**5. Conclusions**

138 Food Industrial Processes – Methods and Equipment

Concrete values were applied in a design matrix. It is evident that increasing reaction temperature enhances the reaction rate, that is way that its affect will be investigated later

During the experiments different, software-defined combinations of the previously selected values of experimental parameters were investigated. Other parameters were fixed: temperature 50 °C, 150 rpm shaking intensity, 5 hours reaction time. Water content of the reaction mixture was also followed as an important parameter of esterification reactions which may shift the equilibrium, but there was not detectable concentration change using Carl-Fischer titration. The aim was to find the optimal parameter values of the isoamyl-

As it shown, increasing amount of ionic liquid results in higher oleate yield, which gives evidence for the advantageous enzyme stabilization effect. Complex investigation of the three chosen key factors shows that the highest ester conversion was obtained in the case of application the highest acid/alcohol molar ratio, amount of enzyme and ionic liquid. Relatively high yield was obtained also around medium values, around the center points.

Fig. 8. Influence of the acid/alcohol molar ratio and amount of ionic liquid on the synthesis

There was no inhibition effect observed neither in 1:16 acid/alcohol molar ratio. As none of the point of parameters could be an optimal value, because the highest yield belonging to the highest values, therefore further investigations will necessary to find the optimal values.

In case of using conventional reaction media it is well known that increasing reaction temperature results the same yield in shorter time, but till a limit due to thermal deactivation of enzyme. Beside of the structure stabilization effect of ionic liquids the enzyme can be resist in the active conformation at higher temperatures (van Rantwijk & Sheldon, 2007). For these investigations that mixture was chosen, in which the highest oleate

separately.

of isoamyl oleate

**4.4 Effect of temperature** 

yield was obtained. The results are shown on Fig. 9.

oleate production. The results are shown on Fig. 8.

Our investigations have proven that an ester type biolubricant could be prepared from fusel oils and oleic acid by lipase enzyme in solvent-free system. Compared to the product obtained by acid catalysis, in the biolubricant there was no trace of oleic acid since complete conversion was achieved by continuous water removal by pervaporation. Our product was then tested in an acute toxicological procedure by zebrafishes, which has verified the assumption: the biolubricant is not toxic for the living water, so it is considered as an environmental safe product.

The tribological study has shown that the features of the biolubricant are similar to the DB 32 type synthetic reference lubricating oil, so it can be applied as a low viscosity lubricant, suitable even for special publication processes, where lubricant loss may accur. Although the biological degradation of the product has not been studied, it was manufactured from initial compounds having biological origin (oleic acid from plant oils and alcohols from a by-product in bioalkohol production), thus it is considered as a completely environmentalfriend product.

The kinetic model containing the parameters determined can be used in the particular enzymatic esterification reaction for calculations of the optimal conditions of various aspects, like highest yield, lowest acid residue, lowest amount of enzyme, shortest reaction time…etc. Moreover we are planning to apply the model for bioreactor design to realise continuous enzymatic i-amyl oleate synthesis by lipase.

Our work has showenn that an ester type biolubricant could be prepared from isoamylalcohol and oleic acid by lipase enzyme in ionic liquid two phase system. Compared to the product obtained in solvent free system, higher conversion in shorter time was achieved. Despite the lack of water removal in the biolubricant there was no trace of oleic

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#### **6. Acknowledgment**

The research work was supported by the projects TAMOP-4.2.2/B-10/1-2010-0025 and TÁMOP 4.2.1 / 4.8 B identification number "Mobility and Environment: automotive, energy and environmental research in Central and West Pannon Region".

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**9** 

137

**Applications** 

*IATA- CSIC Spain* 

María J. Yebra, Vicente Monedero,

*Departamento de Biotecnología de los Alimentos* 

**Genetically Engineered Lactobacilli for** 

Lactic acid bacteria (LAB) are Gram-positive microorganisms that produce lactic acid as a major product of their metabolism. Among them the genus *Lactobacillus* comprises a large heterogeneous group of low G+C DNA content, anaerobic and nonsporulating bacteria, that includes species widely used in the food industry. They play key roles in fermented dairy, meats and vegetables products. Due to their claimed health-promoting properties certain lactobacilli species are used as probiotics and they are commonly applied to dairy and functional foods products. Lactobacilli have a relatively simple fermentative metabolism focused to rapidly convert carbohydrates into lactic acid, and exhibited a limited biosynthetic capacity. In addition, several tools and strategies to manipulate them genetically are available. All those characteristics make lactobacilli specially suited for genetic engineering aimed to increase existing compounds or to produce novel metabolites of interest for the food industry. Regarding probiotic lactobacilli, through genetic manipulation, the health attributes of probiotic strains can be enhanced or new probiotic activities can be developed and additionally, an understanding of the underlying molecular mechanisms can be obtained. Here, we review metabolic engineering strategies in lactobacilli that have successfully been used to efficiently reroute sugar metabolism to compounds such as L-lactic acid, aroma compounds (acetoin, diacetyl), low-calorie sugars (mannitol, sorbitol) and exopolysaccharides. We will also describe strains of probiotic lactobacilli that have been developed to exploit their adherence and immunomodulatory

Because of their fermentative metabolism and global utilization in food fermentations, LAB are specially suited for rerouting sugar metabolism to produce industrially important food compounds. During fermentation, monosaccharides are catabolized through glycolysis (Embden-Meyerhoff pathway) and related pathways (Figure 1). Glycolytic catabolism of sugars involves phosphorylation reactions that drive hexoses to fructose-1,6-bisP, which is then hydrolysed to glyceraldehyde-3P (GADH-3P) and dihydroxyacetone-P. Then, GADH-

properties, and to delivery proteins at the intestinal mucosa.

**1.1 Metabolic potential of lactobacilli** 

**1. Introduction** 

**Technological and Functional Food** 

Gaspar Pérez-Martínez and Jesús Rodríguez-Díaz

Zaidi, A., J.L. Gainer, G. Carta, A. Mrani, T. Kadiri, Y. Belarbi and A. Mir, (2002) Esterification of fatty acids using nylon-immobilized lipase in n-hexane: kinetic parameters and chain-length effects , *J. Biotechnol.* 93, pp. 209–216.
