CN110122578B - Method for preparing fermented soybean milk powder from leuconostoc mesenteroides, prepared fermented soybean milk powder and application - Google Patents
Method for preparing fermented soybean milk powder from leuconostoc mesenteroides, prepared fermented soybean milk powder and application Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C11/00—Milk substitutes, e.g. coffee whitener compositions
- A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
- A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
- A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
- A23C11/106—Addition of, or treatment with, microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/31—Leuconostoc
- A23V2400/321—Mesenteroides
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Abstract
The invention discloses a method for preparing fermented soybean milk powder from leuconostoc mesenteroides, which comprises the following steps: inoculating Leuconostoc mesenteroides into soybean milk for fermentation and metabolism, and freeze drying to obtain fermented soybean milk powder. The fermented soybean milk powder prepared by the method can obviously reduce the formation amount of streptococcus mutans biomembranes, and discloses a new application of leuconostoc mesenteroides fermented soybean milk powder in resisting dental caries, and the fermented soybean milk powder has a good application prospect. Compared with other strains capable of preparing the soybean milk powder and culture mediums produced and propagated by the strains, the fermented soybean milk powder prepared by the leuconostoc mesenteroides has obvious activity of inhibiting the formation of streptococcus mutans biofilms.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of fermented soybean milk powder. In addition, the invention also relates to fermented soybean milk powder 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 inflammation, 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. The decayed tooth 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. Of the above cariogenic bacteria, Streptococcus mutans is recognized by scholars at home and abroad as the most important and major pathogenic bacteria 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 reports in the research on the treatment of dental caries focus on controlling or reducing the quantity of streptococcus mutans in oral cavity, however, the healthy oral environment is generally in a state of balancing harmful bacteria and beneficial bacteria, and excessive reduction of the harmful bacteria can bring about imbalance of oral flora, thereby causing other adverse reactions of human body. 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. While the risk of caries is also reduced by reducing the production of streptococcus mutans biofilm, without disrupting the inherent balance of the oral flora or producing side effects, relatively few reports have been made in this regard, and in particular, there is very limited research relating to microbial fermentation preparations, and fermentation preparations with suppression of streptococcus mutans biofilm formation are relatively lacking.
Therefore, it is urgent to provide a fermented product having an inhibitory effect on the formation of a Streptococcus mutans biofilm. This is a problem that those skilled in the art are at risk for solving.
Disclosure of Invention
Based on the technical problems, the invention provides a preparation method of fermented soybean milk powder, and the fermented soybean milk powder capable of inhibiting the formation of streptococcus mutans biofilm can be prepared by the method.
Specifically, on the one hand, a preparation method of fermented soybean milk powder is provided, which comprises the following steps: inoculating Leuconostoc mesenteroides into soybean milk, fermenting, and freeze drying to obtain fermented soybean milk powder.
Further, the leuconostoc mesenteroides is CGMCC No.6432, ATCC10830a or CGMCC No. 10064.
Wherein, Leuconostoc mesenteroides (CGMCC No.6432 and CGMCC No. 10064) strains are preserved in China general microbiological culture Collection center (CGMCC) at 8-13 days 2012 and 11-26 days 2014 respectively, and the preservation addresses are as follows: west road No.1, north chen, chaoyang district, beijing, zip code: 100101. the preservation information of CGMCC No.6432 strain is disclosed in patent CN 103013891A; the preservation information of the strain CGMCC No.10064 is disclosed in patent CN 105349477A. Leuconostoc mesenteroides ATCC10830a was purchased from ATCC (American type culture Collection).
Further, the amount of the Leuconostoc mesenteroides inoculated was 1X107～5x107cfu/mL。
Further, the soybean milk is soybean milk.
Further, the solid content of the soybean milk is 1-9% by mass.
Further, the temperature of the fermentation is 15 ℃ to 45 ℃.
Further, the fermentation time is 2-48 h.
Further, the fermentation is static culture.
In a second aspect, there is also provided a fermented soy milk powder, prepared by any of the above-mentioned preparation methods.
Furthermore, the half-inhibition concentration IC50 of the fermented soybean milk powder on the formation of the streptococcus mutans biofilm is less than or equal to 44 mg/mL.
In a third aspect, the application of the fermented soybean milk powder in preparing the anti-dental caries food is also provided.
Compared with the prior art, the preparation method in the technical scheme adopts leuconostoc mesenteroides for the first time, and takes the soybean milk as a culture medium for fermentation to prepare the fermented soybean milk powder with the streptococcus mutans biofilm inhibiting activity, and discloses a new application of the leuconostoc mesenteroides fermented soybean milk powder in resisting dental caries. Compared with other strains capable of preparing the soybean milk powder and culture mediums produced and propagated by the strains, the fermented soybean milk powder prepared by the leuconostoc mesenteroides has obvious activity of inhibiting the formation of streptococcus mutans biofilms.
