CN107441121B - Fermentation crude extract of streptococcus salivarius, and preparation method and application thereof - Google Patents
Fermentation crude extract of streptococcus salivarius, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a fermented crude extract of streptococcus salivarius, a preparation method and an application thereof, wherein the preparation method comprises the following steps: (1) inoculating streptococcus salivarius CGMCC NO.13308 into a sucrose-containing M17 culture medium for fermentation to obtain a fermentation liquid, and centrifuging the obtained fermentation liquid to obtain a supernatant; (2) adding ammonium sulfate into the supernatant obtained in the step (1), centrifuging and collecting precipitates separated out when the saturation of the ammonium sulfate is 40-80%, dissolving the obtained precipitates, dialyzing, and freeze-drying to obtain the ammonium sulfate. The invention adopts the streptococcus salivarius CGMCC NO.13308 strain to prepare the fermented crude extract for the first time, the fermented crude extract of the streptococcus salivarius has an inhibiting effect on the formation of a streptococcus mutans biofilm, discloses a new application of the streptococcus salivarius CGMCC NO.13308 in producing the streptococcus mutans biofilm formation inhibitor by fermentation, and widens the source of the streptococcus mutans biofilm formation inhibitor.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a fermented crude extract of streptococcus salivarius, and a preparation method and application thereof.
Background
Dental caries, commonly known as dental caries and decayed tooth, is a bacterial disease which can cause secondary pulpitis and periapical periodontitis, and even inflammation of alveolar bone and jaw bone. If not treated in time, the lesion continues to develop, forming a cavity, eventually the crown is completely destroyed and disappears, the end result of which is the loss of teeth. Dental caries is characterized by high incidence rate and wide distribution. The disease is a main common disease in the oral cavity and is one of the most common diseases of human beings, and the world health organization combines the disease with tumor and cardiovascular diseases as three major key diseases for preventing and treating the diseases of the human beings.
There are many factors that cause dental caries, such as bacteria, oral environment (food, saliva), host, etc., wherein the bacteria are necessary conditions for dental caries, and it is generally considered that there are two types of cariogenic bacteria, one of which is acidogenic bacteria, mainly Streptococcus mutans (s.mutans), actinomyces and lactobacillus, which decompose carbohydrate to produce acid, resulting in inorganic demineralization of teeth; the other is gram-positive coccus, which can destroy organic matters and can form caries in teeth after long-term action. Among the above cariogenic bacteria, Streptococcus mutans is recognized by scholars at home and abroad as the most important and major pathogenic bacterium for caries. However, these pathogenic bacteria do not directly colonize the tooth surface, but indirectly adhere to the tooth surface after forming plaque by binding with biofilm (insoluble polysaccharide) produced by streptococcus mutans, and the degree of caries gradually worsens as cariogenic bacteria accumulate on the biofilm. Therefore, the aim of preventing and treating the decayed tooth can be achieved by reducing the yield of the biological film.
At present, most of the reports in the research on the treatment of the dental caries focus on controlling or reducing the quantity of streptococcus mutans in the oral cavity, however, the healthy oral environment is generally in a state that harmful bacteria and beneficial bacteria reach a balance, and excessive reduction of the harmful bacteria can bring about imbalance of oral flora, so as to cause other adverse reactions of human bodies. In addition, some clinical bactericidal drugs against streptococcus mutans, such as ammonia, silver nitrate and other compounds, have strong side effects, stain teeth after long-term use, and are not suitable for anterior tooth treatment. The risk of caries can also be reduced by reducing the yield of streptococcus mutans biofilm, and the inherent balance of oral flora is not destroyed or side effects are generated, but the reports in the aspect are relatively less, particularly the research related to microorganisms is extremely limited, and the applicable microorganism resources are quite deficient. Therefore, it is urgent to screen and provide novel strains of lactic acid bacteria having the activity of inhibiting the biofilm of Streptococcus mutans and to prepare crude extracts having the corresponding functions therefrom. These are problems that those skilled in the art are faced with.
Disclosure of Invention
Based on the technical problems, the invention provides a crude fermentation extract of streptococcus salivarius, a preparation method and application thereof, wherein the crude fermentation extract of streptococcus salivarius has an effect of inhibiting the formation of streptococcus mutans biofilms.
