Previously, polyunsaturated fatty acids (PUFA) from linseed oil were effectively protected (>80%) against biohydrogenation through polyphenol-oxidase-mediated protein crosslinking of an emulsion, prepared with polyphenol oxidase (PPO) extract from potato tuber peelings. However, until now, emulsions of only 2 wt% oil have been successfully protected, which implies serious limitations both from a research perspective (e.g. in vivo trials) as well as for further upscaling toward practical applications. Therefore, the aim of this study was to increase the oil/PPO ratio. In the original protocol, the PPO extract served both an emulsifying function as well as a crosslinking function. Here, it was first evaluated whether alternative protein sources could replace the emulsifying function of the PPO extract, with addition of PPO extract and 4-methylcatechol (4MC) to induce crosslinking after emulsion preparation. This approach was then further used to evaluate protection of emulsions with higher oil content. Five candidate emulsifiers (soy glycinin, gelatin, whey protein isolate (WPI), bovine serum albumin and sodium caseinate) were used to prepare 10 wt% oil emulsions, which were diluted five times (w/w) with PPO extract (experiment 1). As a positive control, 2 wt% oil emulsions were prepared directly with PPO extract according to the original protocol. Further, emulsions of 2, 4, 6, 8 and 10 wt% oil were prepared, with 80 wt% PPO extract (experiment 2), or with 90, 80, 70, 60 and 50 wt% PPO extract, respectively (experiment 3) starting from WPI-stabilized emulsions. Enzymatic crosslinking was induced by 24-h incubation with 4MC. Ruminal protection efficiency was evaluated by 24-h in vitro batch simulation of the rumen metabolism. In experiment 1, protection efficiencies were equal or higher than the control (85.5% to 92.5% v. 81.3%). In both experiments 2 and 3, high protection efficiencies (>80%) were achieved, except for emulsions containing 10 wt% oil emulsions (<50% protection), which showed oiling-off after enzymatic crosslinking. This study demonstrated that alternative emulsifier proteins can be used in combination with PPO extract to protect emulsified PUFA-rich oils against ruminal biohydrogenation. By applying the new protocol, 6.5 times less PPO extract was required.