Dear Jeremia,

No. First you should make your feed pellets with less fish oil than you have planned to have in the final feed (part of the fish oil will be added later).

You have to know how much probiotic bacteria you are adding into the feed so you can culture it normally in liquid growth medium and measure optical density (OD600) of the culture with spectrophotometer. Then you should make a serial dilution from your culture and plate it on solid growth medium. Then you can evaluate what OD600 of your probiotic culture should be that you have suitable bacterial density for your feed. You may want to repeat this few times to be sure how fast your probiotic reaches wanted bacterial density.

When you have suitable bacterial density (or a bit more than what you actually need) you should centrifuge your bacterial cells and remove the growth medium and wash the cells few times with PBS or 0.9% NaCl and suspend the cells to PBS or 0.9% NaCl so that you will have wanted bacterial density. You may want to check the final bacterial density by plating the dilution series made from the suspension.

To add your bacterial on into your feed you can use for example spray bottle (the kind of you most likely use for 70% ethanol, http://www.opticsplanet.com/control-company-vwr-adjustable-spray-bottle-3345.html).

First spread you suitable and weighed amount of pellets on tray or plastic so that they are monolayer. Then by using fine mist spray (you have adjusted and tested the spray nozzle beforehand) you can add wanted amount of bacteria solution onto feed pellets. You also must know how many ml of your bacterial suspension you are adding, so you can use sterile 50ml centrifuge tube instead of container part of spray bottle. It is essential that you can spray the bacteria evenly on the pellets. Let the pellets dry few minutes and transfer them into food mixer and turn it on. Then add slowly wanted volume of fish oil and continue mixing for few additional minutes.

Make the serial dilution from your probiotic feed and plate on solid growth medium to check probiotic bacteria level in feed.

So there is several work stages which you have to optimize beforehand, but in the end you should have routine method for making your own probiotic feed for your feeding trial.

Dear Jeremia,

If you are using live probiotic bacteria, you cannot add the bacteria prior extrudation. However, you can add it afterwards by spraying the probiotic for example in physiological saline solution on the feed and coating the pellets by additional fish oil. This decreases the loss of probiotic bacteria in water before fish ingests the pellet. Oil coating naturally increases the fat content of the feed, so you may want to start with pellets which contain less fat in basic formula. For our feeding trial we performed the coating with Kenwood food mixer.

Dear Sir Jouni,

Thanks for the advice.

So, in the first time incubation or for starter media, we should use oil-base media?

Because for the application, we use fish oil as a carrier of bacteria.

Dear Jeremia,

No. First you should make your feed pellets with less fish oil than you have planned to have in the final feed (part of the fish oil will be added later).

You have to know how much probiotic bacteria you are adding into the feed so you can culture it normally in liquid growth medium and measure optical density (OD600) of the culture with spectrophotometer. Then you should make a serial dilution from your culture and plate it on solid growth medium. Then you can evaluate what OD600 of your probiotic culture should be that you have suitable bacterial density for your feed. You may want to repeat this few times to be sure how fast your probiotic reaches wanted bacterial density.

When you have suitable bacterial density (or a bit more than what you actually need) you should centrifuge your bacterial cells and remove the growth medium and wash the cells few times with PBS or 0.9% NaCl and suspend the cells to PBS or 0.9% NaCl so that you will have wanted bacterial density. You may want to check the final bacterial density by plating the dilution series made from the suspension.

To add your bacterial on into your feed you can use for example spray bottle (the kind of you most likely use for 70% ethanol, http://www.opticsplanet.com/control-company-vwr-adjustable-spray-bottle-3345.html).

First spread you suitable and weighed amount of pellets on tray or plastic so that they are monolayer. Then by using fine mist spray (you have adjusted and tested the spray nozzle beforehand) you can add wanted amount of bacteria solution onto feed pellets. You also must know how many ml of your bacterial suspension you are adding, so you can use sterile 50ml centrifuge tube instead of container part of spray bottle. It is essential that you can spray the bacteria evenly on the pellets. Let the pellets dry few minutes and transfer them into food mixer and turn it on. Then add slowly wanted volume of fish oil and continue mixing for few additional minutes.

Make the serial dilution from your probiotic feed and plate on solid growth medium to check probiotic bacteria level in feed.

So there is several work stages which you have to optimize beforehand, but in the end you should have routine method for making your own probiotic feed for your feeding trial.

Dear Sir Jouni,

So, the media for spray is PBS or NaCl?

May i can mix probiotic with fish oil after centrifuge?

Thanks

Yes, spraying media is NaCl or PBS, since they do not harm the bacterial cells or affect palatability of the feed when used in low % amounts.

I suppose probiotic could be mixed also with oil, but in that case spraying may not be the best possible choice, since oil, most likely, would not be spread evenly on the pellets. In the case you want to use oil-probiotic mixture, then the mixture could be added with food mixer as described above. However, loss of probiotic bacteria in the water may be greater in this method.

thanks for the advice Sir Jouni...

I'll try your advice

are you work with probiotic too, Sir?

Jeremia, I used to work with fish probiotics but nowadays I work with mostly with production animal feed additive issues

How nice i meet you Sir Jouni,

i have tried to seek the advantages of using bacteria for feedfish. :D

allow me to asking more question, Sir.

I would like to use probiotics for shrimp and fish from freshwater and saltwater. What genus of bacterias that i could use for?

Thanks

That's quite broad field to cover. I've worked only with salmonids so I'm not familiar with shrimp or warm water fish species. In general, you should decide what fish /shrimp species you want to concentrate your attention, what aspects you want enhance (immune parameters, growth parameters, disease resistance, intestinal microbiota, etc). After that, you can check from publications what probiotic species have been tested for that purpose and what kind of results they have achieved. You may want to try to isolate the candidate probiotic strain from your own culture conditions. The advantage of using your own isolate is that the probiotic strain is at least adapted to your culture conditions. There are several publications about screening of probiotic candidates which help you to proceed in your task.

umm, yes Sir, thanks for the advice...

honeslty Sir, i have ordered to make a probiotic concortium and let it pass extruder with high level heat. I know my bacteria will colapse all. I just think about liquid encapsulation to cover my bacteria when in extruder.

once more Sir, PBS addition to fish oil doesn't affect fish oil itself? i am afraid this addition can make an oxidation process and increasing free fatic acid of fish oil?

If you're ordred (by your supervisor?) to add the bacteria in basal diet and pass through the extruder, you could use freeze dried bacteria. Check Panigrahi et al. 2005 in Aquaculture, volume 243, pages 241-254. It nicely compares the effects of addition of live sprayed, freeze dried or heat killed bacteria on immune response in rainbow trout.