CN110012941B - Method for preparing fermented soybean milk by using paenibacillus, fermented soybean milk prepared by method and application of fermented soybean milk - Google Patents
Method for preparing fermented soybean milk by using paenibacillus, fermented soybean milk prepared by method and application of fermented soybean milk Download PDFInfo
- Publication number
- CN110012941B CN110012941B CN201910435064.7A CN201910435064A CN110012941B CN 110012941 B CN110012941 B CN 110012941B CN 201910435064 A CN201910435064 A CN 201910435064A CN 110012941 B CN110012941 B CN 110012941B
- Authority
- CN
- China
- Prior art keywords
- soybean milk
- fermented soybean
- paenibacillus
- streptococcus mutans
- fermentation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 244000068988 Glycine max Species 0.000 title claims abstract description 226
- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 226
- 235000013336 milk Nutrition 0.000 title claims abstract description 222
- 239000008267 milk Substances 0.000 title claims abstract description 222
- 210000004080 milk Anatomy 0.000 title claims abstract description 222
- 241000179039 Paenibacillus Species 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 50
- 241000194019 Streptococcus mutans Species 0.000 claims abstract description 109
- 238000000855 fermentation Methods 0.000 claims abstract description 61
- 230000004151 fermentation Effects 0.000 claims abstract description 61
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 47
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 32
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 32
- 230000005764 inhibitory process Effects 0.000 claims description 62
- 230000015572 biosynthetic process Effects 0.000 claims description 61
- 238000011081 inoculation Methods 0.000 claims description 36
- 239000007787 solid Substances 0.000 claims description 36
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 16
- 240000004922 Vigna radiata Species 0.000 claims description 8
- 235000010721 Vigna radiata var radiata Nutrition 0.000 claims description 8
- 235000011469 Vigna radiata var sublobata Nutrition 0.000 claims description 8
- 230000003068 static effect Effects 0.000 claims description 8
- 240000006677 Vicia faba Species 0.000 claims description 7
- 235000010749 Vicia faba Nutrition 0.000 claims description 7
- 235000002098 Vicia faba var. major Nutrition 0.000 claims description 7
- 235000011453 Vigna umbellata Nutrition 0.000 claims description 7
- 240000001417 Vigna umbellata Species 0.000 claims description 7
- 239000002054 inoculum Substances 0.000 claims description 4
- 235000013305 food Nutrition 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000002131 composite material Substances 0.000 claims description 2
- 239000002068 microbial inoculum Substances 0.000 claims description 2
- 230000000675 anti-caries Effects 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 abstract description 40
- 239000001963 growth medium Substances 0.000 abstract description 22
- 230000032770 biofilm formation Effects 0.000 abstract description 17
- 230000004060 metabolic process Effects 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 description 44
- 241000592795 Paenibacillus sp. Species 0.000 description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 30
- 230000000694 effects Effects 0.000 description 28
- 239000000523 sample Substances 0.000 description 28
- 239000012153 distilled water Substances 0.000 description 14
- 239000000047 product Substances 0.000 description 14
- 239000000725 suspension Substances 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 12
- 208000002925 dental caries Diseases 0.000 description 12
- 239000000463 material Substances 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 11
- 239000007788 liquid Substances 0.000 description 11
- 238000009630 liquid culture Methods 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 244000199866 Lactobacillus casei Species 0.000 description 5
- 241000194020 Streptococcus thermophilus Species 0.000 description 5
- 241001052560 Thallis Species 0.000 description 5
- 238000002791 soaking Methods 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 235000019640 taste Nutrition 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 235000013958 Lactobacillus casei Nutrition 0.000 description 4
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 4
- 229940017800 lactobacillus casei Drugs 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 3
- 230000001013 cariogenic effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 235000021332 kidney beans Nutrition 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000005057 refrigeration Methods 0.000 description 3
- 235000013322 soy milk Nutrition 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000000214 mouth Anatomy 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229960004793 sucrose Drugs 0.000 description 2
- 241000186046 Actinomyces Species 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 201000004328 Pulpitis Diseases 0.000 description 1
- 206010037464 Pulpitis dental Diseases 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 208000008312 Tooth Loss Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002053 acidogenic effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000005115 demineralization Methods 0.000 description 1
- 230000002328 demineralizing effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 208000004480 periapical periodontitis Diseases 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- XNRABACJWNCNEQ-UHFFFAOYSA-N silver;azane;nitrate Chemical compound N.[Ag+].[O-][N+]([O-])=O XNRABACJWNCNEQ-UHFFFAOYSA-N 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
Classifications
-
- 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
-
- 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
- A23C2220/00—Biochemical treatment
- A23C2220/20—Treatment with microorganisms
- A23C2220/204—Use of bacteria which are encapsulated, entrapped or immobilised; Fermentation with these bacteria
-
- 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
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Beans For Foods Or Fodder (AREA)
Abstract
The invention particularly discloses a method for preparing fermented soybean milk by using paenibacillus, the prepared fermented soybean milk and application, wherein the method comprises the following steps: inoculating paenibacillus CGMCC No.10062 into soybean milk for fermentation and metabolism to obtain fermented soybean milk; the invention has the advantages that the paenibacillus CGMCC No. 10062/the paenibacillus CGMCC No.10062 and the lactobacillus plantarum ATCC14917 (purchased from ATCC) are fermented by taking the soybean milk as the culture medium to prepare the fermented soybean milk with the inhibiting activity of the streptococcus mutans biofilm, and compared with other strains capable of fermenting the soybean milk and other fermentation culture media, the obtained fermented soybean milk has more remarkable inhibiting effect on the streptococcus mutans biofilm formation and has good stability.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of fermented soybean milk. In addition, the invention also relates to fermented soybean milk 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 are not directly colonized on the tooth surface, but indirectly adhere to the tooth surface after forming plaque by combining with biofilm (insoluble polysaccharide) produced by streptococcus mutans, and the degree of dental caries gradually worsens as cariogenic bacteria are accumulated 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 for streptococcus mutans, such as compounds like ammonia silver nitrate, have strong side effects, stain teeth after long-term use, and are not suitable for anterior teeth 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, especially the research related to microbial fermentation products is extremely limited, and the fermentation products for inhibiting the formation of the streptococcus mutans biofilm are relatively short.
