When I upgraded to 5.4, the devices that are on the VLANs could not access the pihole for DNS lookups. My pihole is on 192.168.1.21. VLAN 50 is 192.168.50.21--device on that VLAN can't get a DNS lookup from 192.168.50.21 Anything on 192.168.1.x can get DNS lookups on 192.168.1.21.
There seems to be a small bug with the interface listening behavior resetting itself (see here). Please try switching from listen on all interfaces to listen on one specific interface and back to listen on all interfaces. Is the issue now fixed?