技术领域Technical Field
本发明属于功能型糖果技术领域，尤其涉及一种富含果汁和凝结芽孢杆菌的凝胶软糖及其制备方法和应用。The invention belongs to the technical field of functional candy, and in particular relates to a gummy candy rich in fruit juice and Bacillus coagulans and its preparation method and application.
背景技术Background technique
肠道氧化应激也被认为是肠道损伤的重要标志，肠道中的氧化应激是当活性氧(ROS)的产生与身体解毒能力之间存在不平衡时发生的一种现象。这会导致肠道内的细胞、组织和器官受损，并与一系列健康问题有关。Intestinal oxidative stress is also considered an important marker of intestinal damage. Oxidative stress in the intestine is a phenomenon that occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body's detoxification ability. This can lead to damage to cells, tissues and organs in the gut and is linked to a range of health problems.
肠道氧化应激直接影响宿主的健康，可诱发包括炎症性肠病(IBD)、肠易激综合征(IBS)甚至结肠癌等多种疾病。儿童肠道内氧化应激是一种常见的生理现象，其产生的原因、危害和预防策略备受关注。在日常生活中，儿童的肠道受到各种外界因素的影响，包括环境污染、不良饮食习惯以及情绪波动等，这些因素都可能导致肠道内氧化应激的产生。在儿童日常膳食中补充益生菌、VC、水果、蔬菜等复含抗氧化剂的成分的物质有助于缓解儿童肠道内氧化应激。凝胶糖果是一种以明胶为主要成分制成的软糖,具有柔软、易于咀嚼和口感好的特点。深受儿童的喜爱。但因为含糖量高，常规的凝结糖果可能进一步加剧儿童肠道氧化应激。本发明通过体外抗氧化指标筛选出抗氧化性能最高的水果，并通过双氧水诱导结肠癌氧化损伤模型筛选出凝胶芽孢杆菌和水果复配的最佳比例。Intestinal oxidative stress directly affects the health of the host and can induce a variety of diseases including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) and even colon cancer. Oxidative stress in the intestine of children is a common physiological phenomenon, and its causes, hazards and prevention strategies have attracted much attention. In daily life, children's intestines are affected by various external factors, including environmental pollution, bad eating habits, and mood swings. These factors may lead to the generation of oxidative stress in the intestines. Supplementing children's daily diet with probiotics, VC, fruits, vegetables and other substances containing antioxidants can help relieve oxidative stress in the intestines of children. Gel candy is a soft candy made with gelatin as the main ingredient, which is soft, easy to chew and has a good taste. Very popular among children. But because of their high sugar content, regular curdled candies may further exacerbate oxidative stress in children’s guts. The present invention selects fruits with the highest antioxidant properties through in vitro antioxidant indicators, and selects the optimal ratio of Bacillus gelatinosa and fruits through a hydrogen peroxide-induced colon cancer oxidative damage model.
发明内容Contents of the invention
本发明的目的是提供一种富含果汁和凝结芽孢杆菌的凝胶软糖及其制备方法和应用。The object of the present invention is to provide a gummy candy rich in fruit juice and Bacillus coagulans and its preparation method and application.
为达到上述目的，本发明采用的技术方案是：In order to achieve the above object, the technical solution adopted by the present invention is:
一种富含果汁和凝结芽孢杆菌的凝胶软糖的制备方法，包括以下步骤：A preparation method of gelatin gum rich in fruit juice and Bacillus coagulans includes the following steps:
S1.向容器1中加入6.25mL果汁后再加入33.75mL水稀释后加热至85℃后，加入7g明胶至完全溶解；S1. Add 6.25mL of juice to container 1, then add 33.75mL of water to dilute, heat to 85°C, and add 7g of gelatin until completely dissolved;
S2.另取容器2制备葡萄糖浆，将7g葡萄糖浆和0.1g柠檬酸钠加入70mL水，加热浓缩至白利度为86°；S2. Take another container 2 to prepare glucose syrup, add 7g glucose syrup and 0.1g sodium citrate to 70mL water, heat and concentrate until the Brix is 86°;
S3.将容器1中明胶溶液导入容器2混合，并加入2.5mL凝结芽孢杆菌发酵液凝结芽孢杆菌发酵液活菌数为2.0×109CFU/mL，于85℃保温状态下搅拌15min，导入磨具中于25℃烘干48h至水活度为0.70，脱膜，共得到50g软糖，理论活菌数为1.0×107CFU/g，果汁浓度为12.5％。S3. Introduce the gelatin solution in container 1 into container 2 for mixing, and add 2.5 mL of Bacillus coagulans fermentation broth. The viable bacterial count of Bacillus coagulans fermentation broth is 2.0 × 109 CFU/mL. Stir for 15 minutes under insulation at 85°C and introduce into the mill. Dry the container at 25°C for 48 hours until the water activity is 0.70, and remove the film to obtain a total of 50g of soft candy. The theoretical number of viable bacteria is 1.0×107 CFU/g, and the juice concentration is 12.5%.
进一步的，所述果汁为百香果、奇异莓、红李和无花果中的一种水果榨成的汁，榨汁过程为用组织研磨机10000r/min研磨1min，然后于4℃，12000g，离心10min后收集上清果汁，于4℃下储存备用。Further, the juice is the juice squeezed from one of passion fruit, kiwi berry, red plum and fig. The juice extraction process is to grind the juice with a tissue grinder at 10000r/min for 1min, and then centrifuge at 12000g for 10min at 4°C. Collect the supernatant juice and store it at 4°C for later use.
采用上述制备方法制备的功能性糖果。Functional candy prepared using the above preparation method.