Specifically, in one aspect, the invention provides a preparation method of a crude fermentation extract of streptococcus salivarius, which comprises the following steps:
(1) inoculating streptococcus salivarius CGMCC NO.13308 into a sucrose-containing M17 culture medium for fermentation to obtain a fermentation liquid, and centrifuging the obtained fermentation liquid to obtain a supernatant;
(2) adding ammonium sulfate into the supernatant obtained in the step (1), centrifuging and collecting precipitates separated out when the saturation of the ammonium sulfate is 40-80%, dissolving the obtained precipitates, dialyzing, and freeze-drying to obtain the ammonium sulfate.
The Streptococcus salivarius (Streptococcus salivarius) CGMCC No.13308 strain is preserved in China general microbiological culture Collection center (CGMCC) in 2016, 11 and 15 days, and the preservation address is as follows: west road No.1, north chen, chaoyang district, beijing, zip code: 100101.
preferably, in step (1), the inoculation amount of streptococcus salivarius CGMCC NO.13308 is 7x106～3.5x107cfu/mL. When the inoculation amount is too much or too little, the propagation speed of the streptococcus salivarius CGMCC NO.13308 strain is slow, so that the inhibitory activity of the obtained fermentation crude extract on the formation of streptococcus mutans biofilms is reduced.
Preferably, in the step (1), the sucrose content in the M17 culture medium is 0.25-5% (w/v). When the content of the sucrose is too high or too low, the crude extract is not generated by the fermentation of the strains.
Preferably, in the step (1), the fermentation temperature is 26-42 ℃, and the fermentation time is 8-24 h. When the fermentation temperature is too high or too low, the metabolism of the streptococcus salivarius CGMCC NO.13308 is slow, so that the inhibitory activity of the fermentation crude extract on the formation of streptococcus mutans biofilms is reduced.
Preferably, in step (1), the fermentation is an anaerobic culture. Under the anaerobic condition, the streptococcus salivarius CGMCC NO.13308 strain can be rapidly propagated and fermented to generate a crude extract with inhibitory activity on the formation of a streptococcus mutans biofilm.
Preferably, in the step (1), the centrifugation speed is 8000-12000 g, and the centrifugation time is 10-20 min. Within the preferred ranges, a fermentation supernatant having an activity of inhibiting biofilm formation by Streptococcus mutans can be obtained.
Preferably, in the step (2), the centrifugation speed is 8000-12000 g, and the centrifugation time is 10-20 min. Within the preferred ranges, a crude fermentation extract having an activity of inhibiting biofilm formation by Streptococcus mutans can be obtained.
Preferably, in the step (2), the cut-off molecular weight of the dialysis is 8000-12000 Da, and the dialysis time is 1-3 d, so that a fermentation crude extract with the molecular weight of more than 8000-12000 Da can be obtained, and the fermentation crude extract with the molecular weight of more than 8000-12000 Da has the activity of inhibiting the formation of streptococcus mutans biofilms.
In a second aspect, there is also provided a crude fermented extract of streptococcus salivarius prepared by any one of the preparation methods.
In a third aspect, the application of the crude fermentation extract of streptococcus salivarius in preparing a streptococcus mutans biofilm formation inhibitor is further provided. The inhibition rate of 10mg/mL fermentation crude extract solution on the formation of streptococcus mutans biofilm is more than 50%.
The invention has the beneficial effects that: the invention adopts streptococcus salivarius CGMCC NO.13308 strain for the first time to prepare fermented crude extract, the fermented crude extract of streptococcus salivarius has the effect of inhibiting the formation of streptococcus mutans biofilm, discloses a new application of streptococcus salivarius CGMCC NO.13308 in producing the streptococcus mutans biofilm formation inhibitor by fermentation, and widens the source of the streptococcus mutans biofilm formation inhibitor. Meanwhile, the crude fermentation extract of streptococcus salivarius CGMCC NO.13308 has obvious inhibitory activity on the formation of streptococcus mutans biofilm and good stability.
