Effects of Microfluidizer Technology（动态高压微射流技术） on Bacillus licheniformis Spores in Ice Cream Mix

---动态高压微射流应用于非热加工的实验与结果数据分析

本文将简介动态高压微射流技术在冰淇淋高压瞬时杀菌中过程中的应用方法与结果。研究人员通过实验检测研究了动态高压微射流处理对冰淇淋混合物中藓样芽胞杆菌孢子的破碎影响。研究结果表明：动态高压微射流技术在液体食品瞬时杀菌的方法相比于传统的高压等静压灭菌或者高温高压灭菌的方法，它同样能杀死液体食品中的微生物，但是不会带来一般热力杀菌技术导致的食品品质和风味被改变的困扰。

实验样品：

冰激淋混合物

Bacillus licheniformis Spores一种芽孢杆菌孢子（这种微生物的选择，是因为它本身不具有致病性，同时目前40%的生牛乳品种中含有这种微生物，本文将利用这种微生物的杀菌比例来进行数据的呈现）

主要实验设备：

动态高压微射流均质机

图 NanoGenizer动态高压微射流均质机

实验过程与结果简述：

1. 将4批巴氏杀菌冰淇淋混合物预热至33、36、44或50°C；

2. 加入芽孢杆菌孢子，接种获得2.0×104个孢子/ml的孢子冰激凌混合物；

3. 样品分别在50MPa、100MPa、150MPa和200MPa的条件下进行单次样品处理。

不同条件下处理的孢子破坏率分别为6~68%。

表：动态高压微射流处理1次冰激凌样品的杀菌效率统计数据

因此，通过高压微射流作用，加压后的样品在金刚石交互容腔内部产生高速微射流，物料射流速度可以达到500m/s，高压高速的微射流物料在Y型金刚石交互容腔的high shear zone（高频剪切区）受到极高频率的剪切作用力，在Y型金刚石交互容腔high impact zone（高能对撞区）进行高能子弹式的对撞，然后经过压力将与空穴效应，使得微射流高压均质系统加工过程中对孢子破坏产生倍增效应。虽然样品在处理瞬间会有升温作用，但样品的破碎率是在动态高压微射流均质处理1次后，立即冰浴冷却后检测获得，所以动态高压微射流杀菌的作用中包含一定的温度影响，但较主要的还是动态高压微射流的高频剪切、对射、压力降、空穴效应等高能物理作用的结果。数据表明，可以设计一种巴氏杀菌器-动态高压微射流均质杀菌系统，使乳制品中的大多数细菌孢子失活，而不需要目前商业加工操作中需要的极端热处理，可以更好得保存某些品种食品的营养物质与口感。

详细处理过程与结果分享：

Sample Preparation and Inoculation

Unflavored ice cream mix (12% milk fat, 10% milk solids not fat, 12% cane sugar, 5.8% corn syrup, and0.25% stabilizer) was obtained from the Dairy Processing Plant at Mississippi State University(Mississippi State). Mix was pasteurized at 80°C for25 s without homogenization and cooled to 4°C;21-kg portions were delivered to four sanitized stainless steel milk cans (38.8-L capacity). During preliminary trials, it was observed that temperature in the ice cream mix increased as pressure in the Microfluidizer system increased. Therefore, prior to inoculation, the ice cream mix was warmed to temperatures simulating internal temperatures that can be achieved in the raw side of the regenerative section of HTST plate pasteurizers, just before homogenization.Thus, inlet temperatures of ice cream mixes into the Microfluidizer system were set at 33, 36, 44, or 50°C.A 50-ml sample of mix from each can was obtained aseptically for microbiological analysis just prior to inoculation with spores. One milliliter of inoculum of a diluted spore suspension of B. licheniformis was added to the ice cream mix to yield a final concentration of approximately 2.0 × 104 spores/ml of mix.Another sample of the inoculated mix was obtained immediately after spore addition to each can. Inoculated and uninoculated mix samples were kept in an ice-water bath until further analysis

Microfluidizer Processing and Microbial Analysis

After inoculation, the contents of one can at are spective initial temperature were delivered into the stainless steel vessel attached to the Microfluidizer system and homogenized at 50,000, 100,000, 150,000, and200,000 kPa. Two samples were collected aseptically after each high pressure treatment; one served for outlet temperature measurement, and the other was immediately placed into an ice-water bath for further microbiological analysis.

Ice cream samples, before and after microfluidization, were subjected to a spore count. Samples were heat treated at 80°C for 12 min, subsequently plated in plate count agar (Difco Laboratories) containing0.1% soluble starch (4, 11), and incubated at 32°C for24 to 48 h.

Statistical Analysis 

Data were analyzed as a split-plot treatment arrangement in a completely randomized block design,with temperature as main plots and pressure as subplots. Data were subjected to ANOVA, and means were separated by the least significant difference procedure of SAS (15). The experiment was replicated three times, and significance was declared at P< 0.05, unless otherwise stated.

RESULTS AND DISCUSSION

Temperature increased when pressures were increased from atmospheric pressure to 200,000 kPa in50,000-kPa increments in the Microfluidizer system(Table 1). As initial inlet (control) temperatures increased, outlet temperatures also increased. The control mix contained no added spores; the remainder of the pressures shown in Table 1 refer to inoculated ice cream mixes. Temperatures increased as pressure increased, temperatures increased from 46 to 88°C as pressure increased from 50,000 to 200,000 kPa. This result was expected because shear and cavitation effects increase as pressure increased, resulting in increased temperature in the products. This temperature elevation may have a positive effect on destruction of bacterial spores because germination stimulation can be accomplished by sublethal heating(2), rendering the germinated spore sensitive to temperature and forces that are destructive to vegetative cells. Perhaps this combination of heat and pressure was in effect during the microfluidization procedure(3).

Effects of both initial temperature and pressure generated in the Microfluidizer system on B. licheniformis spores in ice cream mix are shown in Table 2. From an initial inoculum of approximately2.0 × 104 spores/g and an initial temperature of 33°C,the number of spores decreased to approximately 1.8× 104 at 50,000 kPa, 1.4 × 104 at 100,000 kPa, 1.2 ×104 at 150,000 kPa, and 1.0 × 104 at 200,000 kPa(48% reduction). At 36°C initial temperature, similar results were obtained; surviving spores ranged from1.5 × 104/g to 9.5 × 103/g as pressure increased from50,000 to 200,000 kPa (50% reduction). At an initial temperature of 44°C, results were again similar,although the rate of destruction was somewhat greater, ranging from approximately 1.5 × 104 survivors at 50,000 kPa to approximately 7.1 × 103 at200,000 kPa (61% reduction). The greatest reduction(68%) occurred at the highest initial temperature(50°C); survivors at 50,000 kPa were approximately1.4 × 104 spores/g and decreased to approximately 5.0× 103 spores/g at 200,000 kPa. No spores were detected in ice cream mix controls before microfluidization treatments.

Our results indicated that numbers of B. licheniformis spores are reduced by forces generated in the Microfluidizer system but are not completely destroyed. Further work needs to be done to determine a feasible mechanism for combining the Microfluidizersystem with HTST pasteurization to reduce further the numbers of spores in fluid dairy products.

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