The study examined the effect of Lactobacillus plantarum together with Saccharomyces cerevisiae on cider quality through simultaneous and sequential inoculation strategies to evoke malolactic fermentation. The antioxidant activities and flavor compound profiles of apple ciders fermented with mixed cultures of commercial wine yeast (S. cerevisia SY) and autochthonous bacteria (L. plantarum SCFF107 and L. plantarum SCFF200) were assessed. The antioxidant ability results indicated that apple ciders fermented with the simultaneous inoculation method had a higher DPPH radical scavenging rate and total antioxidant capacity, especially for SIL107 cider (simultaneous inoculation with S. cerevisiae SY and L. plantarum SCFF107), which exhibited the highest DPPH free radical scavenging activity (78.14% ± 0.78%) and the highest total antioxidant ability (255.92 ± 7.68 mmol/L). The results showed that ciders produced by mixed inoculation with L. plantarum improved flavor because of their higher contents of volatiles such as esters and higher alcohols and higher contents of non-volatile compounds like organic acids and polyphenols in comparison with the single culture of S. cerevisiae, especially for the simultaneous inoculation method. In addition, irrespective of the inoculation mode, compared to the single culture of cider, L-malic acid degraded dramatically in the presence of L. plantarum during alcoholic fermentation, accompanied by increases in lactic acid. What is more, sensory evaluation results demonstrated that ciders produced by mixed cultures gained higher scores than ciders fermented by the single culture of S. cerevisiae, especially in the simultaneous inoculation mode, in terms of the floral, fruity, and overall acceptability of the cider. Therefore, our results indicated that simultaneous inoculation with L. plantarum was found to compensate for some enological shortages of single S. cerevisiae fermented ciders, which could be a potential strategy to enhance the quality of cider products.

Cancer immunotherapy has made recent breakthrough, including immune checkpoint blockade (ICB) that inhibits immunosuppressive checkpoints such as programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1). However, most cancer patients do not durably respond to ICB. To predict ICB responses for patient stratification, conventional immunostaining has been used to analyze the PD-L1 expression level on biopsied tumor tissues but has limitations of invasiveness and tumor heterogeneity. Recently, PD-L1 levels on tumor cell exosomes showed the potential to predict ICB response. Here, we developed a non-invasive, sensitive, and fast assay, termed as exosome-hybridization chain reaction (ExoHCR), to analyze tumor cell exosomal PD-L1 levels. First, using αCD63-conjugated magnetic beads, we isolated exosomes from B16F10 melanoma and CT26 colorectal cancer cells that were immunostimulated to generate PD-L1-positive exosomes. Exosomes were then incubated with a conjugate of PD-L1 antibody with an HCR trigger DNA (T), in which one αPD-L1-T conjugate carried multiple copies of T. Next, a pair of metastable fluorophore-labeled hairpin DNA (H1 and H2) were added, allowing T on αPD-L1-T to initiate HCR in situ on bead-conjugated exosome surfaces. By flow cytometric analysis of the resulting beads, relative to αPD-L1-fluorophore conjugates, ExoHCR amplified the fluorescence signal intensities for exosome detection by 3–7 times in B16F10 cells and CT26 cells. Moreover, we validated the biostability of ExoHCR in culture medium supplemented with 50% FBS. These results suggest the potential of ExoHCR for non-invasive, sensitive, and fast PD-L1 exosomal profiling in patient stratification of cancer immunotherapy.

This study explored the effect of the combination of Saccharomyces yeast, non-Saccharomyces yeast (Pichia kudriavzevii), and Lactiplantibacillus plantarum during cider fermentation on physicochemical properties, antioxidant activities, flavor and aroma compounds, as well as sensory qualities. Ciders fermented with the triple mixed-cultures of these three species showed lower acid and alcohol content than those fermented with the single-culture of S. cerevisiae. The antioxidant activities were enhanced by the triple mixed-culture fermentation, giving a higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rate and total antioxidant capacity; specifically, the SPL5 cider showed the highest DPPH radical scavenging rate (77.28%), while the SPL2 gave the highest total antioxidant capacity (39.57 mmol/L). Additionally, the triple mixed-culture fermentation resulted in improved flavor and aroma with a lower acidity (L-malic acid) and higher aroma compounds (Esters), when compared with the single-culture fermented ciders (Saccharomyces cerevisiae); more specifically, the SPL4 cider resulted in the highest total flavor and aroma compounds. In addition, sensory evaluation demonstrated that ciders produced using the triple mixed-cultures gained higher scores than those fermented using the single-culture of S. cerevisiae, giving better floral aroma, fruity flavor, and overall acceptability. Therefore, our results indicated that the triple mixed-cultures (S. cerevisiae, P. kudriavzevii, and L. plantarum) were found to make up some enological shortages of the single S. cerevisiae fermented cider. This study is believed to provide a potential strategy to enhance cider quality and further give a reference for new industrial development protocols for cider fermentation that have better sensory qualities with higher antioxidant properties.

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