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 method for preparing fermented soybean milk, and the fermented soybean milk with the effect of inhibiting the formation of streptococcus mutans biofilm can be prepared by the method.
Specifically, on the one hand, the preparation method of the fermented soybean milk prepared by the paenibacillus comprises the following steps: the Paenibacillus CGMCC No.10062 is used as a fermentation microbial inoculum and inoculated into the soybean milk for fermentation to obtain the fermented soybean milk.
Through the technical scheme, the application adopts the Paenibacillus sp (CGMCC No. 10062) for the first time, and the soybean milk is used as a culture medium for fermentation to prepare the fermented soybean milk with the streptococcus mutans biofilm inhibition activity, so that the new application of the fermented soybean milk fermented and metabolized by the Paenibacillus sp (CGMCC No. 10062) in resisting decayed teeth is disclosed.
In addition, the Paenibacillus (Paenibacillus sp.) strain of CGMCC No.10062 was deposited in the common microorganism center of the china committee for culture collection management (CGMCC) in 11/26 th 2014, and the deposition address: west road No.1, north chen, chaoyang district, beijing, zip code: the accession information of the strain 100101, CGMCC No.10062 has been disclosed in the patent publication No. CN 106701833A.
Further, the inoculation amount of the paenibacillus CGMCC No.10062 is 2x107～1x108cfu/mL。
By adopting the technical scheme, when the inoculation amount of the Paenibacillus sp (CGMCC No. 10062) is 2x107～1x108When cfu/mL, the fermented soybean milk prepared by Paenibacillus sp (CGMCC No. 10062) can effectively inhibit the formation amount of streptococcus mutans biomembrane; and when the inoculation amount is 4x107～8x107cfu/mL, the achieved inhibition effect is better; when the inoculation amount is 6x107cfu/mL, the best inhibition achieved.
Further, the fermentation inoculant is a composite fermentation inoculant of paenibacillus CGMCC No.10062 and lactobacillus plantarum ATCC14917 (purchased from ATCC) with the inoculation ratio of 1-9: 1-9.
Through the technical scheme, the application firstly inoculates the Paenibacillus (Paenibacillus sp.) CGMCC No.10062 and the lactobacillus plantarum (L.plantarum) ATCC14917 (purchased from ATCC) in the soybean milk to prepare the fermented soybean milk with the streptococcus mutans biofilm inhibiting activity, and discloses the new application of the fermented soybean milk fermented and metabolized by the Paenibacillus (Paenibacillus sp.) CGMCC No.10062 and the lactobacillus plantarum (L.plantarum) ATCC14917 (purchased from ATCC) in resisting decayed teeth.
Further, the soybean milk is one or more of soybean, red bean, mung bean, broad bean, white kidney bean and small white kidney bean.
According to the technical scheme, the effect of the soybean milk prepared from the soybeans is the best in the effect comparison of the soybean milk prepared from the soybeans, red beans, mung beans, broad beans, white kidney beans and small white kidney beans.
Further, the content of solid matters in the soybean milk is 1-9% by mass.
By adopting the technical scheme, when the mass percentage of the solid matters in the soybean milk is 1-9%, the obtained fermented soybean milk has a good inhibition effect on the formation of streptococcus mutans biofilm; when the solid content is 3-7% by mass, the inhibitor has a good inhibition effect on the formation of the streptococcus mutans biofilm, and when the solid content is 5% by mass, the inhibitor has a good inhibition effect on the formation of the streptococcus mutans biofilm; the principle is that the content of solid matters in the soybean milk has a direct relation with the biofilm inhibition 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 a membrane inhibition substance 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 production of the membrane inhibition substance is also influenced.
Further, the temperature of the fermentation is 15 ℃ to 45 ℃.
By adopting the technical scheme, when the fermentation temperature is 15-45 ℃ and the fermentation time is 2-48 h, the obtained fermented soybean milk has a good inhibition effect on the formation of streptococcus mutans biofilm under the condition that the inoculation amount, the soybean milk concentration, the culture temperature and the fermentation time are mutually influenced; the fermentation temperature is preferably 25-40 ℃, and the fermentation temperature is more preferably 37 ℃; preferably, the fermentation time is 12-36 h; more preferably the fermentation time is 24 h.
Further, the fermentation is static culture.
Through the technical scheme, the Paenibacillus (Paenibacillus sp.) CGMCC No.10062 belongs to facultative anaerobe, and the stationary culture has the characteristic of high propagation speed.
In a second aspect, there is also provided a fermented soybean milk prepared by any one of the above-mentioned preparation methods.
Furthermore, the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm is more than or equal to 40%.
In a third aspect, the application of the fermented soybean milk in preparing the anti-dental caries food is also provided.
Compared with the prior art, the application adopts Paenibacillus sp (CGMCC No. 10062)/Paenibacillus sp (CGMCC No. 10062) and Lactobacillus plantarum (L.plantarum) ATCC14917 (purchased from ATCC) for the first time, and takes soybean milk as a culture medium for fermentation to prepare fermented soybean milk with the streptococcus mutans biofilm inhibiting activity, and discloses the fermented soybean milk fermented and metabolized by the Paenibacillus sp (Paenibacillus sp) CGMCC No. 10062/Paenibacillus sp (CGMCC No. 10062) and Lactobacillus plantarum (L.plantarum) ATCC14917 (purchased from ATCC) has the new application of resisting decayed teeth; compared with other strains capable of fermenting soybean milk and other fermentation culture media, the fermented soybean milk fermented and metabolized by the Paenibacillus (Paenibacillus sp.) CGMCC No. 10062/the Paenibacillus (Paenibacillus sp.) CGMCC No.10062 and the Lactobacillus plantarum (L.plantarum) ATCC14917 (purchased from ATCC) has more remarkable effect of inhibiting the formation of streptococcus mutans biofilm and has 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, there is provided a method for preparing fermented soybean milk, comprising the steps of: inoculating Paenibacillus sp (CGMCC No. 10062) into soybean milk for fermentation to obtain fermented soybean milk.
