WO2020233031A1 - 一种由肠膜明串珠菌制备发酵豆浆的方法及制备出的发酵豆浆与应用 - Google Patents

一种由肠膜明串珠菌制备发酵豆浆的方法及制备出的发酵豆浆与应用 Download PDF

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WO2020233031A1
WO2020233031A1 PCT/CN2019/119946 CN2019119946W WO2020233031A1 WO 2020233031 A1 WO2020233031 A1 WO 2020233031A1 CN 2019119946 W CN2019119946 W CN 2019119946W WO 2020233031 A1 WO2020233031 A1 WO 2020233031A1
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soybean milk
fermented soybean
leuconostoc mesenteroides
streptococcus mutans
fermented
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PCT/CN2019/119946
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English (en)
French (fr)
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韩瑨
刘振民
吴正钧
乔祯逸
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光明乳业股份有限公司
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Priority to AU2019425533A priority Critical patent/AU2019425533B2/en
Publication of WO2020233031A1 publication Critical patent/WO2020233031A1/zh

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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
    • 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

Definitions

  • the invention belongs to the technical field of biological fermentation, and specifically relates to a preparation method of fermented soybean milk; in addition, the invention also relates to the fermented soybean milk and its application.
  • Tooth decay commonly known as cavities and tooth decay, is a bacterial disease that can cause pulpitis and periapical periodontitis, and even cause inflammation of the alveolar bone and jaw bone. If it is not treated in time, the disease will continue to develop, forming cavities, and eventually the crown will be completely destroyed and disappear. The final result of its development is tooth loss.
  • Dental caries is characterized by high incidence and wide distribution. The disease is the main common disease in the oral cavity and one of the most common diseases in human beings. The World Health Organization has listed it as the three key human diseases for prevention and treatment alongside tumors and cardiovascular diseases.
  • dental caries there are many factors that cause dental caries, such as bacteria, oral environment (food, saliva), host, etc. Among them, bacteria are a necessary condition for the occurrence of dental caries. It is generally believed that there are two types of cariogenic bacteria. One is the genus Acidogenic bacteria, and the main ones are Streptococcus mutans (S.mutans), Actinomycetes and Lactobacillus, the above-mentioned bacteria can decompose carbohydrates and produce acid, leading to demineralization of tooth minerals; the other is Gram-positive cocci, which can destroy Organic matter, after long-term action, makes teeth form cavities.
  • S.mutans Streptococcus mutans
  • Actinomycetes Actinomycetes
  • Lactobacillus the above-mentioned bacteria can decompose carbohydrates and produce acid, leading to demineralization of tooth minerals
  • Gram-positive cocci which can destroy Organic matter, after long-term action, makes teeth form cavities.
  • Streptococcus mutans is recognized by scholars at home and abroad as the most important and most important pathogen causing dental caries.
  • the above-mentioned pathogenic bacteria do not directly colonize the tooth surface, but form dental plaque by combining with the biofilm (insoluble polysaccharide) produced by Streptococcus mutans, and then indirectly adhere to the tooth surface.
  • biofilm insoluble polysaccharide
  • the present invention provides a method for preparing fermented soybean milk, which can be used to prepare fermented soybean milk with inhibiting the formation of Streptococcus mutans biofilm.
  • a method for preparing fermented soybean milk includes the following steps: Leuconostoc mesenteroides as a fermentation inoculum is inoculated into soybean milk for fermentation metabolism to obtain fermented soybean milk.
  • Leuconostoc mesenteroides is CGMCC No. 6432, ATCC 10830a or CGMCC No. 10064.
  • the Leuconostoc mesenteroides CGMCC No. 6432 and CGMCC No. 10064 strains were deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on August 13, 2012 and November 26, 2014, respectively.
  • CGMCC General Microbiology Center of the China Microbial Culture Collection Management Committee on August 13, 2012 and November 26, 2014, respectively.
  • CGMCC General Microbiology Center of the China Microbial Culture Collection Management Committee on August 13, 2012 and November 26, 2014, respectively.
  • CGMCC The deposit information of CGMCC No. 6432 strain has been disclosed in patent CN103013891A; the deposit information of CGMCC No. 10064 strain has been disclosed in patent CN105349477A.
  • Leuconostoc mesenteroides ATCC 10830a was purchased from ATCC (American Type Culture Collection Center).
  • the inoculum of the fermentation inoculum is 1 ⁇ 10 7 to 5 ⁇ 10 7 cfu/mL.
  • soybean milk is at least one of soybean, red bean, mung bean, broad bean, large white kidney bean or small white kidney bean soybean milk.
  • the mass percentage content of solids in the soybean milk is 1%-9%.
  • the temperature of the fermentation is 15°C to 45°C, and the time is 2 to 48 hours.
  • the fermentation is static culture.
  • a fermented soybean milk is also provided, which is prepared by any of the above preparation methods.
  • the above-mentioned fermented soybean milk has an inhibition rate of ⁇ 62% on the formation of Streptococcus mutans biofilm.
  • the preparation method in the above technical scheme adopts Leuconostoc mesenteroides for the first time, fermenting with soybean milk as a medium, and prepares fermented soy milk with Streptococcus mutans biofilm inhibitory activity, and Leuconostoc mesenteroides Bacteria-fermented soybean milk has a new purpose of anti-caries.
  • the fermented soymilk prepared by Leuconostoc mesenteroides has a significant activity of inhibiting the formation of Streptococcus mutans biofilm.
  • a method for preparing fermented soy milk including the following steps: Leuconostoc mesenteroides is inoculated into soy milk for fermentation to obtain fermented soy milk.