Detailed Description
In order to more clearly illustrate the technical solution of the present invention, the technical solution of the present invention will be further illustrated with reference to the following specific embodiments:
in a specific embodiment, the invention provides a preparation method of a crude fermentation extract of streptococcus salivarius, which comprises the following steps: (1) inoculating streptococcus salivarius CGMCC NO.13308 into a sucrose-containing M17 culture medium for fermentation to obtain a fermentation liquid, and centrifuging the obtained fermentation liquid to obtain a supernatant; (2) adding ammonium sulfate into the supernatant obtained in the step (1), centrifuging and collecting precipitates separated out when the saturation of the ammonium sulfate is 40-80%, dissolving the obtained precipitates, dialyzing, and freeze-drying to obtain the ammonium sulfate.
According to the technical scheme, Streptococcus salivarius (Streptococcus salivarius) CGMCC NO.13308 is adopted for the first time, M17 culture medium is used as a culture medium, and the crude extract with the effect of inhibiting the formation of the Streptococcus mutans biofilm is prepared by centrifugation, ammonium sulfate precipitation, dialysis and freeze drying. Meanwhile, the activity of the fermentation crude extract of streptococcus salivarius prepared by fermenting the culture medium M17 with the streptococcus salivarius CGMCC NO.13308 is remarkable, and the stability is good.
Preferably, in step (1), the inoculation amount of streptococcus salivarius CGMCC NO.13308 is 7x106～3.5x107cfu/mL; preferably 1.4x107～2.8x107cfu/mL, more preferably 2.1X107cfu/mL. When the inoculation amount is too much or too little, the propagation speed of the streptococcus salivarius CGMCC NO.13308 strain is slow, so that the inhibitory activity of the obtained fermentation crude extract on the formation of streptococcus mutans biofilms is reduced.
Preferably, in step (1), the sucrose content in the M17 culture medium is 0.25-5% (w/v), preferably 0.5-3% (w/v), and more preferably 1% (w/v). The sucrose provides a carbon source for the growth of streptococcus salivarius CGMCC NO.13308, and when the content of the sucrose is too high or too low, the fermentation of strains is not favorable for generating a crude extract.
Preferably, in the step (1), the fermentation temperature is 26-42 ℃, preferably 30-38 ℃; more preferably 34 deg.C; the fermentation time is 8-24 h, preferably 30-38 ℃; more preferably 34 deg.c. When the fermentation temperature is too high or too low, the metabolism of the streptococcus salivarius CGMCC NO.13308 is slow, so that the inhibitory activity of the fermentation crude extract on the formation of streptococcus mutans biofilms is reduced.
Preferably, in step (1), the fermentation is an anaerobic culture. The streptococcus salivarius CGMCC NO.13308 is an anaerobic bacterium, and the streptococcus salivarius CGMCC NO.13308 can be rapidly propagated and fermented to generate a crude extract with inhibitory activity on the formation of a streptococcus mutans biofilm under an anaerobic condition.
Preferably, in the step (1), the centrifugal speed is 8000-12000 g, preferably 9000-11000 g, and more preferably 10000 g; the centrifugation time is preferably 10-20 min, more preferably 13-17 min, and still more preferably 15 min. Within the preferred ranges, a fermentation supernatant having an activity of inhibiting biofilm formation by Streptococcus mutans can be obtained.
Preferably, in the step (2), the centrifugation speed is 8000-12000 g, preferably 9000-11000 g, and more preferably 10000 g; the centrifugation time is 10-20 min, preferably 13-17 min, and more preferably 15 min. Within the preferred ranges, a crude fermentation extract having an activity of inhibiting biofilm formation by Streptococcus mutans can be obtained.
Preferably, in step (2), the cut-off molecular weight for dialysis is 8000-12000 Da, more preferably 10000 Da. The preferable dialysis time is 1-3 days, and more preferably 2 days; dialyzing to obtain a crude fermentation extract with the molecular weight of more than 8000-12000 Da, wherein the crude fermentation extract with the molecular weight of more than 8000-12000 Da has the activity of inhibiting the formation of streptococcus mutans biofilms.