一种功能性糖果的应用，凝结芽孢杆菌菌体经培养收集菌体上清液，上清液中活菌数为1.0×106CFU/mL，奇异莓果汁添加量为1.25％时制备的功能性糖果用于制备治疗由人结肠癌Caco-2细胞双氧化诱导损伤引起的儿童肠道内氧化应激药物中。The invention discloses an application of functional candy. The supernatant of Bacillus coagulans is collected after culturing, the number of live bacteria in the supernatant is 1.0×106 CFU/mL, and the functional candy prepared by adding 1.25% kiwi fruit juice is used for preparing a drug for treating oxidative stress in the intestine of children caused by double oxidation-induced damage of human colon cancer Caco-2 cells.
本发明具有的优点是：本发明方法制备得到的功能性糖果，口味好，易于被儿童接受，且当糖果中BCSKB组合为1.0×106CFU/mLBCS+1.25％KB时，对于由人结肠癌Caco-2细胞双氧化诱导损伤的保护效果最为显著。The advantages of the present invention are: the functional candies prepared by the method of the present invention have good taste and are easy to be accepted by children; and when the BCSKB combination in the candies is 1.0×106 CFU/mLBCS+1.25% KB, it has good anti-cancer effects on human colon cancer. The protective effect of Caco-2 cells against damage induced by double oxidation is the most significant.
附图说明Description of drawings
图1是本发明中不同诱导物与Caco-2细胞共培养6h显微观察照片。Figure 1 is a microscopic observation photo of different inducers co-cultured with Caco-2 cells for 6 hours in the present invention.
图2是荧光定量PCR和Western Blotting分析结果。Figure 2 shows the results of fluorescence quantitative PCR and Western Blotting analysis.
具体实施方式Detailed ways
实施例Example
一种富含果汁和凝结芽孢杆菌的凝胶软糖的制备方法，包括以下步骤：A preparation method of gelatin gum rich in fruit juice and Bacillus coagulans includes the following steps:
S1.向容器1中加入6.25mL果汁后再加入33.75mL水稀释后加热至85℃后，加入7g明胶至完全溶解；S1. Add 6.25mL of juice to container 1, then add 33.75mL of water to dilute, heat to 85°C, and add 7g of gelatin until completely dissolved;
S2.另取容器2制备葡萄糖浆，将7g葡萄糖浆和0.1g柠檬酸钠加入70mL水，加热浓缩至白利度为86°；S2. Take another container 2 to prepare glucose syrup, add 7g glucose syrup and 0.1g sodium citrate to 70mL water, heat and concentrate until the Brix is 86°;
S3.将容器1中明胶溶液导入容器2混合，并加入2.5mL凝结芽孢杆菌发酵液凝结芽孢杆菌发酵液活菌数为2.0×109CFU/mL，于85℃保温状态下搅拌15min，导入磨具中于25℃烘干48h至水活度为0.70，脱膜，共得到50g软糖，理论活菌数为1.0×107CFU/g，果汁浓度为12.5％。S3. Introduce the gelatin solution in container 1 into container 2 for mixing, and add 2.5 mL of Bacillus coagulans fermentation broth. The viable bacterial count of Bacillus coagulans fermentation broth is 2.0 × 109 CFU/mL. Stir for 15 minutes under insulation at 85°C and introduce into the mill. Dry the container at 25°C for 48 hours until the water activity is 0.70, and remove the film to obtain a total of 50g of soft candy. The theoretical number of viable bacteria is 1.0×107 CFU/g, and the juice concentration is 12.5%.
进一步的，所述果汁为百香果、奇异莓、红李和无花果中的一种水果榨成的汁，榨汁过程为用组织研磨机10000r/min研磨1min，然后于4℃，12000g，离心10min后收集上清果汁，于4℃下储存备用。Further, the juice is the juice squeezed from one of passion fruit, kiwi berry, red plum and fig. The juice extraction process is to grind the juice with a tissue grinder at 10000r/min for 1min, and then centrifuge at 12000g for 10min at 4°C. Collect the supernatant juice and store it at 4°C for later use.
采用上述制备方法制备的功能性糖果。Functional candy prepared using the above preparation method.
一种功能性糖果的应用，凝结芽孢杆菌菌体经培养收集菌体上清液，上清液中活菌数为1.0×106CFU/mL，奇异莓果汁添加量为1.25％时制备的功能性糖果用于制备治疗由人结肠癌Caco-2细胞双氧化诱导损伤引起的儿童肠道内氧化应激药物中。Application of a functional candy. Bacillus coagulans cells are cultured and the supernatant is collected. The number of viable bacteria in the supernatant is 1.0×106 CFU/mL. The function is prepared when the added amount of kiwi berry juice is 1.25%. Sex candy is used to prepare drugs for the treatment of oxidative stress in children's intestines caused by double oxidation-induced damage to human colon cancer Caco-2 cells.
实验验证Experimental verification
1材料和方法1Materials and methods
1.1实验材料1.1 Experimental materials
凝结芽孢杆菌(Bacillus Coagulans,BC)购自中国科学院普通微生物菌种保藏中心(CGMCC)，人结肠癌Caco-2细胞系购自中国科学院细胞生物所。百香果(中国广西壮族自治区北流市)、红李(中国山东省菏泽市单县)、无花果(中国江苏省南通市)、奇异莓(中国辽宁省丹东市)购自驻马店爱家百货超市。其它试剂均为分析纯或食品级。Bacillus Coagulans (BC) was purchased from the General Microbial Culture Collection Center of the Chinese Academy of Sciences (CGMCC), and the human colon cancer Caco-2 cell line was purchased from the Institute of Cell Biology, Chinese Academy of Sciences. Passion fruits (Beiliu City, Guangxi Zhuang Autonomous Region, China), red plums (Shan County, Heze City, Shandong Province, China), figs (Nantong City, Jiangsu Province, China), and kiwi berries (Dandong City, Liaoning Province, China) were purchased from Zhumadian Aijia Department Store Supermarket. Other reagents are of analytical grade or food grade.