  • the preparation method in the above technical scheme uses Leuconostoc mesenteroides for the first time and fermented with soybean milk as a medium to prepare fermented soy milk with the biofilm inhibitory activity of Streptococcus mutans. It is disclosed that Leuconostoc mesentericus fermented soybean milk has anti-caries The new use of Leuconostoc mesenteroides uses soy milk to synthesize metabolites that have Streptococcus mutans inhibitory activity.
  • the fermented soymilk prepared by Leuconostoc mesenteroides has a more significant inhibitory effect on the formation of Streptococcus mutans biofilm and has better stability.
  • strains used in the preparation method of the above technical scheme are naturally edible strains, and the fermentation medium (various soy milk) used is widely sourced, low in cost, and natural and safe. While reducing material costs, it improves food safety. Sex.
  • Leuconostoc mesenteroides is CGMCC No. 6432, ATCC 10830a or CGMCC No. 10064, preferably CGMCC No. 6432, ATCC 10830a, and more preferably CGMCC No. 6432.
  • inoculation Leuconostoc bacteria was 1x10 7 ⁇ 5x10 7 cfu / mL ; preferably of 2x10 7 ⁇ 4x10 7 cfu / mL , more preferably of 3x10 7 cfu / mL.
  • the soybean milk is at least one of soybean, red bean, mung bean, broad bean, large white kidney bean, or small white kidney bean soybean milk, and more preferably soybean soybean milk.
  • the solid content of the soy milk is preferably 1%-9% by mass; preferably 3%-7%; more preferably 5%.
  • the solid content in soymilk is directly related to the biofilm inhibition effect of the final product.
  • the solid content is too high, it will cause the osmotic pressure of the bacterial habitat to increase, affect the growth of the bacteria and reduce the synthesis of membrane inhibitors.
  • the cost will also increase, and too low solid content will result in poor proliferation of live bacteria due to insufficient nutrients, and will also affect the production of membrane inhibitors.
  • the preferred fermentation temperature is 15°C to 45°C; preferably 25°C to 40°C; more preferably 37°C.
  • the preferred fermentation time is 2 to 48 hours; preferably 12 to 36 hours; more preferably 24 hours.
  • the preferred fermentation method is static culture.
  • Leuconostoc mesenteroides are all facultative anaerobic bacteria, which can grow and reproduce rapidly under static culture conditions.
  • the fermented soymilk prepared by Leuconostoc mesenteroides CGMCC No. 6432 has a significantly reduced inhibitory effect on the formation of Streptococcus mutans biofilm when it is outside the range of the preferred fermentation parameters.
  • the inoculum, soybean milk concentration, culture temperature and fermentation time affect each other, so that the fermented soymilk prepared by Leuconostoc mesenteroides CGMCC No. 6432 has better biofilm formation of Streptococcus mutans The inhibitory effect.
  • a fermented soybean milk is provided, which is prepared by any of the above-mentioned methods for preparing fermented soybean milk.
  • the fermented soybean milk prepared by the preparation method also has corresponding beneficial effects, which will not be repeated here.
  • the above-mentioned fermented soybean milk has an inhibition rate of ⁇ 62% on the formation of Streptococcus mutans biofilm. It can also be known in combination with the examples that the above-mentioned fermented soybean milk has a significantly higher inhibitory effect on the formation of Streptococcus mutans biofilm than other fermentable soybean milk strains.
  • Pretreatment of fermented soybean milk adjust the pH of fermented soybean milk to 6.80 to obtain the sample to be tested.
  • Determination of inhibitory activity immerse a sterile magnetic stirrer A (5 ⁇ 10mm) in the saliva of healthy people after sterile filtration with a 0.22 ⁇ m filter membrane and incubate at 37°C for 4h. Use a sterile magnetic stirrer B (10 ⁇ 50mm) ) Suction the magnetic stirrer A, rinse with 10mm, pH 7.0 PBS, remove the magnetic stirrer A, and place it in the culture well of the 24-well plate.
  • Biofilm inhibition rate (I): I (OD 600nm (blank control)-OD 600nm (sample to be tested))/OD 600nm (blank control) ⁇ 100%.
  • the Leuconostoc mesenteroides CGMCC No. 6432 was aseptically inoculated to a solid content of 5% (w/w, with an inoculum amount of 3% (v/v, the volume percentage of the seed liquid in the fermentation liquid, the same below)
  • v/v the volume percentage of the seed liquid in the fermentation liquid, the same below
  • the percentage by mass of soybeans in water, the same below), fermented soybean milk A is obtained by static cultivation at 37°C for 24 hours.
  • Leuconostoc mesenteroides CGMCC No. 6432 was aseptically inoculated into red soybean milk with a solid content of 9% (w/w) at an inoculation amount of 1% (v/v), and cultured at 15°C for 48 hours Get fermented soy milk B.
  • Leuconostoc mesenteroides CGMCC No. 6432 was aseptically inoculated into mung bean soy milk with a solid content of 1% (w/w) at an inoculum amount of 5% (v/v), and cultured at 45°C for 2 hours. Fermented soy milk C.
  • Leuconostoc mesenteroides CGMCC No. 6432 was aseptically inoculated into broad bean soy milk with a solid content of 7% (w/w) at an inoculum amount of 2% (v/v), and cultured at 25°C for 36 hours. Fermented soy milk D.
  • Leuconostoc mesenteroides CGMCC No. 6432 was aseptically inoculated into white kidney bean milk with a solid content of 3% (w/w) at an inoculum amount of 4% (v/v), and cultured at 40°C for 12 hours Get fermented soy milk E.
  • Leuconostoc mesenteroides ATCC 10830a was aseptically inoculated into soybean milk with a solid content of 5% (w/w) at an inoculum amount of 3% (v/v), and incubated at 37°C for 24 hours to obtain fermented soy milk F.
