I think your way to do it with hexane is safe but you will remove mostly triglycerides. Cholesterol, fatty acids and phospholipids will be still in your hydrolysate. Also if you increase temperature - you will increase extraction of lipids. Beside you can try extraction with diethyl ether. You will remove triglycerides, but partially also cholesterol, fatty acids and phospholipids. It's also safe separates on two layers - upper is ether which easy to aspirate. But proteins in you hydrolysate may coagulate.

Also color change presumably because of oxidation and/or extraction of carotinoids and hemoglobin.

Dear Mr. Artamonov:

I really appreciate for your useful suggestion. No wonder it is still high lipid-content in degreased hydrolysates by total lipid analysis. Maybe diethyl ether is much more suitable. Otherwise, by literature review, we assume the bio-active compounds must be dipeptides or tripeptides, so does the coagulation of protein you mentioned influence the structure of peptides? Is it the formation of simply hydrogen bonds?

Your sincerely

Dear Mr. Artamonov:

I am in the same group with Yi-Hsieng, and is very grateful for your explanation and advice. However i am still confused by some problems.

Actually we compared Control group (without lipid removed) with Hexane-extracted group and we found that the color was darker (brown) in Hexane-extracted after lyophilized (but brighter color in Control group). If oxidation took place in both groups, why the colors we found are different?

Your sincerely

To Yi-hsieng: If you don't care about proteins (peptides) in hydrolysate, you can go and try diethyl ether (DE), also you can combine hexane and DE to improve extraction.

To Po: I'm not sure about scheme of your experiment. What is your goal? Could you explain in detail what is control group and what is hexane extracted.

Why don't you remove fat before starting the protein hydrolysis?

To Mr. Artamonov: Actually, we do care about the peptides which may be investing target in this research, whereas we don't care about the lipid and want to completely remove them. Maybe I'll find appropriated way to solve it in some previous studies. After all, It's really thankful for your help.

To Klinkesorn: The aim of this exp. is to develop functional peptides from chicken liver, and we had successfully gotten it as antioxidant, hepatoprotective agent, hypolipidemic agent. But some reviewers argue on the high lipid content in nutraceutical hydrolysates. For your opinion, we had tried centrifugation and hexane-extraction before hydrolysis, but there is no satisfied results. And we also worry that the outcome peptides will lose due to coagulation during DE-extraction. So, it is embarrassing...

To Mr. Artamonov: really sorry for the unclear illustration. Control group: chicken liver hydrolysates; Hexane extracted group: lyophilized chicken liver homogenates with hexane added in order to remove fat. We repeated it until the supernatant almost became transparent (we centrifuged to remove supernatant ). After hexane volatiled, we started to hydrolyze. we're not sure if this process as described is feasible or not... Actually we found a lot of problems during this experimant. First, after we added hexane and then centrifuged and follwed by removing the supernatant (hexane with fat), we found that chicken liver became sticky and the rest of hexane in chicken liver hardly could be removed (After placing in chemical hood several hours, we still could smell hexane, and we wondered if the rest of fat in chicken liver oxidized during process of hexane volatilization). Second, we found that Hexane goup (chicken liver hydrolysates powder) had darker color then Control group. And the TBARS value in Hexane group is higher than control, we thought it could be attibuted by the darker color disturbing the measurements, or it could be caused by oxidation of remained fat in hexane-extracted chicken liver during the process of hexane volatilization. (Our lyophilize cannot bear organic solvent..) We wonder another way or modification for removing fat, and I really want to try centrifugation, just like the way as production of chicken essence.

thanks for kindly givung us suggestions.

To Po. To prevent oxidation you should run experiments under argon ( better but more expensive) or nitrogen (quite cheap). Also constant blowing of gas under continuos mixing of hydrolysate after hexane extraction will help to remove hexane. For same purpose you can try lyophilizing, also in this case your group temperature will be around 0C. Both processes will speed up drying and prevent oxidation.

Dear Mr. Artamonov:

Thanks for your suggestion:)

we will try.

Your sincerely

Well, you must take cognizance of the objective of your work as regards cost. I think running the expt. under any of the 2 mentioned gases will be a bit expensive. After decanting the hexane, sticky liver residue is not unexpected as the structural lipids especially of the membranes must have been extracted that lead to tissue collapse hence the sticky nature. the residual hexane could be removed by air vacuum. I suggest you try this to completely remove the odour.

Mr. Dairo:

Thank you. I think your opinion is practical, and it makes sense for the sticky nature.

In current, we had found the bug through trouble shooting. Because we used the household blender to make chicken liver homogenous, and it is impossible to destroy the structure of cell membrane. The most important thing is the the lipids in healthy liver tissue are intracellular storage, so we will try ultrasonic agitation after homogenesis. I hope it could aim for majorly lipid-removing via centrifugation, which may instead of any chemical extraction methods.

I think the best way to remove lipids from any solution is using the Folch reaction with methanol dichloromethane, then you can separate the liposolube phase after centrifugation and then evaporate the rest to recover all protein

Did you try Bligh and Dyer method for removing fat in fresh liver?

Dear all:

Thank you for providing useful information to me. Embarrassedly, I do not know Folch method, and Bligh & Dyer method either. I had found a paper to follow them up. If there were any further questions, I will consult with you. Thanks again.

Your proteins in hydrolysate will coagulate in either of methods...

The method is rather simple, you just need to blend a sample with a

mixture of dichloromethane–methanol (2:1, v/v) according Folch

et al. (1957). The mixture is vortex and let it rest for hours, and then repeat three times the same extractions. The solvent supernatants will then contain the fat that you wants to remove. If your sample contains water, then the the lower phase

containing lipids (CH2Cl2) and the higher the water soluble phase. After separating the lipid phase you could also determine the amount of total lipids gravimetrically.

To Artamonov: That is a really bad news. I will think twice before trying them.

To Viana: Thank you for your detail method, that's so sweet! If there was something I could help you, please let me know.

The best way is to first freeze-dry your chicken sample and then extract the dried sample with acetone (see Onuh et al., 2013. Food Res. Int. 53: 260-267). If you need a copy of the paper, let me know.

Extrusion could be a solution. It is used to defat soy beans. See 8.2 in the following book link:

www.lamolina.edu.pe/zootecnia/biblioteca2012/Extrusion%20Cooking%20Techniques[1].pdf

Good luck,

Ana

To Rotimi: If you could give me the copy of that paper, it will be great. Thanks a lot.

To Ana: Thanks your suggestion. I have not thought that extrusion will be the solution before.

See attached file.

Thanks a lot