1.2凝结芽孢杆菌的培养和果汁的制备1.2 Culture of Bacillus coagulans and preparation of juice
凝结芽孢杆菌接种于MRS培养基(g/L)中：蛋白胨10g，酵母提取物10g，葡萄糖20g，吐温80 1mL，K2HPO42 g，乙酸钠5g，柠檬酸钠2g，MgSO40.2g，MnSO40.05 g，pH 6.20-6.60；45℃100r/min培养24h。培养结束后采用平板计数法计数，经检测活菌数为2.0×109CFU/mL。发酵液4℃，12000×g，离心10min，分别收集菌体(BC)和上清(BCS)。百香果、奇异莓、红李和无花果用组织研磨机10000r/min研磨1min，然后4℃，12000×g，离心10min后收集上清果汁，4℃备用。Bacillus coagulans was inoculated into MRS medium (g/L): 10g peptone, 10g yeast extract, 20g glucose, 1mL Tween 80, K2 HPO4 2 g, 5g sodium acetate, 2g sodium citrate, MgSO4 0.2 g, MnSO4 0.05 g, pH 6.20-6.60; culture at 45°C 100r/min for 24h. After the culture, the plate count method was used to count, and the number of viable bacteria was detected to be 2.0×109 CFU/mL. The fermentation broth was centrifuged at 12000 × g at 4°C for 10 min, and the bacterial cells (BC) and supernatant (BCS) were collected. Passion fruit, kiwi berry, red plum and fig were ground with a tissue grinder at 10000 r/min for 1 min, then centrifuged at 12000×g at 4°C for 10 min and the supernatant juice was collected and kept at 4°C for later use.
1.3体外抗氧化性能的评估1.3 Evaluation of antioxidant properties in vitro
4种水果果汁用无菌水稀释10倍配置为10％果汁样品，BCS和VC溶液(浓度0.1mg/mL)，共6个样品溶液进行体外抗氧化性能的评估。6组样品溶液共分为2组，常温组25℃水浴待用，加热组先85℃加热15min，再25℃水浴待用。Four kinds of fruit juices were diluted 10 times with sterile water to prepare 10% juice samples, BCS and VC solutions (concentration 0.1 mg/mL). A total of 6 sample solutions were used to evaluate the antioxidant properties in vitro. The 6 groups of sample solutions were divided into 2 groups. The normal temperature group was placed in a 25°C water bath for later use. The heating group was first heated at 85°C for 15 minutes and then placed in a 25°C water bath for later use.
PTIO(2-苯基-4，4，5，5-四甲基咪唑啉-3-氧代-1-氧，2-phenyl-4，4，5，5-tetramethylimidazolin-3-oxy-1-oxygen，PTIO)自由基清除试验：取PTIO固体30mg溶于200mL蒸馏水中，配置PTIO工作液。在反应试管中加入工作液1.6mL，分别计入不同体积的样品(0.3，0.6，0.9，1.2和1.5mL)，并用蒸馏水补充至反应总体积为3.6mL，37℃水浴2h，并在557nm处测定吸光度值A。PTIO清除率(％)＝(Ao-A)/A0×100％，其中A0表示不加样品时的吸光度值，其数值应在0.2-0.6之间，A表示为加入不同体积样品的吸光度值。以清除率为Y值，以样品体积为X值求出线性回归方程和相关性系数R2，定义SC50为清除率为50％所消耗的样品体积，并以0.1mg/mLVC溶液为参比溶液，计算SC50相对比值，结果以每g鲜果肉或每毫升BCS相当于VC量表示(VCE/g(mL))。PTIO (2-phenyl-4,4,5,5-tetramethylimidazolin-3-oxy-1-oxygen, PTIO) free radical scavenging test: 30 mg of PTIO solid was dissolved in 200 mL of distilled water to prepare PTIO working solution. 1.6 mL of working solution was added to the reaction tube, and different volumes of samples (0.3, 0.6, 0.9, 1.2 and 1.5 mL) were added respectively, and the total reaction volume was supplemented with distilled water to 3.6 mL. The reaction was incubated at 37°C for 2 hours, and the absorbance value A was measured at 557 nm. PTIO scavenging rate (%) = (Ao -A)/A0 × 100%, where A0 represents the absorbance value without adding sample, and its value should be between 0.2-0.6, and A represents the absorbance value after adding different volumes of sample. The linear regression equation and correlation coefficient R2 were obtained with clearance rate as Y value and sample volume as X value. SC50 was defined as the sample volume consumed when the clearance rate was 50%. 0.1 mg/mL VC solution was used as the reference solution to calculate the relative ratio of SC50. The results were expressed as the amount of VC equivalent to each gram of fresh pulp or each milliliter of BCS (VCE/g(mL)).
ABTS(2，2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐，2，2-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)，ABTS)自由基清除试验：将7mMABTS原液与2.45mM过二硫酸钾体积比1:1混合，并在黑暗环境下室温放置12h，用pH 7.4无水乙醇将混合液稀释直至吸光度为0.70±0.02，此溶液即为ABTS+工作液。在反应试管中加入不同体积的样品溶液(0-0.14mL)与3.2mL工作液混合，并用乙醇补充至反应总体积为3.6mL，室温下反应6分钟，并在734nm处测定吸光度值A。ABTS清除率＝(A0-A)/A0×100％，其中A0表示不加样品时的值，A表示为加入不同体积样品的值。ABTS清除率线性回归方程和SC50相对比值计算同PTIO自由基清除试验。ABTS (2,2-Azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, ABTS) free radical scavenging test: 7mM ABTS stock solution was mixed with 2.45mM potassium persulfate in a volume ratio of 1:1, and placed at room temperature in a dark environment for 12 hours. The mixture was diluted with pH 7.4 anhydrous ethanol until the absorbance was 0.70±0.02. This solution was the ABTS+ working solution. Different volumes of sample solution (0-0.14mL) were added to the reaction tube and mixed with 3.2mL of the working solution, and ethanol was added to the total reaction volume of 3.6mL. The reaction was carried out at room temperature for 6 minutes, and the absorbance value A was measured at 734nm. ABTS scavenging rate = (A0 -A)/A0 ×100%, where A0 represents the value without adding sample, and A represents the value with different volumes of sample added. The linear regression equation of ABTS clearance rate and the calculation of SC50 relative ratio were the same as those of PTIO free radical scavenging test.