  • Leuconostoc mesenteroides CGMCC No. 10064 was aseptically inoculated into soybean soy milk with a solid content of 5% (w/w) at an inoculum amount of 3% (v/v), and cultured at 37°C for 24 hours. Fermented soy milk G.
  • the fermented soymilk products A, B, C, D, E, F, and G prepared in the above-mentioned Examples 1-7 were used as experimental subjects to conduct a taste test of the products.
  • the test number is 50 people.
  • Tasting method Tasting is carried out by anonymous scoring; the color, flavor, taste, and nutrition of the above fermented soy milk products A, B, C, D, E, F and G are individually scored, and the full score for each item is 25 Score, calculate the average score and its total score, and record the statistical results in Table 1.
  • the number of people who like each item is counted, and the statistical results are recorded in Table 2.
  • the fermented soybean milk A, B, C, D, E, F, and G prepared in Examples 1-7 were stored in refrigerated conditions (4°C) for 0, 10, 20, and 30 days before being taken out, and the effects of each sample on Streptococcus mutans were determined.
  • Table 3 shows the inhibition rate of biofilm formation.
  • Example 1 The inoculum, soybean milk concentration, culture temperature, and fermentation time in Example 1 were adjusted one by one to obtain the following group of fermented soybean milk prepared by different methods.
  • the inhibitory effects of each group of fermented soybean milk on Streptococcus mutans biofilm are shown in Table 4. Shown.
  • Lactobacillus casei and Lactobacillus bulgaricus seeds Dissolve the freeze-dried powders of Lactobacillus casei ATCC 393 and Lactobacillus plantarum ATCC14917 respectively with a small amount of sterile distilled water, and each use an inoculation loop to take a loop and streak it on the MRS solid medium (purchase (From Merck Co. Germany), anaerobic culture at 37°C for 24 hours, take out a single colony with an inoculation loop and put it into 1mL MRS liquid (purchased from Merck Co.
  • the culture was centrifuged at 9,000rpm for 10 minutes, the supernatant was discarded, and the cells were washed twice with sterile distilled water. Suspend with sterile distilled water of the original culture volume to obtain seeds for fermentation.
  • Each strain was aseptically inoculated into soybean soy milk with a solid content of 5% (w/w) at a 3% (v/v) inoculum amount, and cultured separately (L. mesenteroides, Lactobacillus plantarum and Lactobacillus casei at 37°C) Anaerobic culture, anaerobic culture of Streptococcus thermophilus at 40°C) for 24h to obtain the corresponding fermented soybean milk.
  • Pretreatment of fermentation broth adjust the pH of the fermentation broth to 6.80 to obtain the sample to be tested.
  • Determination of inhibitory activity Refer to the method described in Example 1, with unfermented medium as a blank control for determination.
  • Leuconostoc mesenteroides CGMCC No. 6432, ATCC 10830a and CGMCC No. 10064 were aseptically inoculated into M17 liquid medium containing 1% (w/v) sucrose at an inoculum amount of 3% (v/v). Anaerobic culture at 37°C for 24 hours to obtain fermentation broth.
  • the Leuconostoc mesenteroides fermentation broth was prepared as the sample to be tested by the above method, and its inhibitory activity on the formation of Streptococcus mutans biofilm was measured.
  • the results showed that the fermentation broth of CGMCC No. 6432, ATCC 10830a, and CGMCC No.
  • the inhibition rates of cocci biofilm formation were 8%, 7%, and 5% respectively.
  • Example 1-7 The pH of the fermented soybean milk A, B, C, D, E, F and G prepared in Example 1-7 was adjusted to 6.80, and then centrifuged at 4°C and 10,000 rpm for 10 minutes. The supernatant was the sterile fermented soybean milk ( Sample to be tested). According to the method described in Example 1, the inhibition rate of the above-mentioned samples on the formation of Streptococcus mutans biofilm was measured, and the results are shown in Table 6.
  • the sterile fermented soybean milk still has a strong inhibitory activity on the inhibition rate of Streptococcus mutans biofilm formation, indicating that the inhibitory effect comes from the product after the strain metabolizes the soybean milk.
  • the biofilm inhibition effect of the aseptic fermented soybean milk in each group was lower than that of the sample group before aseptic processing, which indicates that the biofilm inhibition effect of the fermented soybean milk before aseptic processing also comes from the combined action of living bacteria and metabolites.

