Extracellular recombinant production of 4,6 and 4,3 α-glucanotransferases in Lactococcus lactis.

4,6 α-Glucanotransferase (4,6-α-GTase) and 4,3 α-glucanotransferases (4,3-α-GTase) produced by Lactic Acid Bacteria (LAB) in the GH70 enzyme family have become important due to their catalytic effect on starch and maltodextrins. Their high level of production is necessary for their application at industrial scale. In this respect, both enzymes were expressed extracellularly using Lactococcus lactis as GRAS host. 4,6-α-GTase and 4,3-α-GTase genes from Limosilactobacillus reuteri E81 and Limosilactobacillus fermentum PFC282 respectively were transformed into the plasmid pLEB124 vector having the signal peptide usp45 under the P45 continuous promoter and successfully expressed in Lactococcus lactis MG1363. Western blot screening showed that the relevant enzymes were able to be successfully secreted extracellularly. The Vmax and Km of 4,6-α-GTase were 2.58 µmol min-1 and 0054 mg min-1 whereas 3369 µmol min-1 and 0032 mg min-1 for 4,3-α-GTase respectively. NMR analysis demonstrated the formation of new bonds within the corresponding enzymes. Also, both enzymes were active on maltose, maltoheptaose, maltohexaose and starch and produced malto-oligosaccarides observed by TLC analysis. In conclusion, this study demonstrated first time the extracellular production of 4,6-α-GTase and 4,3-α-GTase with GRAS status that can be useful for starch retrogradation delay and glycaemic index reduction.

Occurrence, distribution and contamination levels of heat-resistant moulds throughout the processing of pasteurized high-acid fruit products.

Heat-resistant moulds (HRMs) are well known for their ability to survive pasteurization and spoil high-acid food products, which is of great concern for processors of fruit-based products worldwide. Whilst the majority of the studies on HRMs over the last decades have addressed their inactivation, few data are currently available regarding their contamination levels in fruit and fruit-based products. Thus, this study aimed to quantify and identify heat-resistant fungal ascospores from samples collected throughout the processing of pasteurized high-acid fruit products. In addition, an assessment on the effect of processing on the contamination levels of HRMs in these products was carried out. A total of 332 samples from 111 batches were analyzed from three processing plants (=three processing lines): strawberry puree (n = 88, Belgium), concentrated orange juice (n = 90, Brazil) and apple puree (n = 154, the Netherlands). HRMs were detected in 96.4% (107/111) of the batches and 59.3% (197/332) of the analyzed samples. HRMs were present in 90.9% of the samples from the strawberry puree processing line (1-215 ascospores/100 g), 46.7% of the samples from the orange juice processing line (1-200 ascospores/100 g) and 48.7% of samples from the apple puree processing line (1-84 ascospores/100 g). Despite the high occurrence, the majority (76.8%, 255/332) of the samples were either not contaminated or presented low levels of HRMs (<10 ascospores/100 g). For both strawberry puree and concentrated orange juice, processing had no statistically significant effect on the levels of HRMs (p > 0.05). On the contrary, a significant reduction (p < 0.05) in HRMs levels was observed during the processing of apple puree. Twelve species were identified belonging to four genera - Byssochlamys, Aspergillus with Neosartorya-type ascospores, Talaromyces and Rasamsonia. N. fumigata (23.6%), N. fischeri (19.1%) and B. nivea (5.5%) were the predominant species in pasteurized products. The quantitative data (contamination levels of HRMs) were fitted to exponential distributions and will ultimately be included as input to spoilage risk assessment models which would allow better control of the spoilage of heat treated fruit products caused by heat-resistant moulds.