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 one embodiment, there is provided a method for preparing fermented soybean milk powder, comprising the steps of: inoculating Leuconostoc mesenteroides into soybean milk, fermenting, and freeze drying to obtain fermented soybean milk powder.
According to the preparation method in the technical scheme, the leuconostoc mesenteroides is adopted for the first time, and the soybean milk is used as the culture medium for fermentation to prepare the fermented soybean milk powder with the streptococcus mutans biological membrane inhibition activity. Compared with other strains capable of preparing fermented soybean milk powder or culture media in which the strains can grow and reproduce, the fermented soybean milk powder prepared by leuconostoc mesenteroides has more remarkable inhibiting effect on the formation of streptococcus mutans biofilms and good stability.
In addition, the strain adopted by the preparation method in the technical scheme is a natural edible strain, the adopted fermentation medium (various soybean milks) has wide sources, low cost, nature and safety, and the safety of food is improved while the material cost is reduced.
Further, in the above step, the leuconostoc mesenteroides is CGMCC No.6432, ATCC10830a or CGMCC No.10064, preferably CGMCC No.6432, ATCC10830a, more preferably CGMCC No. 6432.
Further, in the above step, the amount of Leuconostoc mesenteroides inoculated was 1 × 107～5x107cfu/mL; preferably 2x107～4x107cfu/mL, more preferably 3X107cfu/mL。
Further, in the above step, the preferable soybean milk is at least one of soybean, red bean, mung bean, broad bean, white kidney bean and small white kidney bean milk, and more preferably soybean milk.
Further, in the above steps, the preferable content of the soybean milk solid matter is 1-9% by mass; preferably 3% -7%; more preferably 5%. The content of solid matters in the soybean milk has a direct relation with the biofilm inhibiting effect of a final product, when the content of the solid matters is too high, the osmotic pressure of the environment of thalli is increased, the growth of the thalli is influenced, the synthetic amount of the biofilm inhibiting substances is reduced, in addition, the production cost is increased, and when the content of the solid matters is too low, the proliferation effect of viable bacteria is poor due to insufficient nutrient components, and the generation of the biofilm inhibiting substances is also influenced.
Further, in the above step, the preferable fermentation temperature is 15 ℃ to 45 ℃; preferably 25 ℃ to 40 ℃; more preferably 37 deg.c.
Further, in the step, the preferable fermentation time is 2-48 h; preferably 12 to 36 hours; more preferably 24 h.
In the above step, the preferred fermentation method is static culture. Leuconostoc mesenteroides are facultative anaerobes and can grow and reproduce rapidly under the condition of static culture.
Further, in the above step, the preferable freeze-drying is vacuum freeze-drying, and the preferable vacuum freeze-drying conditions are: the ultimate temperature of the plate layer is less than or equal to minus 60 ℃, the ultimate temperature of the cold trap is minus 70 ℃, the thickness of the plate layer loading is 0.5-2.0 mm, and the vacuum degree is 10-30 Pa.
The freeze drying is adopted to prepare the fermented soybean milk into the fermented soybean milk powder, so that the use and the transportation of consumers are more convenient, and meanwhile, the inhibition activity of the freeze-dried fermented soybean milk powder on the streptococcus mutans biomembrane cannot be greatly changed.
It is also clear from the combination of the examples and comparative example 1 that, when the preferred fermentation parameters are out of the range, the inhibitory effect of fermented soybean milk powder prepared from Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.6432 on the formation of Streptococcus mutans biofilm is significantly reduced. In the preferable range, the inoculation amount, the soybean milk concentration, the culture temperature and the fermentation time are mutually influenced, so that the fermented soybean milk powder prepared by Leuconostoc mesenteroides (CGMCC No. 6432) has better inhibition effect on the biofilm formation of streptococcus mutans.
In another embodiment, there is provided a fermented soybean milk powder produced by the method for producing any one of the fermented soybean milk powders described above.
Since the preparation method of the fermented soybean milk powder has the beneficial effects, the fermented soybean milk powder prepared by the preparation method also has corresponding beneficial effects, and the detailed description is omitted.
Furthermore, the half inhibition rate concentration IC50 of the fermented soybean milk powder to the formation of the streptococcus mutans biofilm is not more than 44 mg/mL. The combination of the examples shows that the inhibition effect of the fermented soybean milk powder on the formation of the streptococcus mutans biofilm is obviously higher than that of other strains capable of preparing the fermented soybean milk powder.
The following examples further illustrate the above embodiments, but do not therefore limit the invention within the scope of the examples described. Experimental procedures without specifying specific conditions in the following examples were selected according to the usual procedures and conditions, or according to the commercial instructions.
In the examples below, all the starting materials are commercially available and meet the relevant national standards.