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Abstract

一种由肠膜明串珠菌制备发酵豆浆的方法，包括以下步骤：将肠膜明串珠菌接种于豆浆中进行发酵代谢，得到发酵豆浆，所述肠膜明串珠菌为CGMCC No.6432、ATCC 10830a或CGMCC No.10064。还涉及由上述方法制得的发酵豆浆，以及所属发酵豆浆在制备抗龋齿食品方面的应用。所述发酵豆浆对变形链球菌生物膜形成的抑制率≥62%。

Description

一种由肠膜明串珠菌制备发酵豆浆的方法及制备出的发酵豆浆与应用 技术领域
本发明属于生物发酵技术领域，具体涉及一种发酵豆浆的制备方法；此外，本发明还涉及该发酵豆浆及其应用。
背景技术
龋齿，俗称虫牙、蛀牙，是一种细菌性疾病，可以继发牙髓炎和根尖周炎，甚至能引起牙槽骨和颌骨炎症。如不及时治疗，病变继续发展，形成龋洞，终至牙冠完全破坏消失，其发展的最终结果是牙齿丧失。龋齿特点是发病率高，分布广。该病是口腔主要的常见病，也是人类最普遍的疾病之一，世界卫生组织已将其与肿瘤和心血管疾病并列为人类三大重点防治疾病。
引发龋齿的因素众多，如细菌、口腔环境(食物、唾液)、宿主等，其中，细菌是龋齿发生必要条件，一般认为致龋菌有两种类型，一种是产酸菌属，其中主要为变形链球菌(Streptococcus mutans，简称S.mutans)、放线菌属和乳杆菌，上述菌种可使碳水化合物分解产酸，导致牙齿无机质脱矿；另一种是革兰阳性球菌，可破坏有机质，经过长期作用使牙齿形成龋洞。上述致龋菌中，变形链球菌被国内外学者公认为是引发龋齿的最主要和最重要的病原菌。然而上述这些病原菌并不是直接定植于牙齿表面，而是通过与变形链球菌所产生的生物膜(不溶性多糖)结合形成牙菌斑后，间接地粘附于牙齿表面，随着致龋菌不断在生物膜上的积累，龋齿的程度逐渐恶化。因此，减少其生物膜的产量也可达到防治龋齿的目的。
目前，有关龋齿治疗研究绝大部分集中于控制或降低口腔中变形链球菌的数量，然而，健康的口腔环境一般是处于有害菌与有益菌达到平衡的状态，有害菌过度的减少会带来口腔菌群失衡，进而引起人体的其他不良反应。此外，临床上使用的一些针对变形链球菌的杀菌药物，如氨硝酸银等化合物，副作用强，长期使用会令牙齿染色，故不适用于前牙治疗。而通过减少变形链球菌生物膜产量同样可降低龋齿患病风险，并且不破坏口腔菌群的固有平 衡或产生副作用，但这方面的报道相对较少，尤其是与微生物发酵制品有关的研究极其有限，具有抑制变形链球菌生物膜形成的发酵制品相当匮乏。
因此，提供具有抑制变形链球菌生物膜形成的发酵制品是当务之急。这是本领域技术人员亟待解决的问题。
发明内容
基于上述技术问题，本发明提供了一种发酵豆浆的制备方法，采用该方法可以制得具有抑制变形链球菌生物膜形成的发酵豆浆。
具体的，一方面，提供了一种发酵豆浆的制备方法，包括以下步骤：将肠膜明串珠菌作为发酵菌剂接种于豆浆中进行发酵代谢，得到发酵豆浆。
进一步地，所述肠膜明串珠菌为CGMCC No.6432、ATCC 10830a或CGMCC No.10064。
其中，肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.6432和CGMCC No.10064菌株已分别于2012年8月13日和2014年11月26日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址：北京市朝阳区北辰西路1号院3号，邮编：100101。CGMCC No.6432菌株的保藏信息在专利CN103013891A中已经公开；CGMCC No.10064菌株的保藏信息在专利CN105349477A中已经公开。肠膜明串珠菌(Leuconostoc mesenteroides)ATCC 10830a购买自ATCC(美国模式菌种收集中心)。
进一步地，所述发酵菌剂的接种量为1x10 7～5x10 7cfu/mL。
进一步地，所述豆浆为大豆、赤豆、绿豆、蚕豆、大白芸豆或小白芸豆豆浆中的至少一种。
进一步地，所述豆浆中固形物的质量百分比含量为1％～9％。
进一步地，所述发酵的温度为15℃～45℃，时间为2～48h。
进一步地，所述发酵为静置培养。
第二方面，还提供了一种发酵豆浆，由上述任一种制备方法制得。
进一步地，上述发酵豆浆对变形链球菌生物膜形成的抑制率≥62％。
第三方面，还提供一种发酵豆浆在制备抗龋齿食品方面的应用。
与现有技术相比，上述技术方案中的制备方法，首次采用肠膜明串珠菌，以豆浆作为培养基进行发酵，制备得到具有变形链球菌生物膜抑制活性的发酵豆浆，并且肠膜明串珠菌发酵豆浆具有抗龋齿的新用途。与其它可发酵豆浆的菌株与该菌株可生长繁殖的培养基相比，肠膜明串珠菌所制备的发酵豆浆具有显著的抑制变形链球菌生物膜形成的活性。
具体实施方式
为更清楚的对本发明技术方案予以阐述，下面将结合具体实施方式对本发明的技术方案进行进一步阐述：
在一个具体的实施方式中，提供了一种发酵豆浆的制备方法，包括以下步骤：将肠膜明串珠菌接种于豆浆中进行发酵，得到发酵豆浆。
上述技术方案中的制备方法，首次采用肠膜明串珠菌，以豆浆作为培养基进行发酵，制备得到具有变形链球菌生物膜抑制活性的发酵豆浆，披露了肠膜明串珠菌发酵豆浆具有抗龋齿的新用途，肠膜明串珠菌利用豆浆合成的代谢物质具有变形链球菌抑制活性。与其它可发酵豆浆菌株或该菌株生产繁殖的培养基相比，肠膜明串珠菌所制备的发酵豆浆对变形链球菌生物膜形成的抑制效果更显著，并且稳定性佳。
此外，上述技术方案中的制备方法所采用的菌株为天然可食用菌株，采用的发酵培养基(各类豆浆)来源广泛、成本低廉、天然安全，在降低物料成本的同时，提高了食品的安全性。