According to the preparation method in the technical scheme, Paenibacillus (Paenibacillus sp.) CGMCC No.10062 is adopted for the first time, and the soybean milk is used as a culture medium for fermentation to prepare the fermented soybean milk with the activity of inhibiting the streptococcus mutans biofilm, so that the novel application of the Paenibacillus (Paenibacillus sp.) CGMCC No.10062 fermented soybean milk in resisting dental caries is disclosed. Compared with other strains capable of fermenting soybean milk, the fermented soybean milk prepared by CGMCC No.10062 has more remarkable inhibiting effect on the formation of streptococcus mutans biofilm and has good stability.
Further, in the above steps, the inoculation amount of Paenibacillus (Paenibacillus sp.) CGMCC No.10062 is 2x107～1x108cfu/mL; preferably 4x107～8x107cfu/mL, more preferably 6X107cfu/mL。
Further, in the above step, the preferable soybean milk is one or more of soybean, red bean, mung bean, broad bean, white kidney bean and small white kidney bean, and more preferably soybean milk.
Further, in the above steps, the preferable solid content of the soybean milk is 1-9% by mass; preferably 3% -7%; more preferably 5%.
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 hours.
Further, in the above step, the preferred fermentation method is static culture.
In another embodiment, there is provided a fermented soybean milk produced by any one of the above-mentioned methods for producing fermented soybean milk.
Since the preparation method of the fermented soybean milk has the above-mentioned advantageous effects, the fermented soybean milk prepared by the preparation method also has corresponding advantageous effects, which are not described herein again.
Furthermore, the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm is more than or equal to 40%. In combination with the examples, it can be seen that the inhibition effect of the Paenibacillus (Paenibacillus sp.) CGMCC No.10062 fermented soybean milk on the formation of streptococcus mutans biofilm is significantly higher than that of other strains of fermentable soybean milk, and the inhibition effect of the Paenibacillus (Paenibacillus sp.) CGMCC No.10062 and lactobacillus plantarum (l.plantarum) ATCC14917 (purchased from ATCC) fermented soybean milk on the formation of streptococcus mutans biofilm is significantly higher than that of the Paenibacillus (Paenibacillus sp.) CGMCC No.10062 fermented soybean milk.
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.
In the following examples, all the starting materials are commercially available and meet the relevant national standards.
Example 1
1. Materials and methods
(a) Preparation of Paenibacillus seeds (fermentation strain): dissolving Paenibacillus (Paenibacillus sp.) CGMCC No.10062 lyophilized powder with a small amount of sterile distilled water, taking a ring by using an inoculating loop, scribing on an M17 solid culture medium (purchased from OXOID Co., UK), aerobically culturing at 30 ℃ for 48h, taking out a single colony by using the inoculating loop, putting the single colony into 10mL of M17 liquid culture medium (purchased from OXOID Co., UK), uniformly dispersing the colony in the liquid culture medium by using a vortex oscillator, taking out the colony by performing shaking culture at 30 ℃ and 180rpm for 24h, inoculating the colony into the M17 liquid culture medium (purchased from OXOID Co., UK) in an inoculation amount of 2% (v/v), performing shaking culture at 30 ℃ and 180rpm for 24h, centrifuging the culture at 15000rpm for 10min, discarding supernatant, washing the thallus with sterile distilled water for 2 times, and suspending the thallus with the sterile distilled water in the original culture volume to obtain the seed for fermentation, wherein the bacterial concentration of the seed solution is 2x109cfu/mL。
Preparation of a Streptococcus mutans bacterial suspension: streptococcus mutansDissolving CGMCC No.1.2499 (purchased 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 (purchased from OXOID Co., UK), carrying out anaerobic culture at 37 ℃ for 24h, taking out a single colony by using the inoculating loop, putting the single colony into 10mL BHI 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 the single colony, inoculating the single colony into the BHI liquid culture medium in an inoculation amount of 2% (v/v), carrying out anaerobic culture at 37 ℃ for 24h, centrifuging the culture at 15000rpm for 10min, abandoning supernatant, washing thalli for 2 times by using sterile distilled water, and then re-suspending the thalli in the BHI liquid culture medium to 10 h6CFU/mL.
(b) Preparing soybean milk: weighing dry soybean, adding five times of water, soaking at 37 deg.C for 8 hr, removing water, adding a certain proportion of wet soybean and water 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) And (3) measuring the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm:
pretreatment of fermented soybean milk: and adjusting the pH value of the fermented soybean milk to 6.80 to obtain a sample to be detected.
Determination of inhibitory Activity: immersing a sterile magnetic stirrer A (5 multiplied by 10mM) in saliva of a healthy population after sterile filtration by a 0.22-micron filter membrane for incubation at 37 ℃ for 4h, sucking the magnetic stirrer A by a sterile magnetic stirrer B (10 multiplied by 50mM), rinsing with 10mM PBS (pH7.0), taking down the magnetic stirrer A, and placing the magnetic stirrer A in a culture well of a 24-well plate. To the culture wells, 1.6mL of BHI broth, 200. mu.L of Streptococcus mutans suspension (10. mu.L) was added6CFU/mL) and 400 μ L of test sample solution or blank control solution (unfermented soymilk), again incubated anaerobically at 37 ℃ for 24h (purchased from Greiner, germany). And (3) sucking up the magnetic stirrer A by using a magnetic stirrer B, washing by using PBS, taking down the magnetic stirrer A, placing the magnetic stirrer A in a culture hole of a 24-hole plate, and naturally drying in the air. 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, rinsing by PBS to remove redundant dyeing solution, taking down the magnetic stirrer A, placing the magnetic stirrer A in the culture well of a 24-well plate, and naturally drying in air. Towards the cultureAdding 300 μ L of absolute ethanol into the wells for decolorization for 15min, transferring 200 μ L of absolute ethanol into a 96-well plate (purchased from Greiner, Germany), and measuring OD600nm by using a microplate reader, namely, the quantitative data corresponding to the formation amount of the streptococcus mutans 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%
2. Preparation of fermented soybean milk
The method comprises the following steps of aseptically inoculating Paenibacillus (Paenibacillus sp.) CGMCC No.10062 into soybean milk with solid content of 5% (w/w, soybean in mass percent of water, the same below) by an inoculation amount of 3% (v/v, seed liquid in volume percent of fermentation liquid, the same below), and carrying out static culture at 37 ℃ for 24 hours to obtain fermented soybean milk A.