Example 1
1. Materials and methods
(a) Preparation of leuconostoc mesenteroides (zymocyte): dissolving Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.6432 lyophilized powder with a small amount of sterile distilled water, drawing a ring by using an inoculating ring on an M17 solid culture medium (purchased from Merck Co. Germany) containing 2% (w/v) sucrose, carrying out aerobic culture at 30 ℃ for 24h, picking a single colony by using the inoculating ring, placing the single colony into 20mL of M17 liquid (purchased from Merck Co. Germany) containing 2% (w/v) sucrose, uniformly dispersing the colony in a liquid culture medium by using a vortex oscillator, carrying out shaking culture at 30 ℃ and 180rpm for 24h, centrifuging the culture at 9,000rpm for 10min, discarding the supernatant, washing with sterile distilled water for 2 times, and suspending with sterile distilled water of the original culture volume to obtain seeds for fermentation, wherein the bacterial concentration of the seed liquid is 1x109cfu/mL。
Preparation of a Streptococcus mutans bacterial suspension: dissolving lyophilized powder of Streptococcus mutans CGMCC No.1.2499 (from CGMCC, China) in small amount of sterile distilled water, taking out a loop by using an inoculating loop, streaking on BHI solid culture medium (from OXOID Co., UK), performing anaerobic culture at 37 deg.C for 24h, taking out, picking out a single colony by using the inoculating loop, and placing into 10mL BHI liquid culture medium (from OXOID Co., U.K.,british), uniformly dispersing the colonies in a liquid culture medium by using a vortex oscillator, carrying out anaerobic culture at 37 ℃ for 24h, taking out, inoculating the colonies to a BHI liquid culture medium by using an inoculation amount of 2% (v/v), carrying out anaerobic culture at 37 ℃ for 24h, centrifuging the culture at 15000rpm for 10min, discarding the supernatant, washing the thalli for 2 times by using sterile distilled water, and then re-suspending the thalli to 10 degrees by using the BHI liquid culture medium6CFU/mL.
(b) Preparing soybean milk: weighing dried soybean, adding water with five times of mass, soaking at 37 deg.C for 8 hr, removing water, adding wet soybean and water at a certain ratio into a soybean milk machine to obtain soybean milk with desired solid content, and sterilizing at 121 deg.C for 20min to obtain sterile soybean milk with desired solid content.
(c) The inhibition rate of the fermented soybean milk powder on the formation of the streptococcus mutans biofilm and the determination of IC 50:
pretreating fermented soybean milk powder: and (3) dissolving the fermented soybean milk powder in sterile water, and adjusting the pH to 6.80 to obtain a sample to be detected.
Determination of inhibitory Activity: the sterilized magnetic stirrer A (5X 10mM) was immersed in saliva of a healthy population after sterile filtration through a 0.22 μm filter membrane and incubated at 37 ℃ for 4 hours, the magnetic stirrer A was attracted by a sterilized magnetic stirrer B (10X 50mM), rinsed with 10mM PBS (pH7.0), and the magnetic stirrer A was removed and placed in culture wells of a 24-well plate. To the culture well, 1.6mL of BHI broth and 200. mu.L of Streptococcus mutans suspension (10. mu.L) were added6CFU/mL) and 400 μ L of test sample solution or blank control solution (unfermented soy milk), again incubated anaerobically at 37 ℃ for 24h (purchased from Greiner, germany). And sucking the magnetic stirrer A by using a magnetic stirrer B, washing by using PBS, taking down the magnetic stirrer A, placing in a culture hole of a 24-hole plate, and naturally drying. Adding 1.6mL of 0.1% (w/v) crystal violet solution into the culture well for dyeing for 15min, sucking up the magnetic stirrer A by using a magnetic stirrer B, leaching by using PBS to remove redundant dyeing solution, taking down the magnetic stirrer A, placing the magnetic stirrer A in the culture well of a 24-pore plate, and naturally drying in the air. Adding 300 μ L of absolute ethanol into the culture well for decolorization for 15min, transferring 200 μ L into a 96-well plate (purchased from Greiner, Germany), and measuring OD600nm by using a microplate reader, wherein the data is quantitative data corresponding to the forming amount of the deformed streptococcus biofilm.The OD600nm of the blank control and the sample to be tested were measured by the above method, and the inhibition rate of the biofilm inhibitor was calculated. Biofilm inhibition rate (I):
i ═ OD600nm (blank) -OD600nm (test sample))/OD 600nm (blank) × 100%
Determination of the concentration of semi-inhibitory rate IC50 for the formation of streptococcus mutans biofilm: diluting the sample solution to be detected by a sesquidilution method to obtain a sample group to be detected with different concentrations, determining the inhibition rate of the sample to be detected with different concentrations on the formation of the streptococcus mutans biofilm by using the detection method, and calculating the concentration (IC50) of the sample on the formation of the streptococcus mutans biofilm according to the linear relation formed by the concentration and the inhibition rate of the biofilm formation.
