Nutritional Analysis of Bottarga and Pilot Study Protocol for Bottarga Supplementation in Individuals with Prediabetes

Abstract

Background: Bottarga is a nutrient-dense, marine (“blue”) food produced through sustainable practices. Despite its rich nutritional profile, no clinical studies have investigated its potential health benefits in humans. This study presents a comprehensive nutritional analysis of a commercially available Greek bottarga and outlines the protocol for a pilot clinical investigation to assess its metabolic effects. Methods: The lipid composition of bottarga was analyzed using proton and carbon nuclear magnetic resonance spectroscopy. The clinical protocol consists of two phases: aim 1 is a single-arm, open-label, dose-confirmation study in five overweight and prediabetic adults evaluating the effects of daily bottarga supplementation (20 g/day) over six weeks on metabolic markers; aim 2 is a randomized, open-label, controlled, cross-over pilot study involving 20 overweight and prediabetic participants. Each participant will receive either bottarga or an isocaloric dairy comparator for eight weeks, separated by a two-week washout period. The primary outcome will be selected based on the most clinically relevant findings from Aim 1. Results: According to our nutritional analysis, wax esters are the predominant lipid class in the product, followed by triacylglycerols and free fatty acids. We expect bottarga supplementation to be associated with more beneficial metabolic changes compared to baseline measures and to the calorically equivalent comparator food. Conclusions: This study will provide the first clinical data on the metabolic effects of bottarga in humans, potentially supporting it as a functional food for cardiometabolic health.

1. Introduction

World population growth and increasing animal product consumption have heightened interest in shifting to more sustainable food systems with lower environmental footprints [1]. Food systems contribute to one-third of global greenhouse gas emissions [2], whereas fish production accounts for only 4% of total anthropogenic emissions [3]. “Blue foods”—marine-derived foodstuffs such as fish, mollusks, and aquatic plants—offer excellent alternatives for enhancing global nutrition. These foods have outstanding nutritional profiles, providing proteins along with essential minerals, vitamins, and beneficial fatty acids, while being more sustainable than their land-based counterparts [4]. Innovative cultivation and production technologies have supported healthier and more sustainable diets in various regions [5,6] and could contribute to reducing global hunger [7]. Additionally, increasing blue food consumption can help decrease red meat intake, benefiting both human and planetary health [5].

Another beneficial strategy for sustainability is reducing food waste [8]. Repurposing seafood by-products mitigates environmental impact and prevents the loss of vital nutrients from the food chain [9,10]. For example, undersized or unsalable fish, as well as fish parts not typically destined for human consumption (e.g., viscera, heads, fins), can be used in animal feed and aquaculture, promoting more sustainable agriculture and aquaculture practices [11]. Moreover, some of these so-called waste products, such as fish roe, can be processed into nutritious foods that are regularly consumed in various parts of the world.

Mugil cephalus, a species of mullet, is currently harvested and consumed in the U.S. (Florida, North Carolina) and Europe (Greece, Italy, etc.), and is also well-suited for sustaining populations in developing countries. The roe (egg sac) of this species is used to produce highly nutritious delicacies such as bottarga (Italy) and avgotaracho (Greece), contributing to environmental sustainability [12,13]. Mullet bottarga, sourced from marine or freshwater environments [14], is rich in bioavailable omega-3 (n-3) polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) and eicosatetraenoic acid (EPA) [15], as well as other essential nutrients [16]. Westernized dietary patterns are typically characterized by high intakes of omega-6 (n-6) fatty acids, leading to an elevated n-6:n-3 ratio, which has been associated with an increased risk of inflammatory conditions such as obesity and related metabolic disorders. Current evidence suggests that an optimal n-6:n-3 ratio should range between 1:1 and 5:1 to support metabolic and cardiovascular health [17,18].

Limited animal and in vitro studies (cell lines/cultures) suggest several potential health benefits associated with bottarga consumption, including increased plasma and tissue levels of n-3 polyunsaturated fatty acids [19], possible favorable activity against certain cancer cells [20,21,22], and antithrombotic potential [16]. However, no human clinical studies have been published to date. Given its rich marine omega-3 content, it is reasonable to hypothesize that bottarga may positively impact blood pressure, lipid profiles, inflammation, and possibly insulin resistance [23,24,25,26].

Prediabetes—a condition characterized by impaired glucose metabolism and heightened cardiometabolic risk—affects over one-third of U.S. adults [27] and is considered a key window for preventive intervention. Individuals with prediabetes are at increased risk of progressing to type 2 diabetes, cardiovascular disease, and other chronic conditions. Dietary strategies that can improve metabolic parameters such as lipid profiles, inflammation, and insulin sensitivity are crucial in this high-risk group. Given bottarga’s dense nutrient profile and marine lipid content, it is a promising but underexplored candidate as a functional dietary intervention in individuals with prediabetes.

The goals of this article are twofold: (1) to present the results of lipid analysis of a commercially available Greek bottarga sample and (2) to describe the protocol for a pilot bottarga supplementation study in individuals with prediabetes and obesity. The specific aims of this clinical pilot study are outlined below:

Specific aim 1 is a dose-confirming (open-label, single-arm). The goal is to confirm that a “clinically” recommended daily dose of bottarga (grey mullet fish roe) is associated with beneficial metabolic effects in five participants with overweight and pre-diabetes, comparing metrics before and after daily bottarga consumption (approximately 20 g/day) for 6 weeks.

Hypothesis is that bottarga supplementation is associated with improvements in lipid profiles and reductions in inflammatory and other cardiometabolic markers compared to baseline measures.

