167 2.4 Enzymatic Process for the Synthesis of Cellobiose Birgit Brucher1 and Thomas Häßler2 1c-LEcta GmbH, Perlickstr. 5, Leipzig, Germany 2Pfeifer & Langen GmbH & Co. KG, Dürener Str. 40, Elsdorf, Germany 2.4.1 Enzymatic Synthesis of Cellobiose Cellobiose, a naturally occurring disaccharide consisting of two β1-4 linked glucose monomers, can be produced in an enzymatic cascade reaction using two phosphorylases (Figure 2.4.1). The first enzyme in the enzymatic synthesis of cellobiose is sucrose phosphorylase (SP). This enzyme belongs to the GH-13 family (family assignment can be found at the public Carbohydrate-active enZYme (CAZy) database) and can be classified as a retaining phosphorylase. Interestingly, sucrose phosphorylases can also act as glycosyl transferases and have gained considerable attention in recent years for the synthesis of other carbohydrates, such as kojibiose, nigerose [1, 2], and α- d-glucosides [3]. Cellobiose phosphorylases (CP) catalyze the formation of cellobiose from glu- cose and α-glucose 1-phosphate in the second reaction step. CPs are classified to the GH-94 family. The crystal structure of the cellobiose phosphorylase from Cellulomonas uda has been elucidated [4]. Previous studies involving enzyme engineering of CP enzymes include expanding the substrate spectrum of the enzyme to the phosphorolysis of lactose by enzyme engineering [5]. The production of cellobiose using SP and CP can be performed as a one-pot reaction. Reaction processes using several enzymes in a one-pot reaction for multistep synthesis offer significant advantages compared to sequential reactions such as reduced production costs and no necessity for intermediate product recovery [6]. However, multienzyme reactions are often more challenging concerning process design due to more complex reaction kinetics or the use of enzymes with different optima for reaction parameters such as temperature or pH-value. For this reason, despite significant advances in the development of multienzyme reactions in recent years by enzyme engineering and reaction modeling [7], few processes have found application in industry. Industrial Enzyme Applications, First Edition. Edited by Andreas Vogel and Oliver May. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2019 by Wiley-VCH Verlag GmbH & Co. KGaA. 168 2.4 Enzymatic Process for the Synthesis of Cellobiose OH OH OH O OH HO SP O O HO O + Pi HO + HO OH HO HO HO Fructose O OH OH OPO2– OH OH OH 3 Sucrose Glucose 1-phosphate OH O HO Glucose CP HO OH OH OH O OH HO HO OH HO O + Pi OH O OH Cellobiose Figure 2.4.1 Enzymatic synthesis of cellobiose from sucrose (SP, sucrose phosphorylase; CP, cellobiose phosphorylase). 2.4.2 Cellobiose – Properties and Applications Cellobiose is the basic repeat structural unit of cellulose and thus the main com- ponent of plant cell walls. Cellobiose is a reducing sugar and compared to sucrose it has a lower water activity and a lower sweetness, being rated as 0.2 times relative to sucrose. The properties of cellobiose are very different from sucrose and are much more similar to those of lactose. Hence, cellobiose can be used in similar applications as lactose. Pfeifer & Langen GmbH & Co. KG in cooperation with potential customers from industry investigated a broad range of applications. Selected applications can be found in the field of beverages, meat and sausage products, comprimates (pills), and encapsulation technologies. As a reducing sugar cellobiose can undergo Malliard reactions leading to a browning effect similar to sucrose, which is mostly wanted in baked products. Thus, cellobiose has extensive potential to be used in baking. Furthermore, cellobiose can be used for more efficient encapsulation of sensi- tive compounds in foodstuff such as fats and oils with high amounts of polyun- saturated fatty acids (PUFAs) or flavors in order to prevent oxidation reactions. PUFAs undergo a strong exogenous and endogenous destruction resulting in off-flavor, rancid odor, reduction of nutritionally value, and formation of free radicals [8–10]. The challenge in the production of functional foodstuff is main- taining the general properties and especially the sensory profiles that the con- sumers expect [11]. Linoleic acid, a polyunsaturated omega-6 fatty acid, has a relatively low shelf life and short oxidative induction time [9, 12] and was there- fore chosen as a reference substance for assessing the effect of cellobiose addition to the encapsulation process. It could be shown that the encapsulation efficiency of linoleic acid increased with increasing cellobiose content in the matrix with maltodextrin and starch. Additionally, the peroxide value, which is a measure of the primary oxidation of oils, decreased with increasing amounts of cellobiose in 2.4.2 Cellobiose – Properties