优良干酪发酵剂用菌株性能研究及其在切达干酪中的应用

发布时间：2018-05-21 11:24

本文选题：乳酸乳球菌 + 发酵剂　；参考：《东北农业大学》2013年硕士论文

【摘要】：切达干酪是一种硬质成熟干酪，是将牛乳用凝乳酶浓缩并酸化，随后形成凝胶状态制成的。由于切达干酪的风味温和,更适合我国人民的口味也更容易被我国消费者接受。由于国内乳品企业的发展以及国民饮食结构的优化，人们对干酪的需求日益提高。而我国的干酪工业化生产尚处于起步阶段，当前市面上的天然干酪几乎都是由国外进口的，因此推动我国干酪业的发展迫在眉睫。要实现干酪工业化生产首先要解决的问题是获得性状稳定、性能优良的干酪发酵剂，目前外国企业已经完成直投式发酵剂的研发及生产，其产品种类丰富，垄断了国内外市场。我国直投式发酵剂的研究起步晚且技术水平不高，产品性能与国外产品相比仍相对较差。开发我国自主知识产权，具有活性高、成本低等优点的直投式干酪发酵剂引起国内学者广泛关注。本研究对实验室已鉴定的两株乳酸乳球菌KLDS4.0424和KLDS4.0326进行发酵产酸、产黏和蛋白水解能力测试。并对菌株的高密度培养基进行改良，在单因素研究的基础上进行了响应面优化实验，通过优化得出最佳培养基的成分配比，使菌株活菌数显著提高，达到高密度培养的目的，为菌株开发成直投式干酪发酵剂奠定基础。并对以单菌株、不同比例组合菌株制备的切达干酪在成熟期间进行品质评定。实验结果表明：乳酸乳球菌乳酸亚种KLDS4.0424是主要的产酸菌种，产酸速率为15.0°T/h。与商业发酵剂相比，实验室菌株的蛋白水解能力和产黏能力相对较弱；乳酸亚种与乳脂亚种搭配使用，可弥补不足。乳清是干酪生产的副产品，含有4.5%乳糖、0.8%乳清蛋白、维生素等营养成分，来源广泛且价格低廉，适合作为工业化原料生产发酵剂。采用乳清粉为基础培养基，通过添加酵母粉、酶水解酪蛋白补充有机氮源和多种促生长因子，添加的碳酸钙可以中和菌体增殖过程中产生的乳酸，实现菌株的高密度培养。通过单因素实验确定，菌株KLDS4.0424的活菌数分别在酵母粉浓度为12g/L、酶水解酪蛋白12g/L、碳酸钙5g/L时达到最高。菌株KLDS4.0326的活菌数分别在酵母粉浓度为12g/L、酶水解酪蛋白12g/L、碳酸钙6g/L时达到最高。依据单因素实验结果，确定响应面实验水平的中心点和各水平的步长。使用Design Expert7.0.0软件的Box-Behnken设计三因素三水平响应面优化实验，并通过标准多项式回归方法，对实验数据进行拟合，得到一个二次多项式，进一步建立二次响应面回归模型，可用以下模型方程来表示在乳清粉基础培养基中添加不同量的酵母粉、酶水解酪蛋白、碳酸钙得到12h菌株KLDS4.0424的活菌数：Y1=334.20+9.75*X_3+23.75*X_1+16.75*X_2+4.50*X_3*X_1+4.00*X_2*X_3-24.50*X_1*X_2-65.85*X_32-85.35*X_1~2-54.35*X_2~2 最佳培养基的组成为：碳酸钙5.57g/L，酵母粉9.80g/L，酶水解酪蛋白8.74g/L。在此条件下模型预测的培养12h菌株KLDS4.0424最高活菌数为3.12*109cfu/mL。同理，菌株KLDS4.0326获得的活菌数拟合模型方程为：Y2=166.20+2.38*X_3+7.50*X_1+10.63*X_2+2.50*X_3*X_1+12.25*X_2*X_3+7.00*X_1*X_2-14.98*X_3~2-29.23*X_2~2-20.97*X_2 最佳培养基的组成为：碳酸钙5.28g/L，酵母粉9.60g/L，酶水解酪蛋白9.04g/L。在此条件下模型预测的培养12h菌株KLDS4.0326最高活菌数为1.61*109cfu/mL。按优化出的最优培养基配方配置2.5L培养基，以1%的接种量接种菌体。设置初始pH6.5，搅拌速度100r/min，30℃培养，流加50%（V/V）氨水调节pH，在发酵罐内恒pH发酵。恒pH发酵12h后，，菌株KLDS4.0424活菌数高达2.6*10~(10)cfu/mL，菌株KLDS4.0326活菌数达8.8*109cfu/mL，达到了高密度培养的目的。菌株KLDS4.0424的活菌数较高密度培养前提高一个数量级，菌株KLDS4.0326活菌数亦有显著提高。应用KLDS4.0424（LA）、KLDS4.0326（LC）以及不同比例混合菌（LA∶LC=1∶1，L_A∶L_C=1∶2，L_A∶L_C=2∶1）制作切达干酪，研究这5种发酵剂在干酪成熟过程中对其质构、感官、风味物质形成及蛋白水解程度等方面的影响。以商业发酵剂制作的切达干酪作为对照组。结果表明，菌株KLDS4.0424和KLDS4.0326按比例1:1接种制作的干酪具有良好的成熟度、质构及风味，且理化指标和微生物指标完全符合国家标准。
[Abstract]:Cheddar cheese is a kind of hard matured cheese, which is made of condensed milk with chymase and acidified and then formed by gelation. Because the flavor of Cheddar cheese is mild, it is more suitable for our people's taste and is easier to be accepted by our consumers. The demand for cheese industrial production in China is still in its infancy, and the natural cheese on the market is almost all imported from abroad. Therefore, it is imminent to promote the development of the cheese industry in China. The former foreign enterprises have completed the R & D and production of the direct cast fermentor, whose products are rich in variety and monopolized the domestic and foreign markets. The research of the direct cast fermentor in our country is late and the technical level is not high, and the performance of the product is still relatively poor compared with the foreign products. Cheese fermentative agents have aroused wide attention from domestic scholars.
In this study, two strains of Lactococcus lactis KLDS4.0424 and KLDS4.0326 identified in the laboratory were fermented to produce acid, the ability to produce viscosity and protein hydrolysability test, and to improve the high density medium of the strain. On the basis of the single factor study, the response surface optimization experiment was carried out. The number of bacteria increased significantly and reached the aim of high density culture. It laid the foundation for the strain developed into a direct type cheese starter. The quality of Cheddar cheese prepared with single strain and different proportions of strains was evaluated during maturity.
The experimental results show that the subspecies KLDS4.0424 of Lactococcus Lactococcus lactis is the main acid producing strain. The rate of acid production of 15 T/h. is relatively weak compared with the commercial fermentation agent, and the protein hydrolysis ability and adhesion ability of the laboratory strain are relatively weak; the use of the subspecies of lactic acid and the subspecies of milk fat can make up for the deficiency.