In combination with comparative example 1, it can be seen that, when the fermentation parameters are out of the range of the preferable fermentation parameters, the inhibition effect of the crude extract prepared by fermenting the M17 culture medium with streptococcus salivarius CGMCC NO.13308 on the formation of streptococcus mutans biofilms is obviously reduced. In the preferred range, the inoculation amount, the sucrose concentration, the culture temperature, the fermentation time and the ammonium sulfate saturation degree influence each other, so that the biofilm inhibition activity generated by the fermentation of the streptococcus salivarius CGMCC NO.13308 is better.
The following examples further illustrate the above embodiments, but do not therefore limit the invention within the scope of the examples described. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents used in the examples, unless otherwise specified, were all analytical reagents and were purchased from the national pharmaceutical group. Other test instruments, reagents, and strains, unless otherwise specified, are commercially available.
Example 1
1. Materials and methods
(1) Preparation of seeds (fermentation strain):
streptococcus salivarius CGMCC No. 13308: dissolving freeze-dried powder of Streptococcus salivarius CGMCC No.13308 with small amount of sterile distilled water, taking out an inoculating loop, streaking on M17 solid culture medium (purchased from OXOID Co., UK), anaerobic culturing at 37 deg.C for 24h, taking out a single colony with the inoculating loop, placing into 10mL M17 liquid culture medium (purchased from OXOID Co., UK), uniformly dispersing the colony in the liquid culture medium by using vortex oscillator, anaerobic culturing at 37 deg.C for 24h, taking out, inoculating into M17 liquid culture medium (purchased from OXOID Co., UK) with inoculum size of 2% (v/v), anaerobic culturing at 37 deg.C for 24h, centrifuging culture at 15000rpm for 10min, discarding supernatant, washing with sterile distilled water for 2 times, suspending with sterile distilled water of original culture volume, to obtain seed for fermentation, wherein the bacterial concentration of the seed solution is 7x108cfu/mL。
Streptococcus mutans CGMCC 1.2499: dissolving lyophilized powder of Streptococcus mutans CGMCC 1.2499 (purchased from CGMCC) with a small amount of sterile distilled water, taking out a loop by using an inoculating loop, streaking on BHI solid culture medium (purchased from OXOID Co., UK), carrying out anaerobic culture at 37 ℃ for 24h, taking out a single colony by using an inoculating loop, putting the single colony into 10mLBHI liquid culture medium (purchased from OXOID Co., UK), uniformly dispersing the colony in the liquid culture medium by using a vortex oscillator, carrying out anaerobic culture at 37 ℃ for 24h, taking out, inoculating the single colony into the BHI liquid culture medium (purchased from OXOID Co., UK) in an inoculation amount of 2% (v/v), carrying out anaerobic culture at 37 ℃ for 24h, centrifuging the culture at 15000rpm for 10min, discarding supernatant, washing thallus with sterile distilled water for 2 times, and suspending with sterile distilled water in the original culture volume to obtain seeds for fermentationThe bacterial concentration of the seed liquid is 6x108cfu/mL。
(2) The detection method of the crude extract on the inhibition rate of the biofilm formation amount of streptococcus mutans comprises the following steps: dissolving the crude extract in PBS buffer solution of pH6.80 to obtain 10mg/mL crude extract solution, mixing 100 μ L above solution with 100 μ L Streptococcus mutans (10 μ L) resuspended5cfu/mL) of a sucrose (0.25% w/v) BHI liquid medium was simultaneously added to a 96-well plate, anaerobic culture was carried out at 37 ℃ for 24h, the reaction-involved wells were washed 3 times with deionized water to remove free bacteria, the plate was inverted at room temperature, after it was naturally dried, 100. mu.L of 0.5g/L crystal violet solution was added to the wells, staining was carried out for 15min at room temperature, staining solution was removed, a large amount of deionized water was used to wash away unadsorbed crystal violet, 200. mu.L of absolute ethanol was added to each well after natural drying to dissolve adsorbed crystal violet, and a microplate reader was used to measure OD absorbance values of sample groups at 600nmSample setReplacing the crude extract solution with sterile water, performing the same operation at 600nm to obtain corresponding control group absorbance value ODControl groupUsing the formula "inhibition rate ═ 1-ODSample set/ODControl group) × 100% "the inhibition rate on biofilm formation was calculated.
