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Publication New approaches in salami manufacture with in-situ exopolysaccharide-forming starter cultures(2021) Velasco Cucaita, Lina Maria; Weiss, JochenLactic acid bacteria have always been of great importance in the production of fermented sausages such as salami, as they contribute not only to microbial stability but also to acidity and flavor profiles of such products. Recently, exopolysaccharide (EPS)-forming starter cultures have attracted the interest of the food industry. EPS have water-binding, gelling, viscosity-increasing, as well as emulsifying properties and, due to these technofunctionalities, can contribute to the improvement of existing products as well as to new product developments. However, compared to hydrocolloids, which have similar functionalities, in-situ formed EPS do not have to be legally declared as ingredients on a package. Initial studies looking at the use of such cultures in spreadable, short-ripened raw sausages showed that the use of EPS-forming starter cultures can lead to a significant improvement in the spreadability of fat-reduced tea sausage and deeper acidified onion mettwurst (pH 5.1 instead of 5.6). However, no study to date has comprehensively addressed the use of in-situ EPS-forming starter cultures in sliceable, raw fermented sausage products such as salami, which differ significantly from spreadable raw sausage products in terms of product matrix. Since growth kinetics and acidification depend on the microorganism and the food matrix used, the growth and acidification behavior of selected homo- and heteropolysaccharide (HePS)-forming lactic acid bacteria as a function of different sugar concentrations (2.5 - 10 g/kg) was initially investigated. This was done to obtain an indication of the sugar concentration required in the raw sausage mass to achieve a target pH of 4.8-5.3 in the final product. Subsequently, the performance of two HePS-producing strains L. plantarum TMW 1.1478, and 1.25; and the two homopolysaccharide-producing lactic acid bacteria L. curvatus TMW 1.624 and L. sakei TMW 1.411 was investigated in a raw sausage model system (inoculation concentration 106 CFU/g), which, in addition to 25% pork back fat, 75% lean pork meat, also contained ascorbic acid (0.5 g/kg), nitrite curing salt (28 g/kg), and dextrose or sucrose (5 g/kg). Thereby, the strains to be used were specifically analyzed with regard to their suitability for EPS-formation under typical fermentation conditions prior to use in salami production. The latter was done qualitatively by confocal laser microscopy (CLSM), followed by semi-quantitative data interpretation using MATLAB. The results showed that all selected strains were able to produce EPS in the raw sausage model matrix. There, EPS were located on the surfaces of the proteins. Since presence of HePS, which are more complex in terms of chemical structure and are often charged, can lead to changes in the organization of protein matrices even when used in very small amounts due to e.g. electrostatic interactions, sausages were subsequently prepared with a HePS-forming (L. plantarum 1.1478) and a non-EPS-forming starter culture (L. sakei 1.2037; control). Moreover, the influence of different inoculation concentrations (107 and 109 CFU/g) on fermentation and associated HePS-formation, as well as their effect on quality parameters of the final products, were investigated. The selection of inoculation concentrations was governed by the hypothesis that higher inoculation concentrations could lead to a higher in-situ formed HePS amount in the raw sausage matrix and therefore to enhanced structural and thus organoleptic relevant effects. For this purpose, pork meat and fat-based raw sausages were prepared by adding and mixing spices, 0.5 g/kg Na-ascorbate, 5 g/kg sugar, the appropriate starter culture (107-109 CFU/g), and in the end 28 g/kg nitrite curing salt. Afterwards, the mass was filled, fermented (24 °C), smoked, and dried to a weight loss of 31%. In addition to pH and bacterial plate counts, the formed EPS were detected by CLSM and the influence of the formed HePS on the texture of the raw sausages was analyzed by texture profile analysis (at 16, 23, 27, and 31% weight loss) and further evaluated in a sensory evaluation for the attributes of consistency and taste. Although no significant differences were found with respect to the detected HePS and the inoculation concentration used, dependencies emerged with respect to product quality. Raw sausages produced with the HePS-producing starter culture L. plantarum 1.1478 were significantly (p < 0.05) softer than the corresponding control samples. This effect was more pronounced the higher the inoculation concentrations, which was also reflected in the sensory evaluation of samples. Semi-quantitative data interpretation of the CLSM images revealed that the HePS were predominantly formed during the first 72 h of fermentation at 24 °C, until the final pH of 4.95 ± 0.05 was reached. Although there was no clear preference in the sensory analysis performed, raw sausages with a firmer consistency are generally preferred in Germany. Accordingly, the use of an EPS-forming culture could, depending on the market, also have a negative impact on product properties. To gain a better understanding of the observed results and the influence of process conditions on in-situ HePS-formation and its effects on the quality of sliceable raw fermented sausages, the temperatures of the fermentation phase were varied in a further study. In addition to the 24 °C already examined, an additional incubation temperature of 16 °C, commonly used in the production of raw sausages, and a low temperature incubation of 10 °C were chosen, since increased stress conditions are often associated with increased EPS formation. Raw sausages inoculated with L. plantarum 1.1478 or L. sakei 1.2037 (108 CFU/mL) were fermented at 10, 16, or 24 °C within the first 7 days and then dried under the same conditions (14 °C, controlled relative humidity) until a weight loss of 31% was reached. Microbial growth, pH, and weight loss development were monitored, EPS detected with CLSM, and products further characterized by texture profile analysis and a sensory test. Here, texture profile analysis was performed not only from the final product, but also after 21% and 26% weight loss to better understand the influence of the in-situ produced HePS. Differences were found depending on the starter culture used as well as on the fermentation temperature. Products manufactured with the non-EPS-forming strain L. sakei 1.2037 reached the target weight loss of 31% slightly faster than products manufactured with the HePS-former L. plantarum 1.1478. In both products, the final weight loss of 31% was reached faster at an initial fermentation temperature of 24 °C than at the lower fermentation temperatures. A correlation of temperatures with the amount of HePS formed could not be conclusively proven using semi-quantitative data analysis of CLSM images because matrix effects complicated the determination. However, texture profile analysis results showed a difference between products fermented at 24 °C and those fermented at cooler temperatures. In addition, significant (p < 0.05) differences were again observed between products with (softer) and without (harder) HePS-forming starter cultures at weight losses at or above 21%. These results were confirmed in the final sensory evaluation of the products (pH 4.89 - 5.01; 31% weight loss). In summary, the results of this thesis show that the use of a HePS-forming starter culture in sliceable raw fermented sausage can induce specific structural and textural changes. HePS-formation and associated quality attributes may be modulated via the inoculation concentration and control of processing parameters such as fermentation temperature. The texture softening observed in the present work, can be positively or negatively associated with the product depending on the target country and market. Taken together, results of this work underline the importance of a suitable starter culture selection for the production of fermented sausages.