3. Determination of inhibition rate of fermented soybean milk on formation of streptococcus mutans biofilm
After the fermented soybean milk A is prepared into a sample to be tested by the method, the inhibitory activity of the sample to the formation of the streptococcus mutans biofilm is measured, and the result shows that the inhibition rate I of the fermented soybean milk to the formation of the streptococcus mutans biofilm is 44%.
Example 2
1. Materials and methods
(a) Preparation of paenibacillus seeds (zymophyte) and streptococcus mutans bacterial suspension: 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) Measuring the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm: the same as in example 1.
2. Preparation of fermented soybean milk
Aseptically inoculating Paenibacillus (Paenibacillus sp.) CGMCC No.10062 into red bean milk with solid content of 9% (w/w) at an inoculation amount of 1% (v/v), and performing static culture at 15 ℃ for 48h to obtain fermented soybean milk B.
3. Determination of inhibition rate of fermented soybean milk on formation of streptococcus mutans biofilm
After the fermented soybean milk B is prepared into a sample to be tested by the method, the inhibitory activity of the sample to the streptococcus mutans biofilm formation is measured, and the result shows that the inhibition rate I of the fermented soybean milk to the streptococcus mutans biofilm formation is 42%.
Example 3
1. Materials and methods
(a) Preparation of paenibacillus seeds (zymophyte) and streptococcus mutans bacterial suspension: the same as in example 1.
(b) Preparing soybean milk: weighing dry 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) And (3) measuring the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm: the same as in example 1.
2. Preparation of fermented soybean milk
The bacillus subtilis (Paenibacillus sp.) CGMCC No.10062 is aseptically inoculated into mung bean milk with the solid content of 1% (w/w) by the inoculation amount of 5% (v/v), and is statically cultured for 2h at 45 ℃ to obtain the fermented soybean milk C.
3. Determination of inhibition rate of fermented soybean milk on formation of streptococcus mutans biofilm
After the fermented soybean milk C is prepared into a sample to be tested by the method, the inhibitory activity of the sample to the streptococcus mutans biofilm formation is measured, and the result shows that the inhibition rate I of the fermented soybean milk to the streptococcus mutans biofilm formation is 40%.
Example 4
1. Materials and methods
(a) Preparation of paenibacillus seeds (zymophyte) and streptococcus mutans bacterial 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) And (3) measuring the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm: the same as in example 1.
2. Preparation of fermented soybean milk
The Paenibacillus sp CGMCC No.10062 is aseptically inoculated into broad bean milk with the solid content of 7% (w/w) by the inoculation amount of 2% (v/v), and is statically cultured for 36h at 25 ℃ to obtain the fermented soybean milk D.
3. Determination of inhibition rate of fermented soybean milk on formation of streptococcus mutans biofilm
After the fermented soybean milk 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 measured, and the result shows that the inhibition rate I of the fermented soybean milk to the streptococcus mutans biofilm formation is 41%.
Example 5
1. Materials and methods
(a) Preparation of paenibacillus seeds (fermentation strain) 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 of 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 required solid content, and sterilizing at 121 ℃ for 20min to obtain the sterile white kidney bean soybean milk with the specified solid content.
(c) And (3) measuring the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm: the same as in example 1.
2. Preparation of fermented soybean milk
The bacillus subtilis (Paenibacillus sp.) CGMCC No.10062 is aseptically inoculated into white kidney bean milk with solid content of 3% (w/w) by an inoculation amount of 4% (v/v), and is kept stand and cultured for 12h at 40 ℃ to obtain the fermented soybean milk E.
3. Determination of inhibition rate of fermented soybean milk on formation of streptococcus mutans biofilm
After the fermented soybean milk E is prepared into a sample to be tested by the method, the inhibitory activity of the sample to the formation of the streptococcus mutans biofilm is measured, and the result shows that the inhibition rate I of the fermented soybean milk to the formation of the streptococcus mutans biofilm is 43%.
Example 6
1. Materials and methods
(a) Preparation of paenibacillus seeds (zymophyte) and streptococcus mutans bacterial suspension: the same as in example 1.
(b) Preparation of lactobacillus plantarum (zymophyte): respectively dissolving lyophilized powder of Lactobacillus plantarum ATCC14917 with small amount of sterile distilled water, taking a ring by using an inoculating ring, scribing on 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 oscillator, carrying out anaerobic culture at 37 ℃ for 24h, 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 supernatant, washing thalli with sterile distilled water for 2 times, suspending with the sterile distilled water of the original culture volume to obtain corresponding seed for fermentation, wherein the bacterial concentration of the seed liquid is 1x109cfu/mL。
(c) Preparing soybean milk: the same as in example 1.
(d) And (3) measuring the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm: the same as in example 1.
2. Preparation of fermented soybean milk
Paenibacillus (Paenibacillus sp.) CGMCC No.10062 and Lactobacillus plantarum (L.plantarum) ATCC14917 (purchased from ATCC) are respectively inoculated into soybean milk with solid content of 5% (w/w) in an inoculation amount of 1.5% (v/v) in an aseptic manner, and the soybean milk is subjected to static culture at 37 ℃ for 24 hours to obtain fermented soybean milk F.