进一步地，上述步骤中，肠膜明串珠菌为CGMCC No.6432、ATCC 10830a或CGMCC No.10064，较佳地为CGMCC No.6432、ATCC 10830a，更佳地为CGMCC No.6432。
进一步地，上述步骤中，肠膜明串珠菌的接种量为1x10 7～5x10 7cfu/mL；较佳地为2x10 7～4x10 7cfu/mL，更佳地为3x10 7cfu/mL。
进一步地，上述步骤中，所述豆浆为大豆、赤豆、绿豆、蚕豆、大白芸豆或小白芸豆豆浆中的至少一种，更佳的为大豆豆浆。
进一步地，上述步骤中，优选的豆浆的固形物含量为质量百分比1％～9％；较佳地为3％～7％；更佳地为5％。豆浆中固形物含量与最终产品的生物膜抑制效果有直接关系，当固形物含量过高时，会引起菌体生境渗透压增加，影响菌体的生长而降低抑膜物质的合成量，另外生产成本也会上升，而固形物含量过低则会因营养成分不足导致活菌增殖效果差，同样会影响抑膜物质的产生。
进一步地，上述步骤中，优选的发酵温度为15℃～45℃；较佳地为25℃～40℃；更佳地为37℃。
进一步地，上述步骤中，优选的发酵时间为2～48h；较佳地为12～36h；更佳地为24h。
进一步地，上述步骤中，优选的发酵方式为静置培养。肠膜明串珠菌均为兼性厌氧菌，在静置培养条件下，能快速地生长繁殖。
结合实施例和比较例1亦可知，在优选发酵参数的范围之外时，由肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.6432制备的发酵豆浆对变形链球菌生物膜形成的抑制效果明显下降。而在优选范围之内，接种量、豆浆浓度、培养温度和发酵时间相互影响，使得由肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.6432制备的发酵豆浆具有更佳的变形链球菌生物膜形成的抑制效果。
在另一个具体的实施方式中，提供了一种发酵豆浆，该发酵豆浆由上述任一种发酵豆浆的制备方法制得。
由于发酵豆浆的制备方法具备上述的有益效果，由该制备方法制得的发酵豆浆也具有相应的有益效果，此处不再赘述。
进一步的，上述发酵豆浆对变形链球菌生物膜形成的抑制率≥62％。结合实施例亦可知，上述发酵豆浆对变形链球菌生物膜形成的抑制效果显著高于其他可发酵豆浆的菌株。
下面通过实施例进一步说明上述具体实施方式，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按 照常规方法和条件，或按照商品说明书选择。下述实施例中，所有原料均为市售，并均符合相关的国家标准。
实施例1
1、材料与方法
(a)肠膜明串珠菌(发酵菌种)的制备：将肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.6432的冻干粉用少量无菌蒸馏水溶解，用接种环取一环划线于含2％(w/v)蔗糖的M17固体培养基(购买自Merck Co.德国)上，30℃好氧培养24h取出，用接种环挑取单菌落放入20mL含2％(w/v)蔗糖的M17液体(购买自Merck Co.德国)，运用涡旋震荡器将菌落均匀分散于液体培养基内，30℃、180rpm摇床振荡培养24h取出，培养物9,000rpm离心10分钟，弃去上清，菌体用无菌蒸馏水洗涤2次后，用原培养体积的无菌蒸馏水悬浮，得到发酵用的种子，种子液的菌浓度为1x10 9cfu/mL。
变形链球菌菌悬液的制备：将变形链球菌CGMCC No.1.2499(购自CGMCC，中国)的冻干粉用少量无菌蒸馏水溶解，用接种环取一环划线于BHI固体培养基(购买自OXOID Co.，英国)，37℃厌氧培养24h取出，用接种环挑取单菌落放入10mL BHI液体培养基(购买自OXOID Co.，英国)，运用涡旋振荡器将菌落均匀分散于液体培养基内，37℃厌氧培养24h取出，以2％(v/v)接种量接种于BHI液体培养基，37℃厌氧培养24h后，培养物15000rpm离心10min，弃上清，菌体用无菌蒸馏水洗涤2次后，以BHI液体培养基重悬至10 6CFU/mL即可。
(b)豆浆的制备：将大豆干豆称重后，加入五倍质量的水置于37℃浸泡8h，弃去水，在豆浆机中加入一定比例的湿豆与水，制得所需固形物含量的豆浆，经121℃灭菌20min，得到指定固形物含量的无菌大豆豆浆。
(c)发酵豆浆对变形链球菌生物膜形成的抑制率测定：
发酵豆浆的预处理：将发酵豆浆pH调节至6.80，即得待测样品。
抑制活性的测定：将无菌的磁力搅拌子A(5×10mm)浸没于0.22μm滤膜无菌过滤后的健康人群唾液中37℃孵育4h，用无菌的磁力搅拌子B(10 ×50mm)吸起磁力搅拌子A，以10mm、pH7.0的PBS淋洗，取下磁力搅拌子A，置于24孔板的培养孔中。向培养孔中加入1.6mL BHI液体培养基、200μL变形链球菌菌悬液(10 6CFU/mL)和400μL待测样品溶液或空白对照溶液(未发酵的豆浆)，再次37℃厌氧孵育24h(购买自Greiner公司，德国)。用磁力搅拌子B吸起磁力搅拌子A，以PBS淋洗后，取下磁力搅拌子A，置于24孔板的培养孔中，自然风干。向培养孔中加入1.6mL、0.1％(w/v)的结晶紫溶液染色15min，用磁力搅拌子B吸起磁力搅拌子A，以PBS淋洗去除多余的染色液，取下磁力搅拌子A，置于24孔板的培养孔中，自然风干。向培养孔中加入300μL无水乙醇脱色15min，取200μL转移至96孔板(购买自Greiner公司，德国)内，用酶标仪测定OD 600nm，即为变形链球菌生物膜形成量所对应的量化数据。以上述方法测定空白对照与待测样品的OD600nm，并计算生物膜抑制剂的抑制率。生物膜抑制率(I)：I＝(OD 600nm(空白对照)-OD 600nm(待测样品))/OD 600nm(空白对照)×100％。