2. Preparation method of fermented soybean milk powder
The Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.6432 is aseptically inoculated into soybean milk with solid content of 5% (w/w, soybean mass percent of water, the same below) by an inoculation amount of 3% (v/v, seed liquid accounts for volume percent of fermentation liquid, the same below), and the fermented soybean milk powder A is obtained after standing culture at 37 ℃ for 24 hours, and vacuum freeze drying under the conditions that the limiting temperature of a plate layer is less than or equal to-60 ℃, the limiting temperature of a cold trap is less than-70 ℃, the thickness of the plate layer is 0.5-2.0 mm and the vacuum degree is 10 Pa.
3. Determination of half-inhibitory concentration IC50 of fermented soybean milk powder on formation of streptococcus mutans biofilm
After the fermented soybean milk powder A is prepared into a sample to be tested by the method, the inhibitory activity of the sample to the streptococcus mutans biofilm formation is determined, and the result shows that the half-inhibitory rate concentration IC50 of the fermented soybean milk powder to the streptococcus mutans biofilm formation is 34 mg/mL.
Example 2
1. Materials and methods
(a) Preparation of leuconostoc mesenteroides (zymophyte) and streptococcus mutans suspensions: the same as in example 1.
(b) Preparing soybean milk: weighing dry red bean, adding water with five times of mass, soaking at 37 deg.C for 8 hr, removing water, adding wet bean and water at a certain ratio into a soybean milk machine to obtain soybean milk with desired solid content, and sterilizing at 121 deg.C for 20min to obtain sterile red bean soybean milk with desired solid content.
(c) The inhibition rate of the fermented soybean milk powder on the formation of the streptococcus mutans biofilm and the determination of IC 50: the same as in example 1.
2. Preparation method of fermented soybean milk powder
Inoculating Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.6432 into red bean milk with solid content of 9% (w/w) in an inoculation amount of 1% (v/v) aseptically, standing and culturing at 15 ℃ for 48h, and carrying out vacuum freeze drying under the conditions that the limiting temperature of a plate layer is less than or equal to-60 ℃, the limiting temperature of a cold trap is-70 ℃, the thickness of the plate layer is 0.5-2.0 mm, and the vacuum degree is 30Pa to obtain the fermented bean milk powder B.
3. Determination of half-inhibitory concentration IC50 of fermented soybean milk powder on formation of streptococcus mutans biofilm
After the fermented soybean milk powder B is prepared into a sample to be detected by the method, the inhibitory activity of the sample to the streptococcus mutans biofilm formation is measured, and the result shows that the half-inhibitory rate concentration IC50 of the fermented soybean milk powder to the streptococcus mutans biofilm formation is 36 mg/mL.
Example 3
1. Materials and methods
(a) Preparation of leuconostoc mesenteroides (zymophyte) and streptococcus mutans suspension: the same as in example 1.
(b) Preparing soybean milk: weighing dried mung bean, adding water with five times of mass, soaking at 37 deg.C for 8h, removing water, adding wet mung bean and water at a certain ratio into a soybean milk machine to obtain soybean milk with desired solid content, and sterilizing at 121 deg.C for 20min to obtain sterile mung bean soybean milk with desired solid content.
(c) The inhibition rate of the fermented soybean milk powder on the formation of the streptococcus mutans biofilm and the determination of IC 50: the same as in example 1.
2. Preparation method of fermented soybean milk powder
Inoculating Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.6432 in an inoculation amount of 5% (v/v) in mung bean milk with a solid content of 1% (w/w), standing and culturing for 2h at 45 ℃, and carrying out vacuum freeze drying under the conditions that the limiting temperature of a plate layer is less than or equal to-60 ℃, the limiting temperature of a cold trap is-70 ℃, the thickness of the plate layer is 0.5-2.0 mm and the vacuum degree is 20Pa to obtain the fermented soybean milk powder C.
3. Determination of half-inhibitory concentration IC50 of fermented soybean milk powder on formation of streptococcus mutans biofilm
After the fermented soybean milk powder C is prepared into a sample to be tested by the method, the inhibitory activity of the sample to the biofilm formation of streptococcus mutans is determined, and the result shows that the half inhibition rate concentration IC50 of the fermented soybean milk powder to the biofilm formation of streptococcus mutans is 38 mg/mL.
Example 4
1. Materials and methods
(a) Preparation of leuconostoc mesenteroides (zymophyte) and streptococcus mutans suspension: the same as in example 1.
(b) Preparing soybean milk: weighing dry broad bean, adding water with five times of mass, soaking at 37 ℃ for 8h, removing water, adding a certain proportion of wet bean and water into a soybean milk machine to obtain soybean milk with required solid content, and sterilizing at 121 ℃ for 20min to obtain sterile broad bean soybean milk with specified solid content.
(c) The inhibition rate of the fermented soybean milk powder on the formation of the streptococcus mutans biofilm and the determination of IC 50: the same as in example 1.