DPPH(1，1-二苯基-2-三硝基苯肼，1，1-Diphenyl-2-picrylhydrazylradical，DPPH)自由基清除试验：取DPPH固体1mg溶于24mL乙醇中。取1mL上述步骤配置好的DPPH溶液，加0.5mL 95乙醇稀释，使其吸光度为0.6-1.0之间即为DPPH工作液。若吸光度过大，则继续加溶剂；若吸光度过小，则补加DPPH固体。在反应试管中加入不同体积的样品溶液(0-1.5mL)与3mL工作液混合，并用乙醇补充至反应总体积为4.5mL，室温下反应30分钟，并在519nm处测定吸光度值A。DPPH清除率＝(Ao-A)/A0×100％，其中A0表示不加样品时的值。A表示为加入不同体积样品的值。DPPH清除率线性回归方程和SC50相对比值计算同PTIO自由基清除试验。DPPH (1,1-diphenyl-2-trinitrophenylhydrazine, 1,1-Diphenyl-2-picrylhydrazylradical, DPPH) free radical scavenging test: Dissolve 1 mg of DPPH solid in 24 mL of ethanol. Take 1 mL of the DPPH solution configured in the above steps, add 0.5 mL of 95 ethanol to dilute it, so that the absorbance is between 0.6-1.0, which is the DPPH working solution. If the absorbance is too large, continue to add solvent; if the absorbance is too small, add additional DPPH solid. Add different volumes of sample solution (0-1.5mL) to the reaction tube and mix with 3mL of working solution, and add ethanol to make the total reaction volume 4.5mL. React at room temperature for 30 minutes, and measure the absorbance value A at 519nm. DPPH clearance rate = (Ao -A)/A0 ×100%, where A0 represents the value without adding a sample. A represents the value for adding different volumes of sample. The linear regression equation of DPPH scavenging rate and SC50 relative ratio calculation are the same as those of PTIO free radical scavenging test.
FRAP(铁离子抗氧化能力法，Ferric ion reducing antioxidant power，FRAP)测定试验：FRAP工作液的配置：20mL 0.3M pH 3.6醋酸缓冲液、2mL 10mmol/L TPTZ溶液和2mL20mmol/L FeCl3溶液混合均匀。在反应管中加入0.1mL样品溶液,再加入2.4mL FRAP工作液,混合均匀后37℃条件下水浴10min，于593nm处读取吸光。以0.1-1.6mmol/L的FeSO4的标准液替代样品绘制标准曲线，求得回归方程Y＝0.0606x+0.0241，相关系数为R2＝0.995。将吸光度值带入回归方程求得样品的FRAP值。1FRAP单位＝1mmol/LFeSO4，即样品的抗氧化能力相当于FeSO4的mmol/L数。FRAP (Ferric ion reducing antioxidant power, FRAP) determination test: FRAP working solution configuration: 20mL 0.3M pH 3.6 acetate buffer, 2mL 10mmol/L TPTZ solution and 2mL 20mmol/L FeCl3 solution and mix evenly. Add 0.1 mL of sample solution to the reaction tube, and then add 2.4 mL of FRAP working solution. After mixing evenly, place in a water bath at 37°C for 10 min, and read the absorbance at 593 nm. Use 0.1-1.6mmol/L FeSO4 standard solution instead of the sample to draw a standard curve, and obtain the regression equation Y = 0.0606x + 0.0241, and the correlation coefficient is R2 = 0.995. Put the absorbance value into the regression equation to obtain the FRAP value of the sample. 1FRAP unit = 1mmol/LFeSO4 , that is, the antioxidant capacity of the sample is equivalent to the number of mmol/L of FeSO4 .
1.4细胞试验1.4 Cell test
1.4.1Caco-2细胞的培养1.4.1Cultivation of Caco-2 cells
Caco-2细胞培养液为含20％FBS和1％双抗(青霉素-链霉素)的高糖MEM(含NEAA)培养基，按照1.0×105密度接种于25cm2方瓶中，并放在37℃、5％CO2的培养箱中培养。取对数生长期细胞胰酶消化计数后按1.0×105个/孔密度接种于96孔板或者6孔板培养，当细胞贴壁程度达到60％～70％时，移掉培养基后用PBS清洗，然后进行不同诱导处理。The Caco-2 cell culture medium is a high-glucose MEM (containing NEAA) medium containing 20% FBS and 1% double antibody (penicillin-streptomycin). It is inoculated into a25cm2 square bottle at a density of 1.0×105 and placed in a Culture in an incubator at 37°C and 5%CO2 . Cells in the logarithmic growth phase are trypsinized and counted, and then inoculated into a 96-well plate or a 6-well plate at a density of 1.0×105 cells /well. When the degree of cell adhesion reaches 60% to 70%, remove the culture medium and use Washed with PBS, then subjected to different induction treatments.
2.4.2H2O2氧化损伤模型2.4.2H2 O2 Oxidative Damage Model
使用基础培养基稀释H2O2为终浓度0.31-10mmol/L，对照不含H2O2。使用不同浓度的H2O2在刺激Caco-2细胞，并分别在刺激的2、4、6和8h收获细胞并检测细胞活力。MTT法检测细胞活力：培养结束之后向每孔中添加10μL MTT溶液(5mg/mL)，并孵育4h。此后，小心除去上清液，并向每孔中加入150μL DMSO溶解。在振荡器上震荡10min后用酶标仪检测490nm波长和参考波长630nm下的吸光度值(OD)。相对细胞活力(％)＝(试验组A490nm-A630nm)/(对照组A490nm-A630nm)×100％。Use basal medium to dilute H2 O2 to a final concentration of 0.31-10 mmol/L, and control without H2 O2 . Different concentrations of H2 O2 were used to stimulate Caco-2 cells, and the cells were harvested and cell viability was detected at 2, 4, 6 and 8 hours after stimulation. MTT method to detect cell viability: After the culture, add 10 μL MTT solution (5 mg/mL) to each well and incubate for 4 hours. After this, the supernatant was carefully removed and 150 μL DMSO was added to each well to dissolve. After shaking on the oscillator for 10 minutes, use a microplate reader to detect the absorbance value (OD) at a wavelength of 490 nm and a reference wavelength of 630 nm. Relative cell viability (%) = (test group A490nm - A630nm ) / (control group A490nm - A630nm ) × 100%.
