發(fā)布時間：2018-05-21 11:24

  本文選題：乳酸乳球菌 + 發(fā)酵劑��； 參考：《東北農(nóng)業(yè)大學》2013年碩士論文

【摘要】：切達干酪是一種硬質(zhì)成熟干酪，是將牛乳用凝乳酶濃縮并酸化，隨后形成凝膠狀態(tài)制成的。由于切達干酪的風味溫和,更適合我國人民的口味也更容易被我國消費者接受。由于國內(nèi)乳品企業(yè)的發(fā)展以及國民飲食結(jié)構(gòu)的優(yōu)化，人們對干酪的需求日益提高。而我國的干酪工業(yè)化生產(chǎn)尚處于起步階段，當前市面上的天然干酪?guī)缀醵际怯蓢膺M口的，因此推動我國干酪業(yè)的發(fā)展迫在眉睫。要實現(xiàn)干酪工業(yè)化生產(chǎn)首先要解決的問題是獲得性狀穩(wěn)定、性能優(yōu)良的干酪發(fā)酵劑，目前外國企業(yè)已經(jīng)完成直投式發(fā)酵劑的研發(fā)及生產(chǎn)，其產(chǎn)品種類豐富，壟斷了國內(nèi)外市場。我國直投式發(fā)酵劑的研究起步晚且技術(shù)水平不高，產(chǎn)品性能與國外產(chǎn)品相比仍相對較差。開發(fā)我國自主知識產(chǎn)權(quán)，具有活性高、成本低等優(yōu)點的直投式干酪發(fā)酵劑引起國內(nèi)學者廣泛關(guān)注。 本研究對實驗室已鑒定的兩株乳酸乳球菌KLDS4.0424和KLDS4.0326進行發(fā)酵產(chǎn)酸、產(chǎn)黏和蛋白水解能力測試。并對菌株的高密度培養(yǎng)基進行改良，在單因素研究的基礎(chǔ)上進行了響應面優(yōu)化實驗，通過優(yōu)化得出最佳培養(yǎng)基的成分配比，使菌株活菌數(shù)顯著提高，達到高密度培養(yǎng)的目的，為菌株開發(fā)成直投式干酪發(fā)酵劑奠定基礎(chǔ)。并對以單菌株、不同比例組合菌株制備的切達干酪在成熟期間進行品質(zhì)評定。 實驗結(jié)果表明：乳酸乳球菌乳酸亞種KLDS4.0424是主要的產(chǎn)酸菌種，產(chǎn)酸速率為15.0°T/h。與商業(yè)發(fā)酵劑相比，實驗室菌株的蛋白水解能力和產(chǎn)黏能力相對較弱；乳酸亞種與乳脂亞種搭配使用，可彌補不足。 乳清是干酪生產(chǎn)的副產(chǎn)品，含有4.5%乳糖、0.8%乳清蛋白、維生素等營養(yǎng)成分，來源廣泛且價格低廉，適合作為工業(yè)化原料生產(chǎn)發(fā)酵劑。采用乳清粉為基礎(chǔ)培養(yǎng)基，通過添加酵母粉、酶水解酪蛋白補充有機氮源和多種促生長因子，添加的碳酸鈣可以中和菌體增殖過程中產(chǎn)生的乳酸，實現(xiàn)菌株的高密度培養(yǎng)。通過單因素實驗確定，菌株KLDS4.0424的活菌數(shù)分別在酵母粉濃度為12g/L、酶水解酪蛋白12g/L、碳酸鈣5g/L時達到最高。菌株KLDS4.0326的活菌數(shù)分別在酵母粉濃度為12g/L、酶水解酪蛋白12g/L、碳酸鈣6g/L時達到最高。依據(jù)單因素實驗結(jié)果，確定響應面實驗水平的中心點和各水平的步長。 使用Design Expert7.0.0軟件的Box-Behnken設(shè)計三因素三水平響應面優(yōu)化實驗，并通過標準多項式回歸方法，對實驗數(shù)據(jù)進行擬合，得到一個二次多項式，進一步建立二次響應面回歸模型，可用以下模型方程來表示在乳清粉基礎(chǔ)培養(yǎng)基中添加不同量的酵母粉、酶水解酪蛋白、碳酸鈣得到12h菌株KLDS4.0424的活菌數(shù)：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 最佳培養(yǎng)基的組成為：碳酸鈣5.57g/L，酵母粉9.80g/L，酶水解酪蛋白8.74g/L。在此條件下模型預測的培養(yǎng)12h菌株KLDS4.0424最高活菌數(shù)為3.12*109cfu/mL。 同理，菌株KLDS4.0326獲得的活菌數(shù)擬合模型方程為：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 最佳培養(yǎng)基的組成為：碳酸鈣5.28g/L，酵母粉9.60g/L，酶水解酪蛋白9.04g/L。在此條件下模型預測的培養(yǎng)12h菌株KLDS4.0326最高活菌數(shù)為1.61*109cfu/mL。 按優(yōu)化出的最優(yōu)培養(yǎng)基配方配置2.5L培養(yǎng)基，以1%的接種量接種菌體。設(shè)置初始pH6.5，攪拌速度100r/min，30℃培養(yǎng)，流加50%（V/V）氨水調(diào)節(jié)pH，在發(fā)酵罐內(nèi)恒pH發(fā)酵。恒pH發(fā)酵12h后，，菌株KLDS4.0424活菌數(shù)高達2.6*10~(10)cfu/mL，菌株KLDS4.0326活菌數(shù)達8.8*109cfu/mL，達到了高密度培養(yǎng)的目的。菌株KLDS4.0424的活菌數(shù)較高密度培養(yǎng)前提高一個數(shù)量級，菌株KLDS4.0326活菌數(shù)亦有顯著提高。 應用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）制作切達干酪，研究這5種發(fā)酵劑在干酪成熟過程中對其質(zhì)構(gòu)、感官、風味物質(zhì)形成及蛋白水解程度等方面的影響。以商業(yè)發(fā)酵劑制作的切達干酪作為對照組。結(jié)果表明，菌株KLDS4.0424和KLDS4.0326按比例1:1接種制作的干酪具有良好的成熟度、質(zhì)構(gòu)及風味，且理化指標和微生物指標完全符合國家標準。
[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.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：TS252.53

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