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I'm making tiramisu. Coffee is a large component of the recipe, so I imagine that the choice and preparation of the coffee used will have a big impact on the final taste.

Or will it? I'm not a coffee expert; perhaps all the sugar and other ingredients will overwhelm it to the degree that you can't taste the difference. If that's so, using fancy coffee would just be a waste.

Will there be a noticeable taste difference if I just use instant coffee to soak the ladyfingers in, or is it worth using something of higher quality? If the latter, how should I prepare the coffee that is to be used?

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Someone will be able to notice. In matters of taste, there is always variation. Some people can't taste the difference between stale Folgers and fresh high quality coffee. However, many can.

I the case of your dessert, without more data, I can't tell if you would be able to tell or not. But, for sure, someone would be able to tell. That being the case, it's worth doing with the best ingredients you can reasonably use.

My personal opinion for my cooking is any ingredient that isn't high enough quality to stand on it's own shouldn't get used.

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This does depend substantially on your standards: how sensitive are you to subtle flavors? It also depends on the exact recipe: how much coffee liqueur, how much cocoa powder, and so on.

The difference between instant and real espresso is pretty large, enough that I'd expect the nice flavor notes of real espresso to come through. And you'll certainly notice the difference between coffee and the concentrated flavor of espresso. But noticing details of those flavors, with everything else going on in tiramisu, will probably be tough. The coffee liqueur especially is going to make it hard to notice details; you'll essentially be tasting the combination of its coffee flavor and your espresso as a single thing.

So my very rough answer would be that it's definitely worth using real espresso if you can, but it's most likely not worth obsessing too much over the quality, and definitely not worth obsessing about the exact method of preparation. Keep in mind that tiramisu calls specifically for espresso, not just coffee. Strongly-brewed coffee, like from a moka pot, will work well too. But if all you have is a regular coffee maker, you're probably better off using instant espresso powder, or running out to buy some espresso at a coffee shop.

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Use a med-roast espresso blend and brew it using a Moka pot (stovetop espresso maker).

You're going to buy expensive ingredients for this dessert. Don't skimp on the coffee, it is by far the most aggressive flavor in the dish.

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