2. Preparation method of fermented soybean milk powder
Inoculating Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.6432 in an inoculation amount of 2% (v/v) in broad bean soybean milk with a solid content of 7% (w/w), standing and culturing at 25 ℃ for 36h, and carrying out vacuum freeze drying under the conditions that the limiting temperature of a plate layer is less than or equal to-60 ℃, the limiting temperature of a cold trap is-70 ℃, the thickness of the plate layer is 0.5-2.0 mm, and the vacuum degree is 25Pa to obtain the fermented soybean milk powder D.
3. Determination of half-inhibitory concentration IC50 of fermented soybean milk powder on formation of streptococcus mutans biofilm
After the fermented soybean milk powder D is prepared into a sample to be tested by the method, the inhibitory activity of the sample to the streptococcus mutans biofilm formation is determined, and the result shows that the half-inhibitory rate concentration IC50 of the fermented soybean milk powder to the streptococcus mutans biofilm formation is 37 mg/mL.
Example 5
1. Materials and methods
(a) Preparation of leuconostoc mesenteroides (zymophyte) and streptococcus mutans suspension: the same as in example 1.
(b) Preparing soybean milk: mixing white kidney beans and small white kidney bean dry beans according to a mass ratio of 1: 1, weighing and mixing, adding water with five times the mass, soaking at 37 ℃ for 8 hours, removing the water, adding wet beans and water in a certain proportion into a soybean milk machine to prepare soybean milk with the required solid content, and sterilizing at 121 ℃ for 20 minutes to obtain the sterile white kidney bean soybean milk with the specified solid content.
(c) The inhibition rate of the fermented soybean milk powder on the formation of the streptococcus mutans biofilm and the determination of IC 50: the same as in example 1.
2. Preparation method of fermented soybean milk powder
Inoculating Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.6432 in an inoculation amount of 4% (v/v) in white kidney bean milk with a solid content of 3% (w/w), standing and culturing for 12h at 40 ℃, and carrying out vacuum freeze drying under the conditions that the limiting temperature of a plate layer is less than or equal to-60 ℃, the limiting temperature of a cold trap is-70 ℃, the thickness of the plate layer is 0.5-2.0 mm, and the vacuum degree is 20Pa to obtain the fermented bean milk powder E.
3. Determination of half-inhibitory concentration IC50 of fermented soybean milk powder on formation of streptococcus mutans biofilm
After the fermented soybean milk powder E is prepared into a sample to be detected by the method, the inhibitory activity of the fermented soybean milk powder E on the formation of the streptococcus mutans biofilm is measured, and the result shows that the half-inhibitory rate concentration IC50 of the fermented soybean milk powder on the formation of the streptococcus mutans biofilm is 35 mg/mL.
Example 6
1. Materials and methods
(a) Preparation of leuconostoc mesenteroides (zymocyte): dissolving lyophilized powder of Leuconostoc mesenteroides ATCC10830a with small amount of sterile distilled water, drawing a ring with inoculating loop, inoculating to M17 solid culture medium (purchased from Merck Co. Germany) containing 2% (w/v) sucrose, aerobically culturing at 30 deg.C for 24h, picking single colony with inoculating loop, and placing into 20mL M17 liquid containing 2% (w/v) sucrose(purchased from Merck Co., Germany), uniformly dispersing the bacterial colony in a liquid culture medium by using a vortex oscillator, shaking and culturing for 24h by using a shaking table at 30 ℃ and 180rpm, taking out the bacterial colony, centrifuging the culture for 10min at 9,000rpm, removing the supernatant, washing the thallus for 2 times by using sterile distilled water, and suspending the thallus by using the sterile distilled water with the original culture volume to obtain seeds for fermentation, wherein the bacterial concentration of the seed solution is 1x109cfu/mL。
Preparation of a Streptococcus mutans bacterial suspension: the same as in example 1.
(b) Preparing soybean milk: the same as in example 1.
(c) The inhibition rate of the fermented soybean milk powder on the formation of the streptococcus mutans biofilm and the determination of IC 50: the same as in example 1.
2. Preparation method of fermented soybean milk powder
Inoculating Leuconostoc mesenteroides ATCC10830a in an inoculation amount of 3% (v/v) in soybean milk with a solid content of 5% (w/w) in an aseptic manner, and carrying out standing culture at 37 ℃ for 24h, wherein the temperature of a plate layer is less than or equal to-60 ℃, the temperature of a cold trap is less than-70 ℃, the thickness of the plate layer is 0.5-2.0 mm, and the vacuum degree is 10Pa, so as to obtain the fermented soybean milk powder F.
3. Determination of half-inhibitory concentration IC50 of fermented soybean milk powder on formation of streptococcus mutans biofilm
After the fermented soybean milk powder F is prepared into a sample to be detected by the method, the inhibitory activity of the sample to the streptococcus mutans biofilm formation is measured, and the result shows that the half-inhibitory rate concentration IC50 of the fermented soybean milk powder to the streptococcus mutans biofilm formation is 40 mg/mL.