1.4.3不同浓度的奇异莓(KB)对Caco-2细胞活力的影响1.4.3 Effects of different concentrations of kiwi berry (KB) on Caco-2 cell viability
0.22μm滤膜过滤后的奇异莓原汁使用基础培养分别稀释为终浓度为0.63％、1.25％、2.5％、5％和10％，与Caco-2共培养，对照组为空白培养基，分别在培养的2h，4h，6h和8h检测细胞活力，每组8个重复。The kiwiberry original juice filtered by 0.22 μm filter membrane was diluted to final concentrations of 0.63%, 1.25%, 2.5%, 5% and 10% using basic culture, and co-cultured with Caco-2. The control group was blank culture medium, respectively. Cell viability was detected at 2h, 4h, 6h and 8h of culture, with 8 replicates in each group.
1.4.4不同浓度的BC和BCS对Caco-2细胞活力的影响1.4.4 Effects of different concentrations of BC and BCS on Caco-2 cell viability
BC和BCS分别使用基础培养基进行10倍倍比稀释，浓度范围为2.0×103-2.0×107CFU/mL，含有不同BC和BCS分别与Caco-2共培养，在培养的2、4、6和8h检测细胞活力，对照组为基础培养基。BC and BCS were diluted 10 times using basal culture medium, with a concentration range of 2.0×103 -2.0×107 CFU/mL. Different BC and BCS were co-cultured with Caco-2 at 2 and 4 days of culture. Cell viability was detected at 6, 6 and 8 h, and the control group was the basic culture medium.
1.4.5BCS、KB及BCSKB对Caco-2细胞氧化损伤的保护效果1.4.5 Protective effects of BCS, KB and BCSKB on oxidative damage in Caco-2 cells
根据前期试验结果，分别选取最佳浓度进行保护试验，处理时间为4h。试验分组及终浓度如下，组1：基础培养基；组2：H2O2损伤组(0.625mmol/L)；组3：BCS组(1.0×106CFU/mL)；试验组4：H2O2+BCS保护组；组5：奇异莓组(1.25％)；组6：H2O2+KB保护组；组7：BCSKB(1.0×106CFU/mL BCS+1.25％KB)；组8：H2O2+BCSKB保护组。According to the results of the previous experiments, the optimal concentrations were selected for protection experiments, and the treatment time was 4h. The experimental groups and final concentrations were as follows: Group 1: basal culture medium; Group 2: H2 O2 injury group (0.625mmol/L); Group 3: BCS group (1.0×106 CFU/mL); Experimental group 4: H2 O2 +BCS protection group; Group 5: Kiwi berry group (1.25%); Group 6: H2 O2 +KB protection group; Group 7: BCSKB (1.0×106 CFU/mL BCS+1.25％KB); Group 8: H2 O2 +BCSKB protection group.
1.4.6荧光定量PCR和Western Blotting分析1.4.6 Fluorescence quantitative PCR and Western Blotting analysis
选择对照组、BCSKB组、H2O2损伤组和H2O2+BCSKB保护组4个处理进行试验。4个组处理后的用Trizol(美国，卡尔斯巴德，Invitgen)提取总RNA，然后溶解于50μL无RNase-free水中，保存在-80℃用于下面的试验。RNA浓度由NanoDrop ND-1000分光光度计(Nano-DropTechnologies，威明顿，DE，美国)测量。用TB Green试剂盒(中国大连塔卡拉)将每个样本中约1μg的总RNA反向转录成cDNA。用CFX ConnectTN实时聚合酶链反应检测系统(Bio-Rad，美国加利福尼亚州大力神)进行定量逆转录聚合酶链反应(RTPCR)。以β-actin作为看家基因，用2-ΔΔCT方法分析细胞中的相对基因丰度。Four treatments were selected for the test: control group, BCSKB group, H2 O2 injury group and H2 O2 +BCSKB protection group. After treatment in the four groups, total RNA was extracted with Trizol (Invitgen, Carlsbad, USA), then dissolved in 50 μL of RNase-free water, and stored at -80°C for the following experiments. RNA concentration was measured by a NanoDrop ND-1000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA). About 1 μg of total RNA in each sample was reverse transcribed into cDNA using TB Green kit (Takara, Dalian, China). Quantitative reverse transcription polymerase chain reaction (RTPCR) was performed using the CFX ConnectTN real-time polymerase chain reaction detection system (Bio-Rad, Hercules, CA, USA). Using β-actin as a housekeeping gene, the 2-ΔΔCT method was used to analyze the relative gene abundance in cells.
4个组处理后的细胞将肝组织用RIPA缓冲液(雅酶生物有限公司，上海，中国)提取细胞总蛋白，用BCA蛋白检测试剂盒(北京索莱宝生物有限公司，上海，中国)检测细胞总蛋白浓度。将等量的蛋白质用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分离，并转移到聚偏二氟乙烯(PVDF)膜上。膜用含5％脱脂奶粉的TBST缓冲液封闭2h，然后与一抗在4℃下孵育过夜，然后与二抗在室温下孵育2h。增强化学发光显示免疫阳性条带，用Image J软件进行定量分析。After treatment of the cells in the 4 groups, the total cell protein was extracted from the liver tissue with RIPA buffer (Yase Biotechnology Co., Ltd., Shanghai, China), and detected using the BCA protein detection kit (Beijing Soleba Biotechnology Co., Ltd., Shanghai, China). Total cellular protein concentration. Equal amounts of proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked with TBST buffer containing 5% skimmed milk powder for 2 h, then incubated with primary antibody at 4°C overnight, and then incubated with secondary antibody at room temperature for 2 h. Enhanced chemiluminescence showed immunopositive bands, and Image J software was used for quantitative analysis.