and Applications 169 the matrix, e.g. the peroxide value was reduced by 60% when linoleic acid was encapsulated with 20% cellobiose and stored at 35 ∘C for eight weeks compared to encapsulation without cellobiose. After three weeks of storage, a sensory panel confirmed a significantly less rancid odor of samples encapsulated with cellobiose compared to samples encapsulated without cellobiose. Furthermore, a similar encapsulation procedure for volatile compounds such as flavors, e.g. limonene, resulted in the deceleration of oxidative destruction and occurrence of off-flavors after storage [13]. Another application of cellobiose is in beverages. In general, low-sugar or sugar-free soft drinks have a negative sensory profile, a reduced mouth feeling, frequently perceived as unpleasant by-products and/or aftertaste, and an inferior flavor profile compared to sucrose sweetened beverages. Cellobiose has a positive impact on the sensory profile of carbohydrate reduced and other carbohydrate drinks, i.e. drinks, which in addition to cellobiose contain other carbohydrates or a reduced amount of other carbohydrates. It acts as a texturizing agent and thus gives carbohydrate reduced or carbohydrate drinks a better mouth feeling. Moreover, cellobiose masks the off-flavor of sweeteners and thus enables low sugar compositions with an improved flavor profile. Cellobiose was also tested as an ingredient in jams and baked products. Owing to its low sweetness it was particularly suitable to improve mouth feeling of alternative sweetened or sucrose reduced products with surprisingly improved overall sensory profile in the applications. Cellobiose was also suitable to improve the whitening effect when used as fondant and applications thereof [13, 14]. Carbohydrates are traditionally used in meat, sausages, and offal products. In such food preparations carbohydrates have specific functions. These include a sweet taste, support, and control of fermentation processes as a nutrient for the used microbiological cultures, e.g. in raw sausage and ham, intensification of browning and browning taste due to Maillard reactions and effect on water activ- ity (aw-value). However, the carbohydrates commonly used in food preparations have several drawbacks. Glucose, fructose, and sucrose are comparatively quickly metabolized by microorganisms, which is often undesirable because long fer- mentation times are preferred to allow the complex flavors to fully develop and mature. Other carbohydrates such as maltodextrin ferment too slowly or not at all. For many food preparations the conventionally used carbohydrates glucose, fructose, or sucrose are too sweet whereas lactose, which has much lower sweet- ness, has only limited applicability due to the occurrence of lactose intolerance in an estimated 65% of the global population. In products such as Bratwurst, meat loaf, cooked sausage, grilled sausage, fried and grilled minced meat products, and other meat products, cellobiose leads to an accelerated browning reaction (browning intensity and tan color), which is at least as advantageous as the use of lactose or even better. In addition, cellobiose can be used to lower the aw-value of food preparations. The aw-value has a direct impact on the growth of microorgan- isms. Thus, a decrease in water activity increases the shelf life of food preparation. The low sweetness of cellobiose allows the use of higher amounts in the product without impairing the sensory profile. Most importantly, cellobiose gives a pleas- ant taste profile to processed or fermented meat and sausage products, which was significantly favored in sensory panel trails [15]. 170 2.4 Enzymatic Process for the Synthesis of Cellobiose Cellobiose has also been assigned to be prebiotic, by supporting the growth of probiotic microorganism. During digestion of cellobiose the production of health promoting short-chained fatty acids has been observed [16–25]. Feeding studies with pigs by Otsuka et al. [26] further confirmed the prebiotic effect. The weight gain of pigs fed with a diet containing cellobiose was significant higher than the control group fed without cellobiose. The authors postulated that pigs that were fed with cellobiose are healthier and develop more consistently [26]. Because humans are not able to degrade cellobiose since the β1-4 glycosidic bond is resistant toward enzymatic and chemical degradation in the gastroin- testinal tract, cellobiose in food preparations does not affect the blood glucose and insulin level. Internal studies at Pfeifer & Langen GmbH & Co. KG have shown that cellobiose was resistant to gastric acidity, to hydrolysis by intestinal cells, and to gastrointestinal absorption. Additionally, cellobiose was fermented by the intestinal microflora resulting in the induction of beneficial