3. Determination of inhibition rate of fermented soybean milk on formation of streptococcus mutans biofilm
After the fermented soybean milk F is prepared into a sample to be tested by the method, the inhibitory activity of the sample to the formation of the streptococcus mutans biofilm is measured, and the result shows that the inhibition rate I of the fermented soybean milk to the formation of the streptococcus mutans biofilm is 59%.
Example 7
1. Materials and methods
(a) Preparation of paenibacillus seeds (zymophyte) and streptococcus mutans bacterial suspension: the same as in example 1.
(b) Preparation of lactobacillus plantarum (zymocyte species): the same as in example 6.
(c) Preparing soybean milk: the same as in example 1.
(d) Measuring the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm: the same as in example 1.
2. Preparation of fermented soybean milk
Paenibacillus (Paenibacillus sp.) CGMCC No.10062 is aseptically inoculated into soybean milk with solid content of 5% (w/w) by inoculation amount of 2.7% (v/v), and lactobacillus plantarum (L.plantarum) ATCC14917 (purchased from ATCC) by inoculation amount of 0.3% (v/v), and the soybean milk is statically cultured for 24h at 37 ℃ to obtain fermented soybean milk G.
3. Determination of inhibition rate of fermented soybean milk on formation of streptococcus mutans biofilm
After the fermented soybean milk G is prepared into a sample to be tested by the method, the inhibitory activity of the sample to the formation of the streptococcus mutans biofilm is measured, and the result shows that the inhibition rate I of the fermented soybean milk to the formation of the streptococcus mutans biofilm is 56%.
Example 8
1. Materials and methods
(a) Preparation of paenibacillus seeds (fermentation strain) and streptococcus mutans suspension: the same as in example 1.
(b) Preparation of lactobacillus plantarum (zymophyte): the same as in example 6.
(c) Preparing soybean milk: the same as in example 1.
(d) And (3) measuring the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm: the same as in example 1.
2. Preparation of fermented soybean milk
Paenibacillus (Paenibacillus sp.) CGMCC No.10062 is aseptically inoculated into soybean milk with solid content of 5% (w/w) in an inoculation amount of 0.3% (v/v), and lactobacillus plantarum (L.plantarum) ATCC14917 (purchased from ATCC) in an inoculation amount of 2.7% (v/v), and is subjected to static culture at 37 ℃ for 24 hours to obtain fermented soybean milk H.
3. Determination of inhibition rate of fermented soybean milk on formation of streptococcus mutans biofilm
After the fermented soybean milk H is prepared into a sample to be tested by the method, the inhibitory activity of the sample to the streptococcus mutans biofilm formation is measured, and the result shows that the inhibition rate I of the fermented soybean milk to the streptococcus mutans biofilm formation is 51%.
Example 9
1. Materials and methods
(a) Preparation of paenibacillus seeds (fermentation strain) and streptococcus mutans suspension: the same as in example 1.
(b) Preparation of lactobacillus plantarum (zymophyte): the same as in example 6.
(c) Preparing soybean milk: the same as in example 1.
(d) Measuring the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm: the same as in example 1.
2. Preparation of fermented soybean milk
Paenibacillus (Paenibacillus sp.) CGMCC No.10062 is aseptically inoculated into soybean milk with solid content of 3% (w/w) by inoculation amount of 1.5% (v/v), and lactobacillus plantarum (L.plantarum) ATCC14917 (purchased from ATCC) is aseptically inoculated into soybean milk with solid content of 3% (w/w) by inoculation amount of 1.5% (v/v), and is subjected to static culture at 37 ℃ for 24h to obtain fermented soybean milk I.
3. Determination of inhibition rate of fermented soybean milk on formation of streptococcus mutans biofilm
After the fermented soybean milk I is prepared into a sample to be tested by the method, the inhibitory activity of the sample to the streptococcus mutans biofilm formation is measured, and the result shows that the inhibitory rate I of the fermented soybean milk to the streptococcus mutans biofilm formation is 47%.
Example 10
1. Materials and methods
(a) Preparation of paenibacillus seeds (zymophyte) and streptococcus mutans bacterial suspension: the same as in example 1.
(b) Preparation of lactobacillus plantarum (zymocyte species): the same as in example 6.
(c) Preparing soybean milk: the same as in example 1.
(d) Measuring the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm: the same as in example 1.
2. Preparation of fermented soybean milk
Paenibacillus (Paenibacillus sp.) CGMCC No.10062 with the inoculation amount of 1.5% (v/v) and Lactobacillus plantarum (L.plantarum) ATCC14917 (purchased from ATCC) with the inoculation amount of 1.5% (v/v) are aseptically inoculated into soybean milk with the solid content of 5% (w/w), and the soybean milk J is obtained by standing and culturing at 40 ℃ for 12 h.
3. Determination of inhibition rate of fermented soybean milk on formation of streptococcus mutans biofilm
After the fermented soybean milk J is prepared into a sample to be tested by the method, the inhibitory activity of the sample to the formation of the streptococcus mutans biofilm is measured, and the result shows that the inhibition rate I of the fermented soybean milk to the formation of the streptococcus mutans biofilm is 48%.
Effect example 1Product taste and preference test
The fermented soybean milk product A, B, C, D, E, F, G, H, I, J prepared in examples 1 to 10 was used as a test subject to perform a taste test of the product. The test population was 50. Tasting: tasting in a mode of unmarked scoring; the color, flavor, taste and nutritional items of the fermented soybean milk product A, B, C, D, E, F, G, H, I, J were individually scored, each full score was 25, the average score and the total score thereof were calculated, and the statistical results are recorded in table 1. Meanwhile, according to the opinions 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 the table 2.
TABLE 1 product taste test result data statistics table
TABLE 2 statistical table of product preference test results
As can be seen from the product taste test and the result of the preference degree statistics, the fermented soybean milk with the streptococcus mutans biofilm inhibiting activity prepared by the paenibacillus, the paenibacillus and the lactobacillus plantarum prepared by the method in the technical scheme of the invention is generally accepted by most consumers in terms of product flavor, mouthfeel and nutrition.