2. Preparation and activity detection of streptococcus mutans biofilm inhibitor crude extract
The streptococcus salivarius CGMCC No.13308 seed is inoculated into M17 liquid culture medium containing 1% (w/v) sucrose in 3% (v/v) for aseptic inoculation, and the fermentation broth is obtained after anaerobic culture at 34 ℃ for 16 h.
Slowly adding ammonium sulfate into the supernatant, centrifuging for 15min at 10000g, collecting precipitate separated out when the saturation of the ammonium sulfate is 50-70%, redissolving the precipitate by distilled water, dialyzing for 2d by a dialysis bag with the interception amount of 10000Da, changing water for 3 times every day, and freeze-drying solution in the dialysis bag after dialysis is finished to obtain the crude extract A of the streptococcus mutans biofilm inhibitor.
The 10mg/mL solution of crude extract A was determined to have a 68% inhibition of Streptococcus mutans biofilm formation.
Example 2
1. The material and the method are as follows: same as example 1
2. Preparation and activity detection of streptococcus mutans biofilm inhibitor crude extract
Inoculating 1% (v/v) of Streptococcus salivarius CGMCC No.13308 seed into M17 liquid culture medium containing sucrose 5% (w/v), anaerobically culturing at 42 deg.C for 24h to obtain fermentation broth, and centrifuging 12000g for 10min to obtain supernatant.
Slowly adding ammonium sulfate into the supernatant, centrifuging for 10min at 12000g, collecting precipitate separated out when the saturation of the ammonium sulfate is 40-60%, redissolving the precipitate by distilled water, dialyzing for 1d by a dialysis bag with the interception amount of 9000Da, changing water for 4 times every day, and freeze-drying the solution in the dialysis bag after dialysis is finished to obtain a crude extract B of the streptococcus mutans biofilm inhibitor.
The 10mg/mL crude extract B solution was determined to have 58% inhibition of Streptococcus mutans biofilm formation.
Example 3
1. The material and the method are as follows: same as example 1
2. Preparation and activity detection of streptococcus mutans biofilm inhibitor crude extract
The seed of streptococcus salivarius CGMCC No.13308 with the inoculation amount of 5% (v/v) is aseptically inoculated into M17 liquid culture medium containing 0.25% (w/v) of sucrose, anaerobic culture is carried out for 8h at 26 ℃ to obtain fermentation liquor, and 8000g of the obtained fermentation liquor is centrifuged for 20min to obtain supernatant.
Slowly adding ammonium sulfate into the supernatant, centrifuging for 20min at 8000g, collecting precipitate separated out when the saturation of the ammonium sulfate is 60-80%, redissolving the precipitate with distilled water, dialyzing for 3d with a dialysis bag with the interception amount of 11000Da, changing water for 2 times every day, and freeze-drying the solution in the dialysis bag after dialysis is finished to obtain a crude extract C of the streptococcus mutans biofilm inhibitor.
The 10mg/mL solution of crude extract C was determined to inhibit the formation of Streptococcus mutans biofilm by 61%.
Example 4
1. The material and the method are as follows: same as example 1
2. Preparation and activity detection of streptococcus mutans biofilm inhibitor crude extract
Inoculating 2% (v/v) of Streptococcus salivarius CGMCC No.13308 seed in M17 liquid culture medium containing 2% (w/v) of sucrose aseptically, anaerobically culturing at 38 deg.C for 12h to obtain fermentation broth, centrifuging 11000g of the obtained fermentation broth for 13min, and collecting supernatant.
Slowly adding ammonium sulfate into the supernatant, centrifuging for 13min at 11000g, collecting precipitate separated out when the saturation of the ammonium sulfate is 40-70%, redissolving the precipitate by distilled water, dialyzing for 2D by a dialysis bag with the interception amount of 8000Da, changing water for 3 times every day, and freeze-drying the solution in the dialysis bag after dialysis is finished to obtain a crude extract D of the streptococcus mutans biofilm inhibitor.
The 10mg/mL solution of crude extract D was determined to have a 60% inhibition of Streptococcus mutans biofilm formation.