2、发酵豆浆的制备
将肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.6432以接种量3％(v/v，种子液占发酵液的体积百分比，下同)无菌接种于固形物含量5％(w/w，大豆占水的质量百分比，下同)的大豆豆浆中，37℃静置培养24h得发酵豆浆A。
3、发酵豆浆对变形链球菌生物膜形成抑制率的测定
将发酵豆浆A以上述方法制备为待测样品后测定其对变形链球菌生物膜形成的抑制活性，结果表明该发酵豆浆对变形链球菌生物膜形成的抑制率I＝71％。
实施例2
1、材料与方法
(a)肠膜明串珠菌(发酵菌种)和变形链球菌菌悬液的制备：同实施例1。
(b)豆浆的制备：将赤豆干豆称重后，加入五倍质量的水置于37℃浸 泡8h，弃去水，在豆浆机中加入一定比例的湿豆与水，制得所需固形物含量的豆浆，经121℃灭菌20min，得到指定固形物含量的无菌赤豆豆浆。
(c)发酵豆浆对变形链球菌生物膜形成的抑制率测定：同实施例1。
2、发酵豆浆的制备
将肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.6432以接种量1％(v/v)无菌接种于固形物含量9％(w/w)的赤豆豆浆中，15℃静置培养48h得发酵豆浆B。
3、发酵豆浆对变形链球菌生物膜形成抑制率的测定
将发酵豆浆B以上述方法制备为待测样品后测定其对变形链球菌生物膜形成的抑制活性，结果表明该发酵豆浆对变形链球菌生物膜形成的抑制率I＝67％。
实施例3
1、材料与方法
(a)肠膜明串珠菌(发酵菌种)和变形链球菌菌悬液的制备：同实施例1。
(b)豆浆的制备：将绿豆干豆称重后，加入五倍质量的水置于37℃浸泡8h，弃去水，在豆浆机中加入一定比例的湿豆与水，制得所需固形物含量的豆浆，经121℃灭菌20min，得到指定固形物含量的无菌绿豆豆浆。
(c)发酵豆浆对变形链球菌生物膜形成的抑制率测定：同实施例1。
2、发酵豆浆的制备
将肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.6432以接种量5％(v/v)无菌接种于固形物含量1％(w/w)的绿豆豆浆中，45℃静置培养2h得发酵豆浆C。
3、发酵豆浆对变形链球菌生物膜形成抑制率的测定
将发酵豆浆C以上述方法制备为待测样品后测定其对变形链球菌生物膜形成的抑制活性，结果表明该发酵豆浆对变形链球菌生物膜形成的抑制率I＝69％。
实施例4
1、材料与方法
(a)肠膜明串珠菌(发酵菌种)和变形链球菌菌悬液的制备：同实施例1。
(b)豆浆的制备：将蚕豆干豆称重后，加入五倍质量的水置于37℃浸泡8h，弃去水，在豆浆机中加入一定比例的湿豆与水，制得所需固形物含量的豆浆，经121℃灭菌20min，得到指定固形物含量的无菌蚕豆豆浆。
(c)发酵豆浆对变形链球菌生物膜形成的抑制率测定：同实施例1。
2、发酵豆浆的制备
将肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.6432以接种量2％(v/v)无菌接种于固形物含量7％(w/w)的蚕豆豆浆中，25℃静置培养36h得发酵豆浆D。
3、发酵豆浆对变形链球菌生物膜形成抑制率的测定
将发酵豆浆D以上述方法制备为待测样品后测定其对变形链球菌生物膜形成的抑制活性，结果表明该发酵豆浆对变形链球菌生物膜形成的抑制率I＝66％。
实施例5
1、材料与方法
(a)肠膜明串珠菌(发酵菌种)和变形链球菌菌悬液的制备：同实施例1。
(b)豆浆的制备：将大白芸豆和小白芸豆干豆以质量比1：1称重混合后，加入五倍质量的水置于37℃浸泡8h，弃去水，在豆浆机中加入一定比例的湿豆与水，制得所需固形物含量的豆浆，经121℃灭菌20min，得到指定固形物含量的无菌白芸豆豆浆。
(c)发酵豆浆对变形链球菌生物膜形成的抑制率测定：同实施例1。
2、发酵豆浆的制备
将肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.6432以接种量 4％(v/v)无菌接种于固形物含量3％(w/w)的白芸豆豆浆中，40℃静置培养12h得发酵豆浆E。
3、发酵豆浆对变形链球菌生物膜形成抑制率的测定
将发酵豆浆E以上述方法制备为待测样品后测定其对变形链球菌生物膜形成的抑制活性，结果表明该发酵豆浆对变形链球菌生物膜形成的抑制率I＝70％。
实施例6
1、材料与方法
(a)肠膜明串珠菌(发酵菌种)的制备：将肠膜明串珠菌(Leuconostoc mesenteroides)ATCC 10830a的冻干粉用少量无菌蒸馏水溶解，用接种环取一环划线于含2％(w/v)蔗糖的M17固体培养基(购买自Merck Co.德国)上，30℃好氧培养24h取出，用接种环挑取单菌落放入20mL含2％(w/v)蔗糖的M17液体(购买自Merck Co.德国)，运用涡旋震荡器将菌落均匀分散于液体培养基内，30℃、180rpm摇床振荡培养24h取出，培养物9,000rpm离心10分钟，弃去上清，菌体用无菌蒸馏水洗涤2次后，用原培养体积的无菌蒸馏水悬浮，得到发酵用的种子，种子液的菌浓度为1x10 9cfu/mL。
变形链球菌菌悬液的制备：同实施例1。
(b)豆浆的制备：同实施例1。
(c)发酵豆浆对变形链球菌生物膜形成的抑制率测定：同实施例1。
2、发酵豆浆的制备
将肠膜明串珠菌(Leuconostoc mesenteroides)ATCC 10830a以接种量3％(v/v)无菌接种于固形物含量5％(w/w)的大豆豆浆中，37℃静置培养24h得发酵豆浆F。
