Alternatives to Animal Squalene

 
 

As mentioned in previous blogs, many living things produce squalene. This includes animals, plants, yeasts, bacteria, and even algae! Our hope is for companies to transition from shark-derived squalene to alternatively derived squalene, as it is sustainable and environmentally friendly. Some of the leading alternatively derived squalene companies include, but are not limited to:

  • Amyris

  • Wilshire Tech

  • SynShark

  • EFP Biotech

  • Charkit Chemical Company

  • Caribbean Natural Products Inc.

  • Jedwards International, Inc.

  • EKIZ Olive Oil & Soap

  • Clariant

  • Enepret Inc.

Upon initial research, there was a considerable amount of studies identifying the quantity of squalene that different organisms can produce, but it required some digging to come across research actually comparing its efficacy in cosmetics and vaccines compared to shark squalene. In a timely research paper titled, Synthetic Biology-derived triterpenes as efficacious immunomodulating adjuvants, published in 2020, biosythetic squalene producers, Enepret, compared the vaccine efficacy of yeast-derived squalene to the well known shark-derived adjuvant, MF59. The study found that, “there was no difference in antibody titers elicited between animal and plant sourced squalene.” The results of this study conclude that yeast-derived squalene exhibits equivalent physiochemical properties to the current standards (MF59: shark squalene), with no reactogenic effects, and even match the immunological profiles of current shark-derived adjuvants! The scientists at Enepret even go on to say that, “the key component of the MF59 adjuvant, squalene, harvested from shark liver oil is not a desirable source. In addition to ethical and sustainability issues, there is growing concern for the bioaccumulation and biomagnification of persistent organic pollutants (such as polybrominated diphenyl ethers) in shark-derived squalene.”

It is important to note that the team of scientists tested plant-derived squalene as well (olive), finding that it is less stable, and therefore slightly less pure than shark-derived squalene. This does NOT go for biosynthetically-derived squalene like yeasts. In fact, “injection site morphology and serum cytokine levels did not suggest any reactogenic effects of the yeast-derived squalene or novel triterpenes, suggesting their safety in adjuvant formulations. These results support the advantages of yeast produced triterpene oils to include completely controlled growth conditions, just-in-time and scalable production, and the capacity to produce novel triterpenes beyond squalene.”

Recently, biosynthetic squalene manufacturer, Amyris, has shed light on the topic as well:

“We could make enough squalene for a billion vaccines in about a month. That squalene is pure, it’s consistent, it’s stable, and it can meet the specifications or exceed the specifications of any squalene that would come from a shark. And do it consistently every time. I think that’s the benefit (of sugarcane). It’s fast. From a cost perspective, you could think about squalene from sugarcane as about 1/3 of the cost as squalene from sharks. So its cheaper, its faster, sustainable, doesn’t have the same impurities, and its consistent every single time,” said John Melo, CEO of Amyris.

Nevertheless, it is important to describe potential sources of squalene that exist so that companies and organizations know their options and can pursue safe alternatives to shark squalene. 

In this blog, we will get a little more in depth, listing alternative sources of squalene and the amount of squalene they can actually produce. First, a baseline is needed to demonstrate how much squalene is considered a large amount. Deep-sea sharks have the highest concentrations of squalene known. Sharks have approximately 250 mg of squalene/g of liver oil (Budge and Barry 2019), but the quantity of squalene in sharks can vary greatly, some containing almost no squalene (Bakes and Nichols 1995) and others containing liver oil that is upwards of 80% squalene (Bakes and Nichols 1995; Wetherbee and Nichols 2000). With this in mind, listed below are the alternatives. While the alternatives do not produce as much squalene per unit of measurement as sharks, they have the potential to be grown or cultured at much larger scales, compensating for the smaller squalene concentrations. 

Yeasts

 Of all the studied alternatives, yeasts produce some of the largest concentrations of squalene. They can be grown in large quantities and can be very cost-effective.

  • A yeast species called Kluyveromyces lactis is capable growing in cheap lactose-containing industrial waste, such as waste that comes from the dairy industry, making it very cost effective and reducing the overall price of its squalene. Under certain conditions K. lactis was able to produce approximately 12 mg squalene/g dry cell weight, or 600 μg (micrograms) squalene per 109  cells (Drozdíková et al. 2015).

  • Saccharomyces cerevisiae, commonly known as baker’s yeast, can produce over 1000 μg squalene per 109 cells (Garaiová et al. 2014), or approximately 20 mg squalene per gram dry cell weight.

  • Another yeast, Pseudozyma sp., had a maximum squalene production of 70.32 mg/g of dry cell weight (Mi-Hee Chang et al. 2008).

 

Plants (including vegetable oils) 

Both plants and derived vegetable oils are capable of containing noticeable quantities of squalene. Plants are a good alternative to sharks because they can be grown and harvested sustainably in large quantities.

  • Monkey Jack, or Artocarpus lakoocha, is a plant native to Asia that has been found to produce squalene. Squalene was produced at 10-12% of the dry weight of the plant. Additionally, the squalene from its leaves reached a purity of 99.9% and exhibited remarkable antimicrobial activity, fighting off the bacteria E. coli and S. lutea and three species of aspergillosis, a fungus (Biswas and Chakraborty 2013).

  • There are many different vegetable oils containing squalene (Pokkanta et al. 2019):

    • Olive oil: up to 8.39 ± 0.15 mg/g 

    • Rice bran oil: 3.189 mg/g 

    • Red peanut oil: 1.343 mg/g 

    • Peanut oil: 1.329 mg/g 

    • White sesame oil: 0.607 mg/g 

    • Black sesame oil: 0.573 mg/g

    • Coriander seed oil: 0.451 mg/g 

    • Soybean oil: 0.184 mg/g 

    • Sunflower oil: 0.144 mg/g

 

Bacteria

Squalene was produced under various conditions in both E. coli (Ghimire et al. 2009) and a Synechocystis species named PCC 6803 (Englund et al. 2014). However, E. coli was able to produce 11.8 mg squalene/L (Ghimire et al. 2009), a concentration nearly 18 times that of PCC 6803.

Stramenopiles (Including Algae) 

  • Aurantiochytrium species 18w-13a is a single-cell marine organism that was able to accumulate a whopping 198 mg squalene/g of dry cell weight (Kaya et al. 2011). This is the highest concentration of squalene produced in any of the researched alternatives.

  • Chlamydomonas reinhardtii is a single-cell green alga that was able to produce 0.9-1.1 mg squalene/g of dry cell weight (Kajikawa et al. 2015).

There are many alternatives that can produce comparable concentrations of squalene to sharks, and that are sustainable and environmentally friendly. Aurantiochytrium species 18w-13a and Pseudozyma sp. produced the most squalene of any of the alternative sources. Other alternatives can be beneficial, however, as some can be grown at a low cost while others can produce high-purity squalene. Increased demand for alternatively derived squalene in your products can drive down the cost of this multi-use ingredient.

Laurel Irvine