Example 5
1. The material and the method are as follows: same as example 1
2. Preparation and activity detection of streptococcus mutans biofilm inhibitor crude extract
The streptococcus salivarius CGMCC No.13308 seed is aseptically inoculated in M17 liquid culture medium containing 0.5% (w/v) of sucrose in an inoculum size of 4% (v/v), anaerobically cultured at 30 ℃ for 20h to obtain fermentation liquor, and the obtained fermentation liquor is centrifuged at 9000g for 17min to obtain supernatant.
Slowly adding ammonium sulfate into the supernatant, centrifuging for 17min at 9000g, collecting precipitate separated out when the saturation of the ammonium sulfate is 40-70%, redissolving the precipitate with distilled water, dialyzing for 1d with a dialysis bag with the cut-off of 12000Da, changing water for 4 times every day, and freeze-drying the solution in the dialysis bag after dialysis is finished to obtain a crude extract E of the streptococcus mutans biofilm inhibitor.
The crude extract E solution of 10mg/mL was determined to have a 56% inhibition of Streptococcus mutans biofilm formation.
Effect example 1Stability of crude extract to inhibition effect of Streptococcus mutans biofilm under refrigeration
The crude extracts A, B, C, D and E prepared in examples 1-5 were stored under refrigeration (8 ℃) for 0, 5, 10 and 15 days and then removed, and the inhibition rate of Streptococcus mutans biofilm by each sample was determined, and the results are shown in Table 1.
TABLE 1 stability of crude extracts on the inhibitory effect of Streptococcus mutans biofilm under refrigeration conditions
As can be seen from Table 1, all crude extracts tested were kept at the same level and stable against Streptococcus mutans biofilm-inhibiting activity for 15 days after refrigeration (8 ℃ C.).
Comparative example 1
The amounts of inoculation, sucrose concentration, incubation temperature, fermentation time and ammonium sulfate saturation in example 1 were adjusted one by one to obtain crude extracts prepared by the following different methods, and the inhibitory effects on the formation of Streptococcus mutans biofilm were determined for each of the crude extracts obtained by the methods described in example 1, and the results are shown in Table 2.
TABLE 2 inhibitory Effect of crude extracts prepared by different methods on the biofilm formation of Streptococcus mutans
Referring to the results shown in Table 2, it was found that when the inoculation amount, sucrose concentration, cultivation temperature, fermentation time and ammonium sulfate saturation in the preparation method of the crude extract were adjusted to be out of the preferable ranges, the prepared crude extract could still inhibit the formation of Streptococcus mutans and its biofilm, but the effect was significantly reduced.
Comparative example 2
Referring to the method described in example 1, the inhibitory effects of crude extracts prepared from Streptococcus salivarius CGMCC No.13308, lactococcus lactis CGMCC1.2472 (purchased from CGMCC), Streptococcus thermophilus (S.thermophilus) ST-BODY-3 (supplied by Hansen, Inc. of Ke.) on the biofilm formation of Streptococcus mutans were compared as follows:
1. materials and methods
(a) Preparation of seeds (fermentation strain):
dissolving freeze-dried powder of streptococcus salivarius CGMCC No.13308, streptococcus thermophilus ST-BODY-3 and lactococcus lactis CGMCC1.2472 with a small amount of sterile distilled water, drawing a ring by using an inoculating ring on an M17 solid medium (purchased from Merck Co. Germany), carrying out anaerobic culture at 37 ℃ for 24h, taking a single colony by using the inoculating ring, putting the single colony into 1mL of M17 liquid (purchased from Merck Co. Germany), uniformly dispersing the colony in the liquid medium by using a vortex shaker, carrying out anaerobic culture at 40 ℃ for 24h, taking the colony out, inoculating the single colony into 50mL of M17 liquid in an inoculation amount of 2% (v/v), carrying out 24h culture at 37 ℃, centrifuging a culture at 9000rpm for 10min, discarding a supernatant, washing the thallus for 2 times by using sterile distilled water, and suspending the thallus in the sterile distilled water with the original culture volume to obtain seeds for fermentation.
(2) The method for detecting the inhibition rate of the crude extract prepared by each strain on the formation amount of the streptococcus mutans biofilm comprises the following steps: the same as in example 1.