Example 7
1. Materials and methods
(a) Preparation of leuconostoc mesenteroides (zymophyte): dissolving Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.10064 lyophilized powder with a small amount of sterile distilled water, drawing a ring by using an inoculating ring on an M17 solid culture medium (purchased from Merck Co. Germany) containing 2% (w/v) sucrose, carrying out aerobic culture at 30 ℃ for 24h, taking a single colony by using the inoculating ring, placing the single colony into 20mL of M17 liquid (purchased from Merck Co. Germany) containing 2% (w/v) sucrose, and uniformly dispersing the colony in a vortex shakerShaking culturing in liquid culture medium at 30 deg.C and 180rpm for 24 hr, centrifuging at 9,000rpm for 10min, discarding supernatant, washing thallus with sterile distilled water for 2 times, and suspending with sterile distilled water of original culture volume to obtain seed for fermentation, wherein the bacteria concentration of the seed solution is 1 × 109cfu/mL。
Preparation of a Streptococcus mutans bacterial suspension: the same as in example 1.
(b) Preparing soybean milk: the same as in example 1.
(c) The inhibition rate of the fermented soybean milk powder on the formation of the streptococcus mutans biofilm and the determination of IC 50: the same as in example 1.
2. Preparation method of fermented soybean milk powder
Inoculating Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.10064 with 3% (v/v) of inoculation amount in soybean milk with 5% (w/w) of solid content, standing and culturing at 37 ℃ for 24h, and carrying out vacuum freeze drying under the conditions that the limiting temperature of a plate layer is less than or equal to-60 ℃, the limiting temperature of a cold trap is-70 ℃, the thickness of the plate layer is 0.5-2.0 mm and the vacuum degree is 10Pa to obtain the fermented soybean milk powder G.
3. Determination of half-inhibitory concentration IC50 of fermented soybean milk powder on formation of streptococcus mutans biofilm
After the fermented soybean milk powder G is prepared into a sample to be detected by the method, the inhibitory activity of the sample to the streptococcus mutans biofilm formation is measured, and the result shows that the half-inhibitory rate concentration IC50 of the fermented soybean milk powder to the streptococcus mutans biofilm formation is 44 mg/mL.
Effect example 1Product taste and preference test
The fermented soybean milk powder products A, B, C, D, E, F and G prepared in examples 1 to 7 were dissolved in warm water to obtain reconstituted fermented soybean milk having a concentration of 50mg/mL, which was used as an experimental subject for taste test of the products. The number of test persons was 50. Tasting mode: tasting in a mode of unmarked scoring; the color, flavor, taste and nutrient of the fermented soybean milk powder products A, B, C, D, E, F and G are respectively and independently scored, each full score is 25, the average score and the total score thereof are calculated, and the statistical results are recorded in Table 1. Meanwhile, according to the opinion given to the overall preference degree of the product, the number of people who like each single product is counted, and the counting result is recorded in table 2.
Table 1 data statistics table for taste test results of products
TABLE 2 statistical table of product preference test results
As can be seen from the results of product taste tests and preference degree statistics, in general, the fermented soybean milk powder prepared from leuconostoc mesenteroides and having the streptococcus mutans biofilm inhibiting activity, which is prepared by the method in the technical scheme of the invention, can be accepted by most consumers in the aspects of product flavor, taste and nutrition.
Effect example 2Stability of inhibition activity of fermented soybean milk powder on streptococcus mutans biofilm at normal temperature
The fermented soybean milk powders A, B, C, D, E, F and G prepared in examples 1 to 7 were stored at room temperature (20 ℃) for 0, 10, 20 and 30 days and then taken out, and the half inhibitory concentration IC50 against Streptococcus mutans biofilm formation was determined for each sample, and the results are shown in Table 3.
TABLE 3 stability of inhibitory Effect of fermented Soybean milk powder on the biofilm formation of Streptococcus mutans under Normal temperature conditions
As can be seen from Table 3, the half-inhibitory concentration IC50 of Streptococcus mutans biofilm formation was stably maintained at the same level and the stability was better after all the fermented soybean milk powders tested were stored at room temperature (20 ℃) for 30 days.
Comparative example 1
The amounts of inoculation, soybean milk concentration, culture temperature and fermentation time in example 1 were adjusted one by one to obtain fermented soybean milk powders prepared by the following different methods, and the semi-inhibitory concentration IC50 of the fermented soybean milk powders for streptococcus mutans biofilm formation obtained from each group is shown in table 4.
TABLE 4 half inhibitory concentration IC50 of fermented soybean milk powder prepared by different methods on Streptococcus mutans biofilm formation
As can be seen from the results shown in Table 4, when the inoculation amount, soybean milk concentration, cultivation temperature and fermentation time in the method for preparing fermented soybean milk powder were adjusted to be out of the preferred ranges, the fermented soybean milk powder prepared from Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.6432 could still inhibit the formation of Streptococcus mutans biofilm, but the inhibitory effect was significantly reduced.