Whey is a by-product of cheese production. It contains 4.5% lactose, 0.8% whey protein, vitamin and other nutrients. It has a wide range and low price. It is suitable for the production of fermentative as an industrial raw material. Using whey powder as the base medium, adding yeast powder, enzymatic hydrolysis of casein to fill organic nitrogen source and a variety of growth factors and added calcium carbonate We can neutralize the lactic acid produced during the multiplication of the bacteria and realize the high density culture of the strain. Through the single factor experiment, the living bacteria number of strain KLDS4.0424 is the highest when the yeast powder concentration is 12g/L, the enzyme hydrolyzed casein 12g/L, and the calcium carbonate 5g/L is the highest. The living bacteria number of the strain KLDS4.0326 is 12g/L in the yeast powder concentration, respectively, and the enzyme hydrolysis of casein eggs, respectively White 12g/L and calcium carbonate reached the highest level. According to the result of single factor experiment, the central point and the step length of each level of the response surface experiment were determined. 6g/L
Using the Box-Behnken of Design Expert7.0.0 software, three factors and three level response surface optimization experiments are designed, and the experimental data are fitted by the standard polynomial regression method. A two order polynomial is obtained, and the two response surface regression model is further established. The following model equation can be used to express the addition of the base medium in the whey powder foundation. The same amount of yeast powder, enzyme hydrolyzed casein, calcium carbonate obtained the living bacteria number of 12h strain KLDS4.0424: Y1=334.20+9.75*X_3+23.75*X_1+16.75*X_2+4.50*X_3*X_1+4.00*X_2*X_3-24.50*X_1*X_2-65.85*X_32-85.35*X_1~2-54.35*X_2~2
The composition of the best medium is: calcium carbonate 5.57g/L, yeast powder 9.80g/L, enzyme hydrolyzed casein 8.74g/L. in this condition, the maximum living bacteria number of 12h strain KLDS4.0424 is 3.12*109cfu/mL.
In the same way, the fitting model equation of the living bacteria number obtained by strain KLDS4.0326 is: Y2=166.20+2.38*X_3+7.50*X_1+10.63*X_2+2.50*X_3*X_1+12.25*X_2*X_3+7.00*X_1*X_2-14.98*X_3~2-29.23*X_2~2-20.97*X_2
The composition of the best medium is: calcium carbonate 5.28g/L, yeast powder 9.60g/L, enzyme hydrolyzed casein 9.04g/L. in this condition, the maximum living bacteria number of 12h strain KLDS4.0326 is 1.61*109cfu/mL.
According to the optimized medium of optimum medium, the 2.5L medium was configured to inoculate the bacteria with 1% inoculation. The initial pH6.5 was set up, the stirring speed 100r/min, 30 C, and 50% (V/V) ammonia water were added to pH, and the fermentation in the fermenting tank was constant. The KLDS4.0424 living bacteria number of the strain was as high as 2.6*10~ (10) cfu/mL, and the number of live strain of the strain reached the number of KLDS4.0326. ML, the aim of high density culture was achieved. The number of living bacteria of strain KLDS4.0424 increased by one order of magnitude before high density culture, and the number of KLDS4.0326 living bacteria increased significantly.
Cheddar cheese was made by using KLDS4.0424 (LA), KLDS4.0326 (LC) and different proportions of mixed bacteria (LA: LC=1: 1, L_A: L_C=1: 2, L_A: L_C=2: 1). The effects of these 5 kinds of starters on the texture, sensory, flavour material formation and the range of protein hydrolysis in cheese ripening. The results showed that the cheese produced by the strain KLDS4.0424 and KLDS4.0326 had good maturity, texture and flavor, and the physical and chemical indexes and microbiological indexes were fully in conformity with the national standard.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：TS252.53

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