2. Preparation and activity detection of crude extract of each strain
Inoculating the strain seeds with 3% (v/v) of inoculum size into M17 liquid culture medium containing sucrose 1% (w/v), anaerobically culturing at 34 deg.C for 16h to obtain fermentation liquid, centrifuging the obtained fermentation liquid at 10000g for 15min, and collecting supernatant.
Slowly adding ammonium sulfate into the supernatant, centrifuging for 15min at 10000g, collecting precipitate separated out when the saturation of the ammonium sulfate is 50-70%, redissolving the precipitate by distilled water, dialyzing for 2d by a dialysis bag with the interception amount of 10000Da, changing water for 3 times every day, and freeze-drying solution in the dialysis bag after dialysis is finished to obtain the crude extract of the streptococcus mutans biofilm inhibitor prepared from each strain. The inhibition of Streptococcus mutans biofilm formation by each crude extract is shown in Table 3.
TABLE 3 inhibitory Effect of crude extracts prepared from different strains on the biofilm formation of Streptococcus mutans
As can be seen from Table 3, other conventional M17 fermenting strains do not have the ability of fermenting M17 to produce substances inhibiting the formation of Streptococcus mutans biofilm, whereas Streptococcus salivarius CGMCC No.13308 can ferment M17 medium to finally obtain substances inhibiting the formation of Streptococcus mutans biofilm.
The streptococcus mutans inhibitor and the preparation method thereof provided by the invention are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (10)
1. A method for preparing a crude fermentation extract of streptococcus salivarius, which is characterized by comprising the following steps:
(1) inoculating streptococcus salivarius CGMCC NO.13308 into a sucrose-containing M17 culture medium for fermentation to obtain a fermentation liquid, and centrifuging the obtained fermentation liquid to obtain a supernatant;
(2) adding ammonium sulfate into the supernatant obtained in the step (1), centrifuging and collecting precipitates separated out when the saturation of the ammonium sulfate is 40-80%, dissolving the obtained precipitates, dialyzing, and freeze-drying to obtain the ammonium sulfate.
2. The method for preparing crude fermentation extract of streptococcus salivarius as claimed in claim 1, wherein the inoculation amount of streptococcus salivarius CGMCC NO.13308 in step (1) is 7x106～3.5x107cfu/mL。
3. The method for preparing the fermented crude extract of streptococcus salivarius according to claim 1, wherein in the step (1), the sucrose content in the M17 culture medium is 0.25-5% (w/v).
4. The method for preparing the fermented crude extract of streptococcus salivarius according to claim 1, wherein in the step (1), the fermentation temperature is 26-42 ℃ and the fermentation time is 8-24 h.
5. The method for preparing fermented crude extract of streptococcus salivarius according to claim 1 wherein in step (1), the fermentation is anaerobic culture.
6. The method for preparing the fermented crude extract of streptococcus salivarius according to claim 1, wherein in the step (1), the centrifugation speed is 8000-12000 g, and the centrifugation time is 10-20 min.
7. The method for preparing the fermented crude extract of streptococcus salivarius according to claim 1, wherein in the step (2), the centrifugation speed is 8000-12000 g, and the centrifugation time is 10-20 min.
8. The method for preparing the fermented crude extract of streptococcus salivarius according to claim 1, wherein in the step (2), the cut-off molecular weight of dialysis is 8000-12000 Da, and the dialysis time is 1-3 d.
9. A crude fermentation extract of Streptococcus salivarius prepared by the method of any one of claims 1 to 8.
10. Use of a crude fermented extract of streptococcus salivarius according to claim 9 in the preparation of an inhibitor of streptococcus mutans biofilm formation.
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| Anti-inflammatory properties of Streptococcus salivarius,a commensal bacterium of the oral cavity and digestive tract.;Ghalia Kaci et al.;《Applied and environmental microbiology》;20140116;第80卷(第3期);第928-934页 * |
| Competitive displacement of Mutans Streptococci and inhibition of tooth decay by Streptococcus salivarius TOVE-R;Jason M.Tanzer et al.;《Infection and immunity》;19850430;第48卷(第1期);摘要 * |
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