Comparative example 2
Referring to the method described in example 1, the inhibitory effects of fermented soybean milk powder prepared from Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.6432, lactobacillus plantarum (l.plantarum) ATCC14917 (purchased from ATCC), streptococcus thermophilus (s.thermophilus) ST-BODY-3 (provided by the kohamson company), lactobacillus casei (l.casei) ATCC 393 (purchased from ATCC), Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.10064, Leuconostoc mesenteroides (Leuconostoc mesenteroides) ATCC10830a on the biofilm formation of streptococcus mutans were compared as follows:
1. materials and methods
(a) Preparation of seeds (fermentation strain):
preparation of leuconostoc mesenteroides CGMCC No.6432 seeds: the same as example 1;
preparation of leuconostoc mesenteroides ATCC10830a seeds: the same as in example 6;
preparation of leuconostoc mesenteroides CGMCC No.10064 seeds: the same as in example 7.
Preparation of lactobacillus casei and lactobacillus bulgaricus seeds: respectively dissolving freeze-dried powder of lactobacillus casei ATCC 393 and lactobacillus plantarum ATCC14917 in a small amount of sterile distilled water, respectively drawing a ring by using an inoculating ring on an MRS solid culture medium (purchased from Merck Co. Germany), carrying out anaerobic culture at 37 ℃ for 24h, taking out a single colony by using the inoculating ring, putting the single colony into 1mL of MRS liquid (purchased from Merck Co. Germany), uniformly dispersing the colony in the liquid culture medium by using a vortex shaker, carrying out anaerobic culture at 37 ℃ for 24h, taking out the single colony, inoculating the single colony into 50mL of MRS liquid by using an inoculation amount of 2% (v/v), carrying out culture at 37 ℃ for 24h, centrifuging a culture at 9,000rpm for 10min, discarding a supernatant, washing thalli for 2 times by using sterile distilled water, and suspending the thalli by using sterile distilled water with the original culture volume to obtain corresponding seeds for fermentation.
Preparation of streptococcus thermophilus seeds: dissolving the freeze-dried powder of the streptococcus thermophilus ST-BODY-3 with a small amount of sterile distilled water, drawing a ring by using an inoculating ring, marking on an M17 solid culture medium (purchased from Merck Co. Germany), carrying out anaerobic culture at 40 ℃ for 24h, taking out, picking a single colony by using the inoculating ring, putting the single colony into 1mLM17 liquid (purchased from Merck Co. Germany), uniformly dispersing the colony in the liquid culture medium by using a vortex oscillator, carrying out anaerobic culture at 40 ℃ for 24h, taking out, inoculating the single colony into 50mL of M17 liquid by using an inoculation amount of 2% (v/v), carrying out culture at 40 ℃ for 24h, centrifuging a culture at 9,000rpm for 10min, discarding a supernatant, washing thalli for 2 times by using sterile distilled water, and suspending by using sterile distilled water with the original culture volume to obtain seeds for fermentation.
Preparation of a Streptococcus mutans bacterial suspension: the same as in example 1.
(b) Preparing soybean milk: the same as in example 1.
(c) The inhibition rate of the fermented soybean milk powder on the formation of the streptococcus mutans biofilm and the determination of IC 50: the same as in example 1.
2. Preparation method of fermented soybean milk powder
Inoculating each strain into soybean milk with solid content of 5% (w/w) aseptically at inoculation amount of 3% (v/v), culturing respectively (37 deg.C anaerobic culture of Leuconostoc mesenteroides, Lactobacillus plantarum and Lactobacillus casei, and 40 deg.C anaerobic culture of Streptococcus thermophilus) for 24h, and freeze drying to obtain corresponding fermented soybean milk powder.
3. Determination of half-inhibitory concentration IC50 of fermented soybean milk powder on formation of streptococcus mutans biofilm
The half-inhibitory concentration IC50 of fermented soybean milk powder prepared by the different strains on the formation of streptococcus mutans biofilm is shown in Table 5:
TABLE 5 semi-inhibitory concentration IC50 of fermented soymilk powder prepared from different strains on the biofilm formation of Streptococcus mutans
As can be seen from Table 5, the fermented soybean milk powders prepared from other strains have no inhibitory activity against Streptococcus mutans biofilm, while the fermented soybean milk powders prepared from Leuconostoc mesenteroides (Leuconostoc mesenteroides) CGMCC No.6432, ATCC10830a and CGMCC No.10064 have very significant inhibitory activity against Streptococcus mutans biofilm.
Comparative example 3
1. Materials and methods
(a) Preparation of leuconostoc mesenteroides CGMCC No.6432 seeds: the same as example 1;
preparation of leuconostoc mesenteroides ATCC10830a seeds: the same as in example 6;
preparation of leuconostoc mesenteroides CGMCC No.10064 seeds: the same as in example 7;
preparation of a Streptococcus mutans bacterial suspension: the same as in example 1.