Effect example 2Stability of fermented soybean milk on inhibition activity of streptococcus mutans biofilm under refrigeration condition
The fermented soybean milk A, B, C, D, E, F, G, H, I, J prepared in examples 1 to 5 was stored under refrigeration (4 ℃) for 0, 10, 20 and 30 days and then taken out, and the inhibition rate of each sample against the formation of Streptococcus mutans biofilm was measured, and the results are shown in Table 3.
TABLE 3 stability of inhibitory Effect of fermented Soybean milk on Streptococcus mutans biofilm formation under Cold storage conditions
As can be seen from Table 3, the fermented soybean milk tested in all the tests stably maintained the same level of inhibition rate of Streptococcus mutans biofilm formation after being stored for 30 days under refrigeration condition (4 ℃), and had good stability.
Comparative example 1
The inoculation amount, soybean milk concentration, culture temperature and fermentation time in example 1 were adjusted one by one to obtain fermented soybean milk prepared by the following different methods, and the inhibitory effect of the obtained fermented soybean milk on streptococcus mutans biofilm in each group is shown in table 4.
TABLE 4 inhibitory Effect of fermented Soybean milk prepared by various methods on the formation of Streptococcus mutans biofilm
As can be seen from the results shown in table 4, when the inoculation amount, soybean milk concentration, culture temperature and fermentation time in the method for preparing fermented soybean milk were adjusted to be out of the preferred ranges, it was found by comparing example 1 with comparative example 1 that the preparation of fermented soybean milk from Paenibacillus sp CGMCC No.10062 still inhibited the formation of streptococcus mutans biofilm, but the inhibitory effect was significantly reduced.
Therefore, within 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 prepared by the Paenibacillus sp (CGMCC No. 10062) has better inhibition effect on the formation of the streptococcus mutans biofilm.
Comparative example 2
The inoculation amount, soybean milk concentration, culture temperature and fermentation time in example 6 were adjusted one by one to obtain fermented soybean milk prepared by the following different methods, and the inhibitory effect of the obtained fermented soybean milk on streptococcus mutans biofilm in each group is shown in table 5.
TABLE 5 inhibitory Effect of fermented Soybean milk prepared by different methods on the formation of Streptococcus mutans biofilm
As can be seen from the results shown in table 5, when the inoculation amount, soybean milk concentration, culture temperature and fermentation time were adjusted to be outside the preferred ranges in the method for preparing fermented soybean milk, it was found by comparing example 6 with comparative example 2 that the preparation of fermented soybean milk from Paenibacillus sp CGMCC No.10062 and lactobacillus plantarum (l.plantarum) ATCC14917 (purchased from ATCC) still inhibited the formation of biofilm of streptococcus mutans, but the inhibitory effect was significantly decreased.
Therefore, within the preferred range, the inoculation amount, soybean milk concentration, culture temperature and fermentation time are affected with each other, so that fermented soybean milk prepared from Paenibacillus (Paenibacillus sp.) CGMCC No.10062 and lactobacillus plantarum (l.plantarum) ATCC14917 (purchased from ATCC) has a better inhibition effect on the formation of streptococcus mutans biofilm.
Comparative example 3
Referring to the method described in example 1, the inhibitory effects of fermented soybean milk prepared from Paenibacillus (Paenibacillus sp.) CGMCC No.10062, lactobacillus plantarum (l.plantarum) ATCC14917 (purchased from ATCC), streptococcus thermophilus (s.thermophilus) ST-BODY-3 (provided by hansen, inc., kogaku), lactobacillus casei (l.casei) ATCC 393 (purchased from ATCC) on streptococcus mutans biofilm formation were compared as follows:
1. materials and methods
(a) Preparation of seeds (fermentation strain):
preparation of Paenibacillus seeds (fermentation strain): the same as in example 1.
Preparation of lactobacillus casei and lactobacillus plantarum seeds: respectively dissolving freeze-dried powder of lactobacillus casei ATCC 393 and lactobacillus plantarum ATCC14917 in a small amount of sterile distilled water, respectively taking a ring by using an inoculating ring to streak on an MRS solid culture medium (purchased from Merck Co, Germany), carrying out anaerobic culture at 37 ℃ for 24h, taking a single colony by using the inoculating ring to put in 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 colony, inoculating in 50mL of MRS liquid by using an inoculation amount of 2% (v/v), carrying out culture at 37 ℃ for 24h, centrifuging the culture at 9,000rpm for 10min, discarding 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 corresponding seeds for fermentation.
Preparation of streptococcus thermophilus seeds: dissolving the freeze-dried powder of the streptococcus thermophilus ST-BODY-3 by using a small amount of sterile distilled water, taking a loop by using an inoculating loop, scribing on an M17 solid culture medium (purchased from Merck Co, Germany), carrying out anaerobic culture at 40 ℃ for 24h, taking a single colony by using the inoculating loop, putting the single colony into 1mL of M17 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 40 ℃ for 24h, taking out the single colony, 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 the culture at 9,000rpm for 10min, discarding the supernatant, washing the thallus for 2 times by using the sterile distilled water, and suspending the thallus by using the 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) Measuring the inhibition rate of the fermented soybean milk on the formation of the streptococcus mutans biofilm: the same as in example 1.
2. Preparation of fermented soybean milk
Inoculating each strain in soybean milk with solid content of 5% (w/w) aseptically at inoculation amount of 3% (v/v), and culturing respectively (anaerobic culture of Paenibacillus, Lactobacillus plantarum and Lactobacillus casei at 37 deg.C, and anaerobic culture of Streptococcus thermophilus at 40 deg.C) for 24h to obtain corresponding fermented soybean milk.
3. Determination of inhibition rate of fermented soybean milk on formation of streptococcus mutans biofilm
The inhibitory effect of fermented soymilk prepared from the above different strains on the formation of Streptococcus mutans biofilm is shown in Table 6:
TABLE 6 inhibitory Effect of fermented soymilk prepared from different strains on the formation of Streptococcus mutans biofilm
As can be seen from Table 6, the fermented soybean milk prepared from Paenibacillus sp (Paenibacillus sp.) CGMCC No.10062 showed an inhibitory rate of 40%, while the fermented soybean milk prepared from other strains showed no inhibitory activity against Streptococcus mutans biofilm.
