A few semi-random notes (feel free to ignore/discard them partially or totally):
Winimage was born to manage floppy (and floppy-like) images, the functions relative to partitioned media have been added, but there are some cases (just like it can happen with IMDISK) where they don't work as expected.
You have a typo here (hopefully only on the post and not in the menu):
map --mem /Bios/Bios40.img || map /Bios/Bios40.ima (hd0)
should be:
map --mem /Bios/Bios40.img (hd0) || map /Bios/Bios40.ima (hd0)
In the list of the freedos files the kernel.sys is missing?
ONLY as a side note, there is also the possibility of "truncated" images, see these:
http://reboot.pro/in...showtopic=18312
http://reboot.pro/to...n-many-ramdisk/
About making a new image, personally I would use a VM like Qemu or Virtualbox (for this latter using a RAW image wiith the .vmdk descriptor), basically because this way you have the possibility to directly test it (short of actually flashing the BIOS), boot the VM from freedos and from it partition/format the image, this is advised because when we are talking about small images there could be geometry issues.
Otherwise you can use IMDISK (which "save image with MBR" should be working just fine for your simple needs), or use (the updated version of) my good ol' mbrbatch/mkimg batch (intended for XP, but updated for Vista and later) that should work on newer systems, but you would anyway post process it to installl the grub4dos to the MBR (if you are using it that way).
Or use a driver like the Arsenal one (same Author of IMDISK) or the Microsoft one (though I have no idea if it still exists in a version compatible with newer MS OS's.
You can even do it all manually, the small calculations needed for a simple primary partition are not that difficult.
Or you could get rid of the idea of a partitioned image and see if a floppy (actually a super-floppy) works, the "canonical" sizes are 1.44 and 2.88, but larger sizes are well possible.
Loosely in your case could be *something like* this old idea:
http://reboot.pro/to...-to-g4d-images/
but maybe it is overcomplicated for your uses.
Another idea could be - following the suggestion by Steve6375, automating it - to have a "generic" FreeDOS (floppy sized) image (that you can surely map to memory) and in the grub4dos menu.lst add a command to write on-the-fly, before chainloading the kernel.sys, an autoexec.bat pointing to the specific directory where the .bin/.exe/etc. are (in the stick). ;dubbio:
In any case, with your current approach, you have some 50 entries in the menu.lst to choose from, if I get it right, adding to each of them a dd or echo command wouldn't be an issue, but it remains (I believe) prone to error to have to choose among so many entries, maybe you need a multi-level menu, similar to the ones in (say) the supergrubdisk.
Wonko