1.5富含奇异莓和凝结芽孢杆菌软糖的开发1.5 Development of gummies rich in kiwi berries and Bacillus coagulans
1.5.1凝结软糖制备1.5.1 Preparation of condensed soft candy
细胞试验结果表明BCSKB组合为1.0×106CFU/mL BCS+1.25％KB，因此设计凝结软糖理论活菌数为1.0×107CFU/g+12.5％KB参照Miranda的方法，富含凝结芽孢杆菌和奇异莓复合物凝结软糖(BCKb)制备流程如下：The cell test results show that the BCSKB combination is 1.0×106 CFU/mL BCS+1.25% KB. Therefore, the theoretical viable bacterial count of the designed curdled gummy candy is 1.0×107 CFU/g+12.5%KB. Referring to Miranda’s method, it is rich in coagulated spores. The preparation process of Bacillus and Kiwi Berry Complex Curdled Gummies (BCKb) is as follows:
步骤1：容器1中6.25mL果汁加入33.75mL水稀释后加热至85℃后，加入7g明胶至完全溶解；Step 1: Dilute 6.25mL of juice in container 1 with 33.75mL of water and heat to 85°C, then add 7g of gelatin until completely dissolved;
步骤2：另取容器2制备葡萄糖浆，7g葡萄糖浆和0.1g柠檬酸钠加入70mL水，加热浓缩至白利度为86°；Step 2: Take another container 2 to prepare glucose syrup, add 7g glucose syrup and 0.1g sodium citrate to 70mL water, heat and concentrate until the Brix is 86°;
步骤3：容器1种明胶溶液导入容器2混合,并加入2.5mL凝结芽孢杆菌发酵液(活菌数为2.0×109CFU/mL)搅拌15min(85℃保温)，导入磨具中于25℃烘干48h至水活度为0.70，脱膜。共得到50g软糖，理论活菌数为1.0×107CFU/g，奇异莓浓度12.5％。并分别配制只含凝结芽杆菌或奇异莓凝胶软糖以及对照组，原料步骤差异见表1，其它操作相同。Step 3: The gelatin solution in container 1 is introduced into container 2 for mixing, and 2.5 mL of Bacillus coagulans fermentation broth (number of viable bacteria is 2.0 × 109 CFU/mL) is added, stirred for 15 minutes (insulated at 85°C), introduced into a grinder, and dried at 25°C. After 48 hours until the water activity reaches 0.70, remove the film. A total of 50g of soft candy was obtained, with a theoretical viable bacterial count of 1.0×107CFU/g and a kiwi berry concentration of 12.5%. The gummies containing only Bacillus coagulans or kiwi berry and the control group were prepared respectively. The differences in raw material steps are shown in Table 1, and other operations are the same.
表1不同凝胶软糖原料组成差异Table 1 Differences in raw material composition of different gel gums
1.5.2理化分析1.5.2 Physical and chemical analysis
4组样品进行质构分析(Texture Profile Analysis，TPA):采用P/50柱型探头，测前速度为1mms-1、测试以及测后速度均为5mm·s-1，压缩率为50％，触发力为5g，停留时间为5s。样品取中间平整部分(1.4cm×1.4cm)进行测定，每组样品平行测定3次。每次测完一个样品用纸巾将探头擦拭干净后再进行下一次测试。Texture Profile Analysis (TPA) of 4 groups of samples: P/50 column probe is used, the pre-test speed is 1mms-1 , the test and post-test speeds are both 5mm·s-1 , and the compression rate is 50%. The triggering force is 5g and the dwell time is 5s. The middle flat part of the sample (1.4cm×1.4cm) was taken for measurement, and each group of samples was measured three times in parallel. After each sample is measured, wipe the probe clean with a paper towel before performing the next test.
色度测定：使用MSC-S型色差计测定糖果的L(亮值)、a(红值)和b(蓝值)值。剪切力的测定：使用Salter剪切力仪测定剪切力。凝结芽孢杆菌活菌数测定：分别取不同处理软糖1g加入9mL无菌水然后水浴加热65℃至糖果全部融化后，立即进行梯度稀释采用平板计数测定活菌数。Colorimetry: Use the MSC-S colorimeter to measure the L (brightness value), a (red value) and b (blue value) values of the candy. Determination of shear force: Shear force was measured using a Salter shear force meter. Determination of the number of viable bacteria of Bacillus coagulans: Take 1g of soft candy from different treatments, add 9 mL of sterile water, and heat in a water bath at 65°C until all the candy melts. Immediately perform gradient dilution and use plate counting to determine the number of viable bacteria.
1.5.3微生物计数和抗氧化测定1.5.3 Microbial enumeration and antioxidant determination
分别取不同处理的四种软糖1g加入9mL无菌水然后水浴加热65℃至糖果全部融化。取融化液梯度稀释后涂MRS固体平板，37℃培养48h，计数。Take 1g of four types of soft candies with different treatments, add 9mL of sterile water, and heat in a water bath at 65°C until the candies are completely melted. Gradient dilute the melted solution and apply it to MRS solid plates, incubate at 37°C for 48 hours, and count.
融化液12000g离心10min后取上清，检测ABTS、DPPH、PTIO清除率和FRAP值，检测和计算方法同上。Centrifuge the melted liquid at 12,000g for 10 minutes and take the supernatant to detect ABTS, DPPH, PTIO clearance rate and FRAP value. The detection and calculation methods are the same as above.