As can be seen from example 6, the inhibition ratio of the fermented soybean milk prepared by combining Paenibacillus (Paenibacillus sp.) CGMCC No.10062 and lactobacillus plantarum (l.plantararum) ATCC14917 (purchased from ATCC) was 59%, and the inhibition activity of the fermented soybean milk prepared by combining Paenibacillus (Paenibacillus sp.) CGMCC No.10062 and lactobacillus plantarum (l.plantararum) ATCC14917 (purchased from ATCC) on streptococcus mutans biofilm was significant when compared with the fermented soybean milk prepared by combining Paenibacillus (Paenibacillus sp.) CGMCC No. 10062.
Comparative example 4
1. Materials and methods
(a) Preparation of paenibacillus seeds (zymophyte) and streptococcus mutans bacterial suspension: the same as in example 1. Preparation of lactobacillus plantarum (zymophyte): the same as in example 6.
(b) And (3) determining the inhibition rate of the fermentation liquor on the formation of the streptococcus mutans biofilm:
pretreatment of fermentation liquor: and adjusting the pH value of the fermentation liquor to 6.80 to obtain a sample to be detected.
Determination of inhibitory Activity: the assay was carried out as described in reference to example 1, using the unfermented medium as a blank.
2. Paenibacillus single-bacterium fermentation broth and preparation of paenibacillus and lactobacillus plantarum mixed fermentation broth
The method comprises the following steps of (1) aseptically inoculating Paenibacillus (Paenibacillus sp.) CGMCC No.10062 into an M17 liquid culture medium containing 1% of cane sugar by an inoculation amount of 3% (v/v), and carrying out anaerobic culture at 37 ℃ for 24h to obtain a fermentation liquid;
paenibacillus (Paenibacillus sp.) CGMCC No.10062 and Lactobacillus plantarum (L.plantarum) ATCC14917 (purchased from ATCC) were aseptically inoculated in a 1.5% (v/v) M17 liquid medium containing sucrose at 1% (w/v), respectively, and anaerobically cultured at 37 ℃ for 24 hours to obtain fermentation broth.
3. Determination of inhibition rate of fermentation liquor on formation of streptococcus mutans biofilm
The inhibition activity of the fermentation broth to the formation of the streptococcus mutans biofilm is measured after the fermentation broth is prepared into the sample to be detected by the method, and the results show that the inhibition rates of the single-bacterium fermentation broth of the paenibacillus and the mixed fermentation broth of the paenibacillus plantarum on the formation of the streptococcus mutans biofilm are 8% and 10% respectively.
Comparative example 5
The pH of the fermented soybean milk A, B, C, D, E, F, G, H, I and J prepared in examples 1 to 10 was adjusted to 6.80, and then centrifuged at 10,000rpm at 4 ℃ for 10min to obtain a supernatant as a sterilized fermented soybean milk (sample to be tested). The inhibition rate of biofilm formation by Streptococcus mutans was determined for the above samples according to the method described in example 1, and the results are shown in Table 7.
TABLE 7 inhibitory Effect of aseptically fermented Soybean milk on the biofilm formation of Streptococcus mutans
As can be seen from Table 7, the aseptic 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 products of the strains after metabolizing the soybean milk. However, the biofilm inhibition effect of each group of the aseptic fermented soybean milk is lower than that of the sample group before the aseptic treatment, which shows that the effect of inhibiting the biofilm bacteria membrane of the fermented soybean milk before the aseptic treatment comes from the combined action of the viable bacteria and the metabolite.
The fermented soybean milk, the preparation method of the fermented soybean milk and the application thereof provided by the present invention are described in detail above. The principles and embodiments of the present invention have been described herein using specific examples, which are presented only to assist in understanding the method and its core concepts of the present invention. 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 (6)
1. A method for preparing fermented soybean milk from Paenibacillus is characterized by comprising the following steps: inoculating paenibacillus CGMCC No.10062 as a fermentation microbial inoculum into the soybean milk for fermentation to obtain fermented soybean milk;
the fermentation inoculant is a composite fermentation inoculant of paenibacillus CGMCC No.10062 and lactobacillus plantarum ATCC14917 with the inoculation ratio of 1-9: 1-9;
the mass percentage content of the solid matters in the soybean milk is 1-9%;
the inoculation amount of the paenibacillus CGMCC No.10062 is 2 multiplied by 107～1×108cfu/mL；
The fermentation temperature is 15-45 ℃, and the fermentation time is 2-48 h.
2. The method for preparing fermented soybean milk according to claim 1, wherein the soybean milk is one or more of soybean, red bean, mung bean, broad bean, white kidney bean and small white kidney bean.
3. The method for preparing fermented soybean milk according to claim 1, wherein the fermentation is static culture.
4. A fermented soybean milk produced by Paenibacillus, characterized by being produced by the production method according to any one of claims 1 to 3.
5. The fermented soybean milk according to claim 4, wherein the inhibition rate of the fermented soybean milk on the formation of Streptococcus mutans biofilm is not less than 40%.
