in real time. Analysis of PCR reactions was performed on 2% agarose gels. Densitometry (% and ng 160 values) was calculated from the imaged PCR gels using a GS-900 densitometer and Image Lab 161 software (Version 6.1, Bio-Rad). Bands were gel extracted (Qiagen Qiaquick Gel Extaction Kit) and 162 sequenced to confirm species identity. ZymoBiomics Gut Microbiome Standard (negative control) 163 was used to demonstrate lack of nonspecific amplification. 164
165
2.4
Biomarker Analysis
Serum SCFA levels were measured by GC-MS at Creative Proteomics (Shirley, NY). The following 166 SCFAs were analyzed: acetic acid (C2:0), propionic acid (C3:0), butyric acid (C4:0), isobutyric acid 167 (C4:0i), valeric acid (C5:0); isovaleric acid (C5:0i), hexanoic acid (C6:0). Free short chain fatty acids 168 were derivatized using methyl chloroformate in 1-propanol yielding propyl esters before subsequent 169 liquid-liquid extraction into hexane and analysis on a SLB- 5ms (30x0.25x1.0μm) (Supelco) column 170 and detection using GC-EI-MS in SIM-mode. Instrumental analysis was performed on an Agilent 171 7890 GC coupled to an Agilent 5977 MSD (Agilent Technologies). Quantification was performed 172 against a 5-point calibration curve. 173
174
3
Results
3.1 Oral supplementation with probiotic blend is safe for human consumption 175
Forty total participants were screened to randomize 22 participants with asthma (n=11) or were 176 healthy (n=11) who met study eligibility criteria (Table 1, Figure 1). Smoking status was noted 177 (healthy smokers n=3, asthmatic smokers n=4) because of its well-documented impact on lung health 178 and susceptibility to disease. Three asthmatic participants also reported having a history of allergic 179 rhinitis. Participants who met the eligibility criteria and successfully completed the Screening Visit 180 were enrolled in the trial. Subjects took the probiotic blend twice a day for 4 weeks, and assessments 181 were conducted at Baseline and the end of Week 2 and Week 4. 182 No SAEs or withdrawals due to AEs occurred during this study in the healthy and asthmatic 183 populations, successfully satisfying our primary endpoint (Table 2). There was only one mild 184 gastrointestinal complaint of bloating, which required no action, reported during the trial 185 (Supplemental Table 1). Individual results for vital signs and safety blood parameters were clinically 186 reviewed by the medical doctor and deemed to be safe at all timepoints across all participants 187 (Supplemental Tables 2-3). No participants reported changes in their alcohol habits during the course 188 of the study. 189
190
3.2 Probiotic blend improves lung function in asthmatic subjects
Spirometry measurements were taken at Baseline and Week 4 to assess changes in lung function. 191 Data was normally distributed as measured by D’Agostino & Pearson test of normality: FEV1 192 (K2=1.097, P=0.5778), FVC (K2=0.4601, P=0.7945). In asthmatic participants, average FEV1% 193 increased significantly ( P =0.018) and FVC trended up ( P =0.082) from Baseline to Week 4 (Table 3). 194 The healthy population did not see a change in FEV1% ( P =0.099) or FVC ( P =0.387), and neither 195 group had a significant improvement in FEV/FVC ratio (healthy P =0.113, asthma P =0.284). Three 196 patients showed a decrease in average FEV1 or FVC, but the change was <10% (Supplemental Table 197 4). 198
3.3 Probiotic blend improves quality of life scores as measured by SGRQ 199
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