3、发酵豆浆对变形链球菌生物膜形成抑制率的测定
将发酵豆浆F以上述方法制备为待测样品后测定其对变形链球菌生物膜形成的抑制活性，结果表明该发酵豆浆对变形链球菌生物膜形成的抑制率I＝66％。
实施例7
1、材料与方法
(a)肠膜明串珠菌(发酵菌种)的制备：将肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.10064的冻干粉用少量无菌蒸馏水溶解，用接种环取一环划线于含2％(w/v)蔗糖的M17固体培养基(购买自Merck Co.德国)上，30℃好氧培养24h取出，用接种环挑取单菌落放入20mL含2％(w/v)蔗糖的M17液体(购买自Merck Co.德国)，运用涡旋震荡器将菌落均匀分散于液体培养基内，30℃、180rpm摇床振荡培养24h取出，培养物9,000rpm离心10分钟，弃去上清，菌体用无菌蒸馏水洗涤2次后，用原培养体积的无菌蒸馏水悬浮，得到发酵用的种子，种子液的菌浓度为1x10 9cfu/mL。
变形链球菌菌悬液的制备：同实施例1。
(b)豆浆的制备：同实施例1。
(c)发酵豆浆对变形链球菌生物膜形成的抑制率测定：同实施例1。
2、发酵豆浆的制备
将肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.10064以接种量3％(v/v)无菌接种于固形物含量5％(w/w)的大豆豆浆中，37℃静置培养24h得发酵豆浆G。
3、发酵豆浆对变形链球菌生物膜形成抑制率的测定
将发酵豆浆G以上述方法制备为待测样品后测定其对变形链球菌生物膜形成的抑制活性，结果表明该发酵豆浆对变形链球菌生物膜形成的抑制率I＝62％。
效果实施例1产品口味与喜好程度测试
以上述实施例1-7所制备的发酵豆浆产品A、B、C、D、E、F和G为实验对象，进行产品的口味测试。测试人数50人。品尝方式：采用不记名打分的方式进行品尝；分别对上述发酵豆浆产品A、B、C、D、E、F和G的色泽、风味、口感、营养项进行单独打分，每一项满分是25分，计算平均分及其总分，统计结果记录于表1。同时，根据对产品的整体喜好程度给 出的意见，统计对每个单品的喜好人数，统计结果记录于表2。
表1产品口味测试结果数据统计表
Figure PCTCN2019119946-appb-000001
表2产品喜好程度测试结果数据统计表
Figure PCTCN2019119946-appb-000002
从表2可以看出，总体而言，通过本发明技术方案中的方法所制得的由肠膜明串珠菌制备的、具有变形链球菌生物膜抑制活性的发酵豆浆在产品风味、口感、营养方面可被大部分消费者所接受。
效果实施例2冷藏条件下发酵豆浆对变形链球菌生物膜抑制活性的稳定性
将实施例1-7制备的发酵豆浆A、B、C、D、E、F和G置于冷藏条件(4℃)保存0、10、20和30天后取出，分别测定各样品对变形链球菌生物膜形成抑制率，结果如表3所示。
表3冷藏条件下发酵豆浆对变形链球菌生物膜形成抑制效果的稳定性
Figure PCTCN2019119946-appb-000003
Figure PCTCN2019119946-appb-000004
由表3可知，所有测试的发酵豆浆在冷藏条件(4℃)保存30天后，对变形链球菌生物膜形成抑制率稳定保持在同一水平，稳定性较好。
对比例1
将实施例1中的接种量，豆浆浓度，培养温度以及发酵时间逐一进行调整，获得了以下一组不同方法制备的发酵豆浆，各组所得发酵豆浆对变形链球菌生物膜的抑制效果如表4所示。
表4不同方法制备所得发酵豆浆对变形链球菌生物膜形成的抑制效果
Figure PCTCN2019119946-appb-000005
从表4所示的结果中可以得出，将所述发酵豆浆的制备方法中接种量，豆浆浓度，培养温度以及发酵时间调整到优选范围之外的时候，由肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.6432制备的发酵豆浆依然可以抑制变形链球菌生物膜的形成量，但是其抑制效果明显下降。
对比例2
参考实施例1所述方法，比较由肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.6432、植物乳杆菌(L.plantarum)ATCC 14917(购买自ATCC)、嗜热链球菌(S.thermophilus)ST-BODY-3(由科.汉森公司提供)、干酪乳杆菌(L.casei)ATCC 393(购买自ATCC)、肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.10064、肠膜明串珠菌(Leuconostoc mesenteroides) ATCC 10830a制备的发酵豆浆对变形链球菌生物膜形成的抑制效果，具体操作如下：
1、材料与方法
(a)种子(发酵菌种)的制备：
肠膜明串珠菌CGMCC No.6432种子的制备：同实施例1；
肠膜明串珠菌ATCC 10830a种子的制备：同实施例6；
肠膜明串珠菌CGMCC No.10064种子的制备：同实施例7。
干酪乳杆菌和保加利亚乳杆菌种子的制备：将干酪乳杆菌ATCC 393和植物乳杆菌ATCC14917的冻干粉分别用少量无菌蒸馏水溶解，各自用接种环取一环划线于MRS固体培养基(购买自Merck Co.德国)上，37℃厌氧培养24h取出，用接种环挑取单菌落放入1mL MRS液体(购买自Merck Co.德国)，运用涡旋震荡器将菌落均匀分散于液体培养基内，37℃厌氧培养24h取出，以2％(v/v)接种量接种于50mL MRS液体，37℃培养24h后，培养物9,000rpm离心10分钟，弃去上清，菌体用无菌蒸馏水洗涤2次后，用原培养体积的无菌蒸馏水悬浮，得到相应的发酵用的种子。
嗜热链球菌种子的制备：将嗜热链球菌ST-BODY-3的冻干粉用少量无菌蒸馏水溶解，用接种环取一环划线于M17固体培养基(购买自Merck Co.德国)上，40℃厌氧培养24h取出，用接种环挑取单菌落放入1mL M17液体(购买自Merck Co.德国)，运用涡旋震荡器将菌落均匀分散于液体培养基内，40℃厌氧培养24h取出，以2％(v/v)接种量接种于50mL M17液体，40℃培养24h后，培养物9,000rpm离心10分钟，弃去上清，菌体用无菌蒸馏水洗涤2次后，用原培养体积的无菌蒸馏水悬浮，得到发酵用的种子。
变形链球菌菌悬液的制备：同实施例1。
(b)大豆豆浆的制备：同实施例1。
(c)发酵豆浆对变形链球菌生物膜形成的抑制率测定：同实施例1。
1、发酵豆浆的制备
将各菌株以3％(v/v)接种量无菌接种于固形物含量5％(w/w)的大豆豆 浆中，分别培养(肠膜明串珠菌、植物乳杆菌和干酪乳杆菌37℃厌氧培养，嗜热链球菌40℃厌氧培养)24h，获得相应的发酵豆浆。
2、发酵豆浆对变形链球菌生物膜形成抑制率的测定
上述不同菌株制备的发酵豆浆对变形链球菌生物膜形成的抑制效果如表5所示：
表5不同菌株制备的发酵豆浆对变形链球菌生物膜形成的抑制效果
Figure PCTCN2019119946-appb-000006
由表5可知，由其他菌株制备的发酵豆浆不具有变形链球菌生物膜抑制活性，而肠膜明串珠菌(Leuconostoc mesenteroides)CGMCC No.6432、ATCC 10830a、CGMCC No.10064制备的发酵豆浆对变形链球菌生物膜的抑制活性非常显著。
对比例3
1、材料与方法
(a)肠膜明串珠菌CGMCC No.6432种子的制备：同实施例1；
肠膜明串珠菌ATCC 10830a种子的制备：同实施例6；
肠膜明串珠菌CGMCC No.10064种子的制备：同实施例7；
变形链球菌菌悬液的制备：同实施例1。
(b)发酵液对变形链球菌生物膜形成的抑制率测定：
发酵液的预处理：将发酵液pH调节至6.80，即得待测样品。
抑制活性的测定：参照实施例1所述方法，以未发酵的培养基作为空白对照进行测定。
2、肠膜明串珠菌发酵液的制备
将肠膜明串珠菌CGMCC No.6432、ATCC 10830a和CGMCC No.10064种子以接种量3％(v/v)分别无菌接种于含蔗糖1％(w/v)的M17液体培养基中，37℃厌氧培养24h，得到发酵液。
3、肠膜明串珠菌发酵液对变形链球菌生物膜形成抑制率的测定
将肠膜明串珠菌发酵液以上述方法制备为待测样品后测定其对变形链球菌生物膜形成的抑制活性，结果表明CGMCC No.6432、ATCC 10830a、CGMCC No.10064的发酵液对变形链球菌生物膜形成的抑制率分别为8％、7％、5％。
对比例4
将实施例1-7制备的发酵豆浆A、B、C、D、E、F和G的pH调节至6.80，再以4℃、10,000rpm离心10min，上清部分即为无菌的发酵豆浆(待测样品)。根据实施例1中所述方法测定上述样品进行对变形链球菌生物膜形成抑制率，结果如表6所示。
表6无菌的发酵豆浆对变形链球菌生物膜形成抑制效果
Figure PCTCN2019119946-appb-000007
由表6可知，无菌的发酵豆浆对变形链球菌生物膜形成抑制率依然有较强的抑制活性，说明抑制作用来自菌株代谢豆浆后的产物。但各组无菌发酵豆浆的生物膜抑制效果均低于无菌处理前的样品组，这表明无菌处理前的发酵豆浆的抑制生物膜菌膜的效果也来自活菌与代谢产物共同作用。
以上对本发明所提供的发酵豆浆的制备方法、制备出的发酵豆浆与应用进行了详细介绍。本文中应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提 下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  1. 一种由肠膜明串珠菌制备发酵豆浆的方法，其特征在于，该方法包括以下步骤：将肠膜明串珠菌接种于豆浆中进行发酵代谢，得到发酵豆浆。
  2. 根据权利要求1所述的发酵豆浆的制备方法，其特征在于，所述肠膜明串珠菌为CGMCC No.6432、ATCC 10830a或CGMCC No.10064。
  3. 根据权利要求1所述的发酵豆浆的制备方法，其特征在于，所述肠膜明串珠菌的接种量为1x10 7～5x10 7cfu/mL。
  4. 根据权利要求1所述的发酵豆浆的制备方法，其特征在于，所述豆浆为大豆、赤豆、绿豆、蚕豆、大白芸豆或小白芸豆豆浆中的至少一种。
  5. 根据权利要求1所述的发酵豆浆的制备方法，其特征在于，所述豆浆中固形物的质量百分比含量为1％～9％。
  6. 根据权利要求1所述的发酵豆浆的制备方法，其特征在于，所述发酵的温度为15℃～45℃，时间为2～48h。
  7. 根据权利要求1所述的发酵豆浆的制备方法，其特征在于，所述发酵为静置培养。
  8. 一种由肠膜明串珠菌制备的发酵豆浆，其特征在于，由权利要求1～7任一种制备方法制得。
  9. 根据权利要求8所述的发酵豆浆，其特征在于，所述发酵豆浆对变形链球菌生物膜形成的抑制率≥62％。
  10. 一种如权利要求8或9所述的发酵豆浆在制备抗龋齿食品方面的应用。
PCT/CN2019/119946 2019-05-23 2019-11-21 一种由肠膜明串珠菌制备发酵豆浆的方法及制备出的发酵豆浆与应用 WO2020233031A1 (zh)

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