(b) And (3) determining the inhibition rate of the fermentation liquid freeze-dried powder on the formation of the streptococcus mutans biofilm:
pretreatment of fermentation liquid freeze-dried powder: and (4) dissolving the fermentation liquid freeze-dried powder in sterile water, and adjusting the pH to 6.80 to obtain a sample to be detected.
Determination of inhibitory Activity: the assay was performed with unfermented medium as a blank, according to the method described in example 1.
(c) Determination of the concentration of semi-inhibitory rate IC50 for the formation of streptococcus mutans biofilm: the same as in example 1.
2. Preparation of leuconostoc mesenteroides fermentation liquid freeze-dried powder
And (2) aseptically inoculating leuconostoc mesenteroides seeds CGMCC No.6432, ATCC10830a and CGMCC No.10064 in an M17 liquid culture medium containing 1% (w/v) of cane sugar by an inoculation amount of 3% (v/v), and carrying out anaerobic culture at 37 ℃ for 24h, wherein the freeze-dried powder of the fermentation liquid is obtained after vacuum freeze-drying under the conditions that the limiting temperature of a plate layer is less than or equal to-60 ℃, the limiting temperature of a cold trap is less than-70 ℃, the loading thickness of the plate layer is 0.5-2.0 mm and the vacuum degree is 10 Pa.
3. Determination of half inhibition rate concentration IC50 of fermentation liquid freeze-dried powder on streptococcus mutans biofilm formation
The leuconostoc mesenteroides fermentation liquid freeze-dried powder is prepared into a sample to be detected by the method, and then the inhibitory activity of the leuconostoc mesenteroides fermentation liquid freeze-dried powder on the formation of the streptococcus mutans biofilm is measured, and the result shows that the semi-inhibition rate concentration IC50 of the fermentation liquid freeze-dried powder of CGMCC No.6432, ATCC10830a and CGMCC No.10064 on the formation of the streptococcus mutans biofilm can not be measured (the maximum concentration solution to be detected prepared from the fermentation liquid freeze-dried powder has no half inhibition effect on the streptococcus mutans).
Comparative example 4
The fermented soybean milk powders A, B, C, D, E, F and G prepared in examples 1 to 7 were dissolved in sterile water, pH was adjusted to 6.80, and then centrifuged at 10,000rpm at 4 ℃ for 10min to obtain a supernatant, i.e., sterile reconstituted fermented soybean milk (sample to be tested). The half-inhibitory concentration IC50 of the above samples on the formation of Streptococcus mutans biofilm was determined according to the method described in example 1 and the results are shown in Table 6.
TABLE 6 inhibitory Effect of sterile reconstituted fermented soymilk on Streptococcus mutans biofilm formation
As can be seen from Table 6, the sterilized reconstituted fermented soybean milk still has strong inhibitory activity on the biofilm formation inhibition rate of Streptococcus mutans, which indicates that the inhibitory effect is derived from the product of the strain after metabolizing soybean milk. However, the biofilm inhibition effect of each group of the sterilized restored fermented soybean milk is lower than that of the sample group before the sterilized treatment, which shows that the biofilm inhibition effect of the restored fermented soybean milk before the sterilized treatment comes from the combined action of the live bacteria and the metabolite.
The preparation method of the fermented soybean milk powder, the prepared fermented soybean milk powder and the application 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 (7)
1. A method for preparing fermented soybean milk powder from leuconostoc mesenteroides is characterized by comprising the following steps: inoculating Leuconostoc mesenteroides into soybean milk for fermentation and metabolism, and freeze-drying to obtain fermented soybean milk powder;
the leuconostoc mesenteroides is CGMCC No.6432, ATCC10830a or CGMCC No. 10064;
the mass percentage content of the solid in the soybean milk is 1-9%.
2. The method for preparing fermented soybean milk powder according to claim 1, wherein the amount of leuconostoc mesenteroides CGMCC No.6432 inoculated is 1x107～5x107cfu/mL。
3. The method for preparing fermented soybean milk powder according to claim 1, wherein the soybean milk is at least one of soybean, red bean, mung bean, broad bean, white kidney bean or small white kidney bean milk.
4. The preparation method of the fermented soybean milk powder according to claim 1, wherein the fermentation temperature is 15-45 ℃ and the fermentation time is 2-48 h, and the fermentation is static culture.
5. The method for preparing fermented soybean milk powder according to claim 1, wherein the freeze-drying conditions are: the ultimate temperature of the plate layer is less than or equal to minus 60 ℃, the ultimate temperature of the cold trap is minus 70 ℃, the loading thickness of the plate layer is 0.5-2.0 mm, and the vacuum degree is 10-30 Pa.
6. A fermented soybean milk powder prepared from Leuconostoc mesenteroides, which is prepared by the preparation method of any one of claims 1 to 5.
7. The fermented soybean milk powder according to claim 6, wherein the half-inhibitory concentration IC50 of the fermented soybean milk powder on the formation of streptococcus mutans biofilm is less than or equal to 44 mg/mL.
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