1.6数据处理和分析1.6 Data processing and analysis
试验数据用平均数±标准差表示，用SPSS20.0进行方差分析(ANOVA)，采用邓肯多量程检验比较均值的差异性。用P<0.05表示有显著性差异。The experimental data were expressed as mean ± standard deviation, and SPSS 20.0 was used for variance analysis (ANOVA), and Duncan's multi-range test was used to compare the differences in means. P < 0.05 indicated a significant difference.
2结果与分析2 Results and analysis
2.1不同水果果汁和凝结芽孢杆菌上清抗氧化性能的评估2.1 Evaluation of the antioxidant properties of different fruit juices and Bacillus coagulans supernatants
表1BCS和4种水果抗氧化性能的评估Table 1 Evaluation of antioxidant properties of BCS and four fruits
注：BCS凝结芽孢杆菌上清，对应菌体浓度2.0×109CFU/mL，VCE:每g果肉或每毫升溶液相当于VitaminC的量，单位VCE/g(mL)。Note: The supernatant of BCS Bacillus coagulans corresponds to a cell concentration of 2.0×109 CFU/mL. VCE: The amount of Vitamin C per g of pulp or per milliliter of solution, unit VCE/g (mL).
2.2细胞试验2.2 Cell test
2.2.1不同浓度的H2O2对Caco-2细胞相对细胞活力的影响2.2.1 Effects of different concentrations of H2O2 on relative cell viability of Caco-2 cells
表2不同浓度的H2O2对Caco-2细胞相对活力的影响(％)Table 2 Effects of different concentrations of H2 O2 on the relative viability of Caco-2 cells (%)
注：同行小写字母不同表示差异显著(P＜0.05)，相同表示差异不显著(P＞0.05)，下同。Note: Different lowercase letters in the same row indicate significant differences (P＜0.05), while the same lowercase letters indicate no significant differences (P＞0.05), the same below.
不同浓度的H2O2对Caco-2细胞相对活力的影响见表2，由表2可知随着H2O2浓度和作用时间的增加，细胞相对活力下降。其中H2O2浓度为0.625mmol/L，在作用的2、4、6和8h细胞活力均显著低于对照组(P＜0.05)。因此选择0.625mmol/L H2O2，诱导6h作为损伤模型。显微观察照片(100×)见图1A，有图1A可知对照组细胞呈不规则鳞片状，边界清晰，贴壁紧实，随着浓度增加，细胞间隙逐渐增大，细胞变圆，底部出现大面积脱落。The effects of different concentrations of H2 O2 on the relative viability of Caco-2 cells are shown in Table 2. It can be seen from Table 2 that with the increase of H2 O2 concentration and action time, the relative viability of cells decreased. Among them, when the H2 O2 concentration was 0.625mmol/L, the cell viability at 2, 4, 6 and 8h of action was significantly lower than that of the control group (P < 0.05). Therefore, 0.625mmol/L H2 O2 and 6h of induction were selected as the injury model. Microscopic observation photos (100×) are shown in Figure 1A. As shown in Figure 1A, the cells in the control group were irregularly scaly, with clear boundaries and tight adhesion to the wall. As the concentration increased, the intercellular gap gradually increased, the cells became round, and a large area of detachment appeared at the bottom.
2.2.2不同浓度的奇异莓对Caco-2细胞相对活力的影响2.2.2 Effects of different concentrations of kiwi berries on the relative viability of Caco-2 cells
表3不同浓度的奇异莓对Caco-2细胞相对活力的影响(％)Table 3 Effects of different concentrations of kiwi berries on the relative viability of Caco-2 cells (%)
不同浓度的奇异莓对Caco-2细胞相对活力的影响见表3，由表3可知随着奇异莓浓度和作用时间的增加，细胞相对活力下降。在2、4、6和8h诱导时间点当奇异莓浓度不高于1.25％时，对细胞没有损伤(P＞0.05)。显微观察照片(100×)见图1中B，图1中B随着KB浓度增加，细胞间隙逐渐增大，底部出现大面积脱落。The effects of different concentrations of kiwiberry on the relative viability of Caco-2 cells are shown in Table 3. It can be seen from Table 3 that as the concentration and action time of kiwiberry increase, the relative viability of the cells decreases. When the kiwiberry concentration was not higher than 1.25% at the 2, 4, 6 and 8 h induction time points, there was no damage to the cells (P>0.05). The microscopic observation photo (100×) is shown in B in Figure 1. As the concentration of KB in B in Figure 1 increases, the intercellular space gradually increases, and a large area of the bottom appears to fall off.
2.2.3不同浓度的凝结芽孢杆菌菌体和上清对Caco-2细胞相对活力的影响2.2.3 Effects of different concentrations of Bacillus coagulans cells and supernatant on the relative viability of Caco-2 cells
表4不同浓度的BC和BCS对Caco-2细胞相对活力的影响(％)Table 4 Effects of different concentrations of BC and BCS on the relative viability of Caco-2 cells (%)
不同浓度的BC和BCS对Caco-2细胞相对活力的影响见表4，由表4可知BC和BCS浓度不高于1×106 CFU/mL，对细胞活力无抑制(P＞0.05)。BCS浓度1×106 CFU/mL，在诱导的4、6和8h时间点，相对细胞活力均显著高于对照组(P＜0.05)。BCS与Caco-2细胞共培养6h显微照片(100×)见图1中C，BCS浓度1×107 CFU/mL时细胞出现间隙变大，脱落但细胞形态无明显变化。BC与Caco-2细胞共培养6h显微照片(200×)见图1中D，黑色箭头表示菌体，菌体可吸附在细胞表面或者间隙中。The effects of different concentrations of BC and BCS on the relative viability of Caco-2 cells are shown in Table 4. It can be seen from Table 4 that the concentration of BC and BCS is not higher than 1×106 CFU/mL and has no inhibition on cell viability (P>0.05). The BCS concentration was 1×106 CFU/mL. At the 4, 6 and 8 h time points of induction, the relative cell viability was significantly higher than that of the control group (P<0.05). The micrograph (100×) of BCS and Caco-2 cells co-cultured for 6 hours is shown in C in Figure 1. When the BCS concentration was 1×107 CFU/mL, the cells appeared to have enlarged gaps and fell off, but the cell morphology did not change significantly. The micrograph (200×) of BC and Caco-2 cells co-cultured for 6 hours is shown in D in Figure 1. The black arrow indicates the bacterial cells, which can be adsorbed on the cell surface or in the gaps.