6. Use of the fermented soybean milk of claim 4 for preparing an anti-caries food.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910435064.7A CN110012941B (en) | 2019-05-23 | 2019-05-23 | Method for preparing fermented soybean milk by using paenibacillus, fermented soybean milk prepared by method and application of fermented soybean milk |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910435064.7A CN110012941B (en) | 2019-05-23 | 2019-05-23 | Method for preparing fermented soybean milk by using paenibacillus, fermented soybean milk prepared by method and application of fermented soybean milk |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110012941A CN110012941A (en) | 2019-07-16 |
| CN110012941B true CN110012941B (en) | 2022-07-15 |
Family
ID=67194329
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910435064.7A Active CN110012941B (en) | 2019-05-23 | 2019-05-23 | Method for preparing fermented soybean milk by using paenibacillus, fermented soybean milk prepared by method and application of fermented soybean milk |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110012941B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110338228B (en) * | 2019-07-26 | 2022-10-14 | 广东轻工职业技术学院 | Soybean milk powder without abdominal distension and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2482666A1 (en) * | 2012-10-09 | 2014-08-04 | Universidade De Santiago De Compostela | Use of the cect 7426 strain for generating quorum quenching of the autoinducer-2 signal (ai-2) |
| CN106148251A (en) * | 2016-09-21 | 2016-11-23 | 光明乳业股份有限公司 | One Bacillus species fermentation liquid and its preparation method and application |
| CN106701833A (en) * | 2015-11-18 | 2017-05-24 | 光明乳业股份有限公司 | Fungistatic paenibacillus sp. fermentation liquor extract |
| CN108728497A (en) * | 2017-04-24 | 2018-11-02 | 光明乳业股份有限公司 | A kind of streptococcus mutans inhibitor and preparation method thereof |
-
2019
- 2019-05-23 CN CN201910435064.7A patent/CN110012941B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2482666A1 (en) * | 2012-10-09 | 2014-08-04 | Universidade De Santiago De Compostela | Use of the cect 7426 strain for generating quorum quenching of the autoinducer-2 signal (ai-2) |
| CN106701833A (en) * | 2015-11-18 | 2017-05-24 | 光明乳业股份有限公司 | Fungistatic paenibacillus sp. fermentation liquor extract |
| CN106148251A (en) * | 2016-09-21 | 2016-11-23 | 光明乳业股份有限公司 | One Bacillus species fermentation liquid and its preparation method and application |
| CN108728497A (en) * | 2017-04-24 | 2018-11-02 | 光明乳业股份有限公司 | A kind of streptococcus mutans inhibitor and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| "益生菌防治龋病的机制及应用"，马婧娴，等，医学综述，第24卷第8期，第1493-1497页，2018年12月;马婧娴，等;《医学综述》;20181231;第24卷(第8期);第1493-1497页 * |
| Hideaki Tsumori，等.Mutanase of Paenibacillus humicus from Fermented Food Has a Potential for Hydrolysis of Biofilms Synthesized by Streptococcus mutans，Hideaki Tsumori，等，Journal of Health Science，第57卷第5期，第420–424页，2011年12月.《Journal of Health Science》.2011,第57卷(第5期),第420–424页. * |
| Mutanase of Paenibacillus humicus from Fermented Food Has a Potential for Hydrolysis of Biofilms Synthesized by Streptococcus mutans，Hideaki Tsumori，等，Journal of Health Science，第57卷第5期，第420–424页，2011年12月;Hideaki Tsumori，等;《Journal of Health Science》;20111231;第57卷(第5期);第420–424页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110012941A (en) | 2019-07-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110100899B (en) | Method for preparing fermented soybean milk powder from lactobacillus fermentum, prepared fermented soybean milk powder and application | |
| CN110122576B (en) | Method for preparing fermented soybean milk by lactobacillus fermentum, prepared fermented soybean milk and application | |
| KR100471631B1 (en) | Bifidobacterium breve and fermented soymilk prepared with the same | |
| FI76373B (en) | FOERFARANDE FOER FRAMSTAELLNING AV EN ODLING SOM INNEHAOLLER LEVANDE CELLMASSA AV BIFIDOBAKTERIER OCH MJOELKSYRABAKTERIER. | |
| CN113736716B (en) | Lactobacillus paracasei, compound biological leavening agent for yellow storage and yellow storage method | |
| KR102000475B1 (en) | Bacillus amyloliquefaciens NY12-2 strain having antibiotic activity against Bacillus cereus and inhibitory activity on biogenic amine and compositions thereof | |
| CN110012941B (en) | Method for preparing fermented soybean milk by using paenibacillus, fermented soybean milk prepared by method and application of fermented soybean milk | |
| CN110122581B (en) | Method for preparing fermented soybean milk from leuconostoc mesenteroides, prepared fermented soybean milk and application | |
| CN115141860A (en) | Method for producing gamma-aminobutyric acid and fermentation culture prepared by same | |
| US4210672A (en) | Preparation of yogurt | |
| CN110122578B (en) | Method for preparing fermented soybean milk powder from leuconostoc mesenteroides, prepared fermented soybean milk powder and application | |
| CN107441121B (en) | Fermentation crude extract of streptococcus salivarius, and preparation method and application thereof | |
| CN110122577B (en) | Method for preparing fermented soybean milk from paenibacillus bovis, prepared fermented soybean milk and application | |
| CN107410479B (en) | Fermented milk and preparation method thereof | |
| Sanni et al. | Microbiological evaluation of Ghanaian maize dough co-fermented with cowpea | |
| CN110331104A (en) | A kind of lactobacillus plantarum CV10D1 and its application | |
| CN110150387B (en) | Method for preparing fermented soybean milk powder from paenibacillus, fermented soybean milk powder and application | |
| CN108813045A (en) | A kind of preparation method of pectase honey lemon tea | |
| CN114369546A (en) | Pediococcus acidilactici, fermentation microbial inoculum and application thereof, and preparation method of coarse cereal fermented food | |
| JPH02268644A (en) | Preparation of fermented milk | |
| CN110951654A (en) | Lactobacillus plantarum proliferation agent and application thereof | |
| CN110122580A (en) | A kind of fermented soybean milk powder for being prepared the method for fermented soybean milk powder by streptococcus salivarius and being prepared and application | |
| CN110122575A (en) | A kind of fermented soybean milk powder for being prepared the method for fermented soybean milk powder by ox series bacillus and being prepared and application | |
| CN110122579A (en) | It is a kind of the method for fermented soybean milk to be prepared by streptococcus salivarius and the fermented soybean milk prepared and application | |
| CN116064311A (en) | Lactobacillus plantarum resistant to high-concentration tartaric acid and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |