C for 10 hours, use it as a plate for nematode subculture, and store in a sealed container at 4°C for later use.

Under sterile conditions, use a scalpel to cut a small piece of culture medium about 1cm×1cm from the NGM plate with nematodes growing on it, buckle the front side down on the plate for nematode passage, and culture it at 25°C, and the 60mm plate usually lasts for 7 days Passage once to maintain nematode activity.

The plate on which most of the adult worms are in the egg-laying stage after subculture is used for nematode synchronization:

Add 2mL of LM9 buffer solution to the plate, pipette with a sterile pipette, wash down the worms on the plate as much as possible, transfer the worms to a 1.5mL centrifuge tube, centrifuge at 800×g for 1min, discard the supernatant to collect the worms, add 1mL of LM9 and centrifuge again Wash the worms and suck up the supernatant as much as possible.

Add 1 mL of fresh nematode lysate, immediately vortex for 10 s, centrifuge at 800×g for 1 min, and pipette away the supernatant.

Immediately add 1mL LM9 to wash the parasites, and centrifuge at 800×g for 1min twice in a row to remove residual lysate. Finally, about 100 μL of the M9 suspension containing eggs remained in the tube.

Using a sterile pipette, the eggs were dropped onto the OP50-coated NGM plate, cultured at 25° C. for 48 hours, and nematodes synchronized for 48 hours were obtained.

(3) Place the nematodes synchronized for 48 hours under a stereomicroscope, and use a platinum shovel to pick L4 stage hermaphrodites with transparent half-moon-shaped genital openings on the mNGM plates obtained in step (1). Pick 10 per plate. Only, the plates were cultured at 25°C. Observe the living conditions of nematodes every day. If the nematodes have no active response to the light touch of the platinum shovel, the nematodes are considered dead. Exclude missing, internal hatching and unnatural death nematodes. When all nematodes died, the experiment ended. The average lifespan of nematodes is calculated in Table 1.

Example 1.3

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

(1) Inoculate Escherichia coli OP50 into LB liquid medium with 1% inoculum amount, shake and culture in a constant temperature shaker at 37°C and 220rpm for 8 hours to the end of the logarithm, and obtain Escherichia coli OP50 culture fluid; inoculate Lactobacillus salivarius A with 1% The inoculation amount of the inoculum was inoculated into MRS liquid medium, and cultured in nitrogen-filled anaerobic bottle at 37°C for 12 hours to the end of logarithm to obtain Lactobacillus salivarius A culture solution.

Centrifuge the above-mentioned Escherichia coli OP50 and Lactobacillus salivarius A culture solution at 4000×g for 15 minutes to collect the bacteria, repeat centrifugation and washing twice with M9 buffer, then centrifuge at 16000×g at 4°C for 15 minutes, remove all supernatant, Weigh the wet weight of the bacteria. Resuspend the bacteria with a certain amount of M9, so that the final concentration of the bacteria is 400mg/mL, and distribute them in 1.5mL centrifuge tubes as concentrated bacteria.

Heat inactivate the above two concentrated bacteria at 85°C for 0.5h, respectively, and mix Escherichia coli OP50 and Lactobacillus salivarius A at a weight ratio of 1:1 (that is, Lactobacillus salivarius is 50% by weight) after cooling to obtain a mixture. Suspend in M9 buffer at a ratio of 400 mg/mL, spread on the surface of mNGM plates with a diameter of 60 mm, so that the amount of bacteria per plate is 10 mg, a total of 6 plates, and seal and store at 4 °C for later use.

(2) On the surface of NGM medium with a diameter of 90 mm, evenly spread 200 μL of Escherichia coli OP50 culture solution, incubate at 37°C for 10 hours, use it as a plate for nematode subculture, and store in a sealed container at 4°C for later use.

Under sterile conditions, use a scalpel to cut a small piece of culture medium about 1cm×1cm from the NGM plate with nematodes growing on it, buckle the front side down on the plate for nematode passage, and culture it at 25°C, and the 60mm plate usually lasts for 7 days Passage once to maintain nematode activity.

The plate on which most of the adult worms are in the egg-laying stage after subculture is used for nematode synchronization:

Add 2mL of LM9 buffer solution to the plate, pipette with a sterile pipette, wash down the worms on the plate as much as possible, transfer the worms to a 1.5mL centrifuge tube, centrifuge at 800×g for 1min, discard the supernatant to collect the worms, add 1mL of LM9 and centrifuge again Wash the worms and suck up the supernatant as much as possible.

Add 1 mL of fresh nematode lysate, immediately vortex for 10 s, centrifuge at 800×g for 1 min, and pipette away the supernatant.

Immediately add 1mL LM9 to wash the parasites, and centrifuge at 800×g for 1min twice in a row to remove residual lysate. Finally, about 100 μL of the M9 suspension containing eggs remained in the tube.

Using a sterile pipette, the eggs were dropped onto the OP50-coated NGM plate, cultured at 25° C. for 48 hours, and nematodes synchronized for 48 hours were obtained.

(3) Place the nematodes synchronized for 48 hours under a stereomicroscope, and use a platinum shovel to pick L4 stage hermaphrodites with transparent half-moon-shaped genital openings on the mNGM plates obtained in step (1). Pick 10 per plate. Only, the plates were cultured at 25°C. Observe the living conditions of nematodes every day. If the nematodes have no active response to the light touch of the platinum shovel, the nematodes are considered dead. Exclude missing, internal hatching and unnatural death nematodes. When all nematodes died, the experiment ended. The average lifespan of nematodes is calculated in Table 1.

Example 1.4

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

Carry out the nematode lifespan test according to the method of embodiment 1.1, the difference is that in step (1), after inactivation and cooling, Escherichia coli OP50 and Lactobacillus salivarius A are mixed in a weight ratio of 3:7 (that is, Lactobacillus salivarius is 70% by weight) )mix. The average lifespan of nematodes is calculated in Table 1.

Example 1.5

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

Carry out nematode lifespan test according to the method of embodiment 1.1, difference is, in step (1), inactivated and cooled Escherichia coli OP50 and Lactobacillus salivarius A by weight ratio of 9:1 (that is, Lactobacillus salivarius is 10% by weight )mix. The average lifespan of nematodes is calculated in Table 1.

Example 1.6

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

The nematode longevity test was carried out according to the method in Example 1.1, except that the inactivation temperature was 90° C. and the inactivation time was 0.3 h. The average lifespan of nematodes is calculated in Table 1.

Example 1.7

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

The lifespan test of nematodes was carried out according to the method in Example 1.1, except that the inactivation temperature was 60° C. and the inactivation time was 2.0 h. The average lifespan of nematodes is calculated in Table 1.

Comparative example 1.1

The nematode lifespan test was carried out according to the method of Example 1.1, except that Lactobacillus salivarius A was replaced by the strain disclosed in CN101538545A. The average lifespan of nematodes is calculated in Table 1.

Comparative example 1.2

Carry out the nematode lifespan test according to the method of Example 1.1, the difference is that after inactivation and cooling, Escherichia coli OP50 is suspended in the M9 buffer according to the ratio of 400 mg/mL, and coated on the surface of the mNGM plate, that is, the surface of the mNGM plate is only coated with Cloth inactivated cooled E. coli OP50. The average lifespan of nematodes is calculated in Table 1.

Table 1

Average lifespan of nematodes (days) Example 1.1 24.1±0.4 Example 1.2 23.1±0.6 Example 1.3 24.3±0.5 Example 1.4 18.9±0.4 Example 1.5 18.7±0.9 Example 1.6 19.1±0.2 Example 1.7 20.0±0.3 Comparative example 1.1 17.8±0.2 Comparative example 1.2 17.2±1.0

Comparing Example 1.1 with Comparative Example 1.1 and Comparative Example 1.2, it can be seen that the Lactobacillus salivarius of the present invention has the effect of delaying aging, and can be used to feed nematodes after inactivation, which can significantly prolong the lifespan of nematodes.

Comparing Example 1.1 with Example 1.4 and Example 1.5 respectively, it can be seen that Escherichia coli OP50 and Lactobacillus salivarius A are mixed after inactivation and cooling, based on the total weight of the mixture, the addition of Lactobacillus salivarius A is 30-50% by weight, can significantly prolong the lifespan of nematodes; comparing Example 1.1 with Example 1.6 and Example 1.7, it can be seen that inactivation at 65-85°C for 0.5-1.5h is more conducive to prolonging nematodes lifespan.

(2) Using mice as subjects

In the following examples and comparative examples:

Kunming male mice: purchased from Beijing Weitong Lihua Company, weighing 20±2g.

Lactobacillus salivarius A is the Lactobacillus salivarius of the present invention (this bacterial strain was preserved on June 25, 2012 in the General Microbiology Center of China Microbiological Culture Collection Management Committee (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101) (the abbreviation of the depository unit is CGMCC), and the deposit number is CGMCCNo.6288).

MRS medium: purchased from Beijing Land Bridge Company.

Coomassie Brilliant Blue G250: purchased from Shanghai Huyu Biological Co., Ltd.

For the determination of liver lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity, see "Anti-aging effect of water-soluble yam polysaccharide on mice" (Zhan Tong, Tao Jing, Wang Shuru , Advances in Pharmacy, Volume 23, Issue 6, 1999):

Determination of liver lipofuscin content: Take the liver, make a 5% homogenate with chloroform-methanol mixture, bathe in warm water at 40°C for 5 minutes, centrifuge at 3000rpm for 10 minutes, take the supernatant to measure fluorescence, the emission wavelength is 435nm, and the excitation wavelength 365nm, set the sensitivity × 1, the slit 10nm, adjust the fluorescence intensity of the instrument to 50 with 0.1μg/mL quinine sulfate.

Brain MAO-B (type B monoamine oxidase) activity measurement: take brain tissue, add ice-cold 0.2mol/L pH7.4 phosphate buffer (0.1mol/LK ₂ HPO ₄ 80.2ml mixed with 0.1mol/LKH ₂ PO ₄ 19.8ml ), ultrasonically homogenate twice, and centrifuge at 3000rpm for 10 minutes at 4°C, suspend the precipitate in PBS solution (pH7.4), shake and mix in a water bath, centrifuge at 3000rpm for 10 minutes, measure the cyclohexane layer at 242nm Absorbance value and protein concentration of crude enzyme solution were measured with Coomassie Brilliant Blue G250, bovine serum albumin was used as standard protein, and enzyme activity unit was defined as the amount of enzyme that produced a change in optical density of 0.01/3h at 37°C.

Determination of brain SOD (superoxide dismutase) activity: brain tissue was taken, and the tissue sample was determined using the SOD ultra-trace rapid determination reagent of the Department of Diseases and Research of Nanjing Railway Medical College. The enzyme activity is expressed in U/mg enzyme amount, and the calculation formula is: [ (optical density of control tube-optical density of measurement tube)/optical density of control tube]/50/100×sample dilution/sample protein concentration. The protein concentration of the sample was determined with Coomassie brilliant blue G250, and calf serum was used as the standard protein.

Determination of brain Na ⁺ /K ⁺ -ATPase activity: take 0.1 g of brain tissue, and quickly add 1 ml of extract (containing 250 mmol/L sucrose, 30 mmol/L histidine, 5 mmol/L EDTA, and 10% by weight of deoxycorticosterone. Homogenize in ice bath, filter with three layers of gauze to get crude enzyme solution, and measure protein concentration with Coomassie Brilliant Blue G250. Enzymatic reaction is divided into two groups: Group A, add 0.4mL medium ATP solution (MgCl ₂ 6mmol/L, NaCl 100mmol /L, KCl20mmol/L, Tris30mmol/L, ATP3mmol/L), 50μL medium solution (MgCl ₂ 6mmol/L, NaCl100mmol/L, KCl20mmol/L, Tris30mmol/L) and crude enzyme solution 50μL; group B, add Quabain to inhibit For Na ⁺ /K ⁺ -ATPase, replace 50 μL of medium with 50 μL of 5 mmol/L Quabain, and the rest are the same. React in a water bath at 37°C for 20 minutes, add 0.3 mL of 30% ice-cold trichloroacetic acid, transfer to an ice bath, and operate at 1500 rpm Centrifuge for 5 minutes, take supernatant 0.5ml, and measure its inorganic phosphorus content. The steps are: take 0.5mL measurement solution, add 2mL chromogen (1 volume of 10% ammonium molybdate + 9 volumes of freshly prepared 9.15% FeSO ₄ 7H ₂ O) and 2mL of distilled water, react at 25°C for 8 minutes, measure the absorbance at 652nm, use the standard phosphorus solution as a standard curve to obtain the content of inorganic phosphorus, and finally the Na ⁺ /K ⁺ -ATPase decomposed Inorganic phosphorus content is the amount of group A-B. Enzyme activity is expressed as μmolpi/h·rmg protein, and the protein concentration is measured with Coomassie brilliant blue G250, and calf serum is used as the standard protein.

Example 2.1

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

(1) Lactobacillus salivarius A was inoculated into 10 L of MRS medium with a sterile inoculation loop, and cultured at 37° C. for 12 hours under anaerobic conditions to obtain a culture solution. The obtained culture solution was centrifuged at a speed of 4000×g for 10 minutes, and then the supernatant was discarded to obtain live cells of Lactobacillus salivarius A.

(2) Randomly select 15 Kunming male mice, inject D-galactose 0.12mg/g behind the eyeballs every day, and feed drinking water containing 1×10 ⁹ CFU/g of live Lactobacillus salivarius A, for one month in a row. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Example 2.2

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

(1) Lactobacillus salivarius A was inoculated into 10 L of MRS medium with a sterile inoculation loop, and cultured at 37° C. for 12 hours under anaerobic conditions to obtain a culture solution. The obtained culture solution was centrifuged at a speed of 4000×g for 10 minutes, and then the supernatant was discarded to obtain live cells of Lactobacillus salivarius A.

(2) Randomly select 15 Kunming male mice, inject D-galactose 0.12 mg/g behind the eyeballs every day, and feed drinking water containing 1×10 ⁸ CFU/g of live Lactobacillus salivarius A, for one month in a row. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Example 2.3

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

(1) Lactobacillus salivarius A was inoculated into 10 L of MRS medium with a sterile inoculation loop, and cultured at 37° C. for 12 hours under anaerobic conditions to obtain a culture solution. The obtained culture solution was centrifuged at a speed of 4000×g for 10 minutes, and then the supernatant was discarded to obtain live cells of Lactobacillus salivarius A.

(2) Randomly select 15 Kunming male mice, inject D-galactose 0.12 mg/g behind the eyeballs every day, and feed drinking water containing 1×10 ⁷ CFU/g live bacteria of Lactobacillus salivarius A, for one month in a row. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Example 2.4

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

The experiment was carried out according to the method of Example 2.1, except that the content of live bacteria of Lactobacillus salivarius A in the drinking water was 1×10 ¹⁰ CFU/g. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Example 2.5

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

The experiment was carried out according to the method of Example 2.1, except that the content of live bacteria of Lactobacillus salivarius A in the drinking water was 1×10 ⁵ CFU/g. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Example 2.6

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

The experiment was carried out according to the method of Example 2.1, except that the living cells of Lactobacillus salivarius A obtained in step (1) were heat-inactivated at 75°C for 1 hour to obtain cell substances, which were added to drinking water Without adding live bacteria, the content of bacteria in drinking water is 40% by weight. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Example 2.7

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

The experiment was carried out according to the method of Example 2.1, except that the live cells of Lactobacillus salivarius A obtained in step (1) were heat-inactivated at 65°C for 1.5 hours to obtain cell substances, which were added to drinking No live bacteria are added to the water, and the content of the bacteria in the drinking water is 50% by weight. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Example 2.8

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

The experiment was carried out according to the method of Example 2.1, the difference was that the live cells of Lactobacillus salivarius A obtained in step (1) were heat-inactivated at 85°C for 0.5h to obtain cell substances, which were added to drinking No live bacteria are added to the water, and the content of the bacteria in the drinking water is 30% by weight. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Example 2.9

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

The experiment was carried out according to the method of Example 2.6, except that the content of bacterial substances in the drinking water was 65% by weight. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Example 2.10

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

The experiment was carried out according to the method of Example 2.6, except that the content of bacterial substances in the drinking water was 15% by weight. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Example 2.11

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

The test was carried out according to the method of Example 2.6, except that the inactivation temperature was 90°C and the inactivation time was 0.3h. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Example 2.12

This example is used to illustrate the anti-aging effect of Lactobacillus salivarius of the present invention.

The test was carried out according to the method of Example 2.6, except that the inactivation temperature was 60° C. and the inactivation time was 2.0 h. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Comparative example 2.1

The experiment was carried out according to the method of Example 2.1, except that the bacterial strain disclosed in CN101538545A was used instead of Lactobacillus salivarius A. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Comparative example 2.2

The experiment was carried out according to the method of Example 2.6, except that Lactobacillus salivarius A was replaced by the bacterial strain disclosed in CN101538545A. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Comparative example 2.3

The experiment was carried out according to the method of Example 2.1, the difference was that no strains were added to the drinking water. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Comparative example 2.4

The experiment was carried out according to the method of Example 2.1, except that D-galactose was not injected and strains were not added to the drinking water. The hepatic lipofuscin content, brain MAO-B activity, brain SOD activity and brain Na ⁺ /K ⁺ -ATPase activity were measured in Kunming male mice. The results are shown in Table 2.

Table 2

Free radicals produced during metabolism can act on polyunsaturated fatty acids in biomembrane phospholipids to generate lipid peroxides, such as malondialdehyde (MDA), and then cross-link with phosphatidylethanolamine and proteins to form macromolecular complexes- Lipofuscin (senile pigment), which is a sign of aging in the body.

The activity of MAO-B in the human brain increases with age after the age of 45. Fowier found that the activity of MAO-B in the 19th region of the human brain is positively correlated with age. The change of MAO-B in brain tissue with age can cause the disorder of catecholamine content in the brain , Promoting the imbalance of physiological activities, leading to aging.

SOD is an important class of oxygen free radical scavenging enzymes in the body, but with age, the activity of SOD in body tissues gradually decreases.

Na ⁺ /K ⁺ -ATPase, also known as sodium-potassium pump, is an extremely important membrane enzyme that exists widely in organisms. This enzyme plays an important role in maintaining physiological activities, body temperature, normal metabolism, and ion balance inside and outside the cell membrane. During biological aging, various biochemical changes will occur, among which the changes in brain cell function are more obvious, which may be related to the reduction of brain cell energy. It has been reported that the Na ⁺ /K ⁺ -ATPase activity in some tissues of aged rats was lower than that of young rats.

Comparing Comparative Example 2.3 with Comparative Example 2.4, it can be seen that injecting D-galactose behind the eyeballs of mice can accelerate the aging of mice. Comparing Example 2.1 to Example 2.12 with Comparative Example 2.3, it can be seen that Lactobacillus salivarius of the present invention has the effect of delaying aging; Example 2.1 is compared with Comparative Example 2.1, and Example 2.6 is carried out with Comparative Example 2.2 It can be seen from the comparison that the Lactobacillus salivarius of the present invention has a more significant anti-aging effect than the strain disclosed in CN101538545A.

Comparing Example 2.1 with Example 2.4 and Example 2.5 respectively, it can be seen that the content of live bacteria of Lactobacillus salivarius in drinking water is 10 ⁷ -10 ⁹ CFU/g, which is more conducive to delaying aging; 2.6 Compared with Example 2.9 and Example 2.10 respectively, it can be seen that the content of the thalline substance obtained after the inactivation of the live thalline of Lactobacillus salivarius in drinking water is 30-50% by weight, which is more conducive to delaying aging; Comparing Example 2.6 with Example 2.11 and Example 2.12, it can be seen that inactivation at 65-85°C for 0.5-1.5h is more beneficial to delay aging.

Both the live cells and the inactivated cells of Lactobacillus salivarius provided by the invention have the effect of delaying aging.

Claims (12)

1. A Lactobacillus salivarius (Lactobacillus salivarius), characterized in that the preservation number of the Lactobacillus salivarius is CGMCCNo.6288. 2. A method for preparing a functional food composition, the method comprising: inactivating the living cells of Lactobacillus salivarius according to claim 1 to obtain cell substances. 3. The method according to claim 2, wherein the inactivation conditions include: a temperature of 65-85° C. and a time of 0.5-1.5 h. 4. The method according to claim 2 or 3, wherein the method further comprises adding the bacterial substance to food. 5. The method according to claim 4, wherein, based on the total weight of the functional food composition, the added amount of the bacterial substance is 10-70% by weight. 6. The method according to claim 4, wherein, based on the total weight of the functional food composition, the added amount of the bacterial substance is 30-50% by weight. 7. A functional food composition prepared by the method according to any one of claims 2-6. 8. A functional food composition, characterized in that, the functional food composition contains the living cells of Lactobacillus salivarius according to claim 1. 9. The functional food composition according to claim 8, wherein, based on the total weight of the functional food composition, the content of the living cells of Lactobacillus salivarius is 10 ⁵ -10 ¹⁰ CFU/g. 10. The functional food composition according to claim 8, wherein, based on the total weight of the functional food composition, the content of the live cells of Lactobacillus salivarius is 10 ⁷ -10 ⁹ CFU/g. 11. The functional food composition according to any one of claims 8-10, wherein the functional food composition further contains food. 12. The application of Lactobacillus salivarius according to claim 1 in the preparation of anti-aging functional food composition.