2.2.4 BCS、奇异莓及其复合物对双氧水诱导Caco-2细胞保护作用2.2.4 The protective effects of BCS, kiwiberry and their complexes on hydrogen peroxide-induced Caco-2 cells
表5 BCS、奇异莓和复合物对双氧水诱导Caco-2细胞活力的影响Table 5 Effects of BCS, kiwiberry and complex on hydrogen peroxide-induced Caco-2 cell viability
注：H202浓度0.625mmol/L,BCS浓度1.0×106CFU/mL，KB浓度1.25％，BCSKB终浓度1.0×106CFU/mLBCS+1.25％KB。Note: H2 02 concentration is 0.625 mmol/L, BCS concentration is 1.0×106 CFU/mL, KB concentration is 1.25%, BCSKB final concentration is 1.0×106 CFU/mL BCS+1.25% KB.
BCS、奇异莓及其复合物对双氧水诱导Caco-2细胞保护作用见表5，由表5可知在最佳浓度条件下，双氧化诱导损伤的保护效果大小顺序为：BCSKB＞BCS＞KB(P＜0.05)。因此选择1.0×106 CFU/mLBCS+1.25％KB作为最佳保护剂模型，用于后续的试验。The protective effects of BCS, kiwi berries and their complexes on Caco-2 cells induced by hydrogen peroxide are shown in Table 5. From Table 5, it can be seen from Table 5 that under the optimal concentration conditions, the order of the protective effects of double oxygenation-induced damage is: BCSKB>BCS>KB (P <0.05). Therefore, 1.0×106 CFU/mLBCS+1.25% KB was selected as the best protective agent model for subsequent tests.
2.2.5荧光定量PCR和Western Blotting分析2.2.5 Fluorescence quantitative PCR and Western Blotting analysis
荧光定量PCR和Western Blotting分析结果见图2.由图2可知BCS通过激活Nrf2抗氧化通路缓解双氧水显著诱导的Caco-2细胞的氧化损伤。The results of fluorescence quantitative PCR and Western Blotting analysis are shown in Figure 2. Figure 2 shows that BCS alleviates the oxidative damage of Caco-2 cells significantly induced by hydrogen peroxide by activating the Nrf2 antioxidant pathway.
3.3凝胶软糖的分析3.3 Analysis of gelatin gummies
3.3.1质构和颜色分析3.3.1 Texture and color analysis
表6质构和颜色分析Table 6 Texture and color analysis
注：同行小写字母相同表示差异不显著(P＞0.05)；同列小写字母不同表示差异显著(P＜0.05)，下同。BC：凝结芽孢杆菌，KB：奇异莓。Note: The same lowercase letters in the same column indicate no significant difference (P>0.05); different lowercase letters in the same column indicate significant differences (P<0.05), the same below. BC: Bacillus coagulans, KB: Kiwi berry.
质构和颜色分析结果见表6,由表6可知BCKB在口感和颜色方面优于对照组。The texture and color analysis results are shown in Table 6. From Table 6, it can be seen that BCKB is better than the control group in terms of taste and color.
3.3.2微生物和抗氧化分析3.3.2 Microbial and antioxidant analysis
表7不同组合凝胶软糖微生物和抗氧化分析Table 7 Microbial and antioxidant analysis of different combinations of jelly candies
BC组：凝结芽孢杆菌软糖，KB组：奇异莓软糖，BCKB组富含凝结芽孢杆菌和奇异莓软糖组BC group: Bacillus coagulans soft candy, KB group: kiwi berry soft candy, BCKB group is rich in Bacillus coagulans and kiwi berry soft candy group
不同组合凝胶软糖微生物和抗氧化分析见表7，由表7可知奇异莓和凝结芽孢杆菌复合制备的凝胶软糖具有最高的抗氧化性能。The microbial and antioxidant analysis of different combinations of jelly candies are shown in Table 7. It can be seen from Table 7 that the jelly candies prepared by combining kiwi berry and Bacillus coagulans have the highest antioxidant property.
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| CN202311834200.2ACN117796466A (en) | 2023-12-28 | 2023-12-28 | A gel candy rich in juice and bacillus coagulans and its preparation method and application |
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| CN202311834200.2ACN117796466A (en) | 2023-12-28 | 2023-12-28 | A gel candy rich in juice and bacillus coagulans and its preparation method and application |
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| WO2011130487A1 (en)* | 2010-04-14 | 2011-10-20 | Ganeden Biotech, Inc. | Probiotic confection and lipid compositions |
| CN111557366A (en)* | 2020-05-13 | 2020-08-21 | 江苏微康生物科技有限公司 | Bacillus coagulans BC99 soft sweets capable of improving immunity and preparation method thereof |
| CN114196726A (en)* | 2020-09-17 | 2022-03-18 | 仙乐健康科技股份有限公司 | Detection method of probiotics in gelatin system |
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2011130487A1 (en)* | 2010-04-14 | 2011-10-20 | Ganeden Biotech, Inc. | Probiotic confection and lipid compositions |
| CN111557366A (en)* | 2020-05-13 | 2020-08-21 | 江苏微康生物科技有限公司 | Bacillus coagulans BC99 soft sweets capable of improving immunity and preparation method thereof |
| CN114196726A (en)* | 2020-09-17 | 2022-03-18 | 仙乐健康科技股份有限公司 | Detection method of probiotics in gelatin system |
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