Specific aim 2 is a randomized, controlled, cross-over (open label, two-arm) pilot study. The goal is to investigate the metabolic effects of daily bottarga supplementation (versus consumption of a calorically equal dairy product) in 20 participants with overweight and prediabetes, examining intra-participant metabolic changes before and after 8 weeks of each treatment with a 2-week “washout” period between the two supplementation conditions. The primary study outcome will be determined based on the most clinically important results observed during aim 1.

Hypothesis is that 8 weeks of bottarga supplementation will lead to more beneficial changes in lipid profiles, inflammatory markers, and other cardiometabolic markers compared to 8 weeks of calorically equivalent cream cheese supplementation.

2. Materials and Methods

2.1. Lipid Analysis and Nutritional Profile of a Commercial Bottarga Sample

Commercial, processed mullet roe products (wax-covered, dried, and salted Mugil cephalus roe, known as avgotaracho), manufactured in 2024 by the Trikalinos company in Athens, Greece, were analyzed at the Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, National and Kapodistrian University of Athens. In addition to the laboratory-based lipid analysis, which focused on determining the detailed fatty acid composition and total lipid content, we also present the nutritional information as declared on the product’s packaging label for consumers.

2.1.1. Lipid Extraction

Lipids were extracted from mullet avgotaracho by the Folch et al. [28] procedure. An aliquot of total lipid extract recovered from the lower chloroform phase was analyzed by ¹H and ¹³C NMR.

2.1.2. NMR Analysis

The extracted lipids (10 or 100 mg for the ¹H or ¹³C-NMR measurements, respectively) were dissolved in 0.6 mL of CDCl₃. All NMR experiments were performed at 298 K on a Bruker AVANCE III (Bruker Corporation, Billerica, MA, USA) spectrometer equipped with a BBI 5 mm probe operating at 400.13 MHz for ¹H and 100.03 MHz for ¹³C (Bruker Corporation, Billerica, MA, USA). The spectra were processed using Topspin (Bruker Corporation, Billerica, MA, USA) and Mestrenova (Mestrelab Research S.L., Santiago de Compostela, Spain) software.

2.2. Pilot Study Sample

Aim 1: We will recruit 5 local adults with prediabetes and overweight from Massachusetts, aged 18–60 years, who are otherwise healthy, not pregnant, and interested in a nutritional supplement through clinical colleagues and flyers at local clinical sites and graduate schools. Further inclusion criteria will include confirmation of hemoglobin A1c (HbA1c) > 5.7–6.9% and body mass index (BMI) > 27 kg/m². Exclusion criteria will include the use of any medications for diabetes, dyslipidemia, or immunosuppression; current use of any supplements containing n-3 fatty acids; current use of tobacco/nicotine products, marijuana, or illicit drugs; self-reported consumption of a diet rich in plant- or marine-derived fats (e.g., fatty fish, nuts, seeds, or olive oil); use of hormone therapy (except oral contraceptives); known allergies to fish, seafood, or any fish-derived products, including bottarga; pregnancy; and clinical evidence or history of cardiac, pulmonary, hepatic, or renal insufficiency, immunodeficiency conditions, or current non-skin cancer. Additionally, individuals participating in other clinical research studies will be excluded from this study. Via interviews with individuals expressing interest, we will obtain informed consent for participation.

Aim 2: We will recruit 20 local adults with overweight and prediabetes from Massachusetts using the same inclusion and exclusion criteria outlined in aim 1.

This study will be conducted in accordance with the Declaration of Helsinki and has been approved by the Institutional Review Board of the Cambridge Health Alliance (CHA-IRB-24-25-346, 13 March 2025). The trial has been registered on ClinicalTrials.gov (NCT06988462).

2.3. Study Screening Assessment

Participants who express interest in this study will receive an email with a link to an online screening survey, administered via the Cambridge Health Alliance (CHA) Research Electronic Data Capture (REDCap) platform. The screening survey will assess eligibility based on the inclusion and exclusion criteria for both aim 1 and aim 2.

2.4. Baseline Clinical Eligibility Assessment

Screened participants who meet the initial eligibility criteria will undergo a clinical eligibility in person assessment at the Cambridge Health Alliance’s Occupational Medicine clinic until five eligible and willing participants are identified for the dose-confirming study (aim 1), and 20 eligible and willing participants are enrolled for the randomized, controlled, cross-over pilot study (aim 2).

2.5. Bottarga Supplementation

The bottarga used in this study is sourced from the Messolonghi-Aitolikon lagoons in Greece, where ten fishing cooperatives employ sustainable, traditional methods to create semi-enclosed, low-intensity fish-farming areas, often referred to as “natural fish farms.” In these lagoons, mullet are fed naturally in the ecosystem without supplementation with grain or artificial feed. Known as “avgotaracho,” Greek bottarga is a sustainably produced, semi-dried mullet roe and is recognized as a traditional marine food product with a Protected Designation of Origin (PDO). It is nutrient-rich and has been noted for its potential nutraceutical properties. Participants will receive prepackaged daily doses of bottarga, each containing the required amount of 20 g per day, provided by Trikalinos Co., (Athens, Greece), Participants will be encouraged to maintain the same dietary habits practiced prior to supplementation.

Aim 1, open-label, single-arm study: The first five eligible participants will be enrolled in the open-label study involving the daily consumption of bottarga for six weeks, following the completion of online informed consent via the CHA REDCap platform. Participants will be provided with a three-week supply of bottarga (20 g per day) at the start of the intervention. Weekly check-ins will be conducted via phone or Google Meet during the first two weeks. At Week 3, participants will receive an additional three-week supply of bottarga, and a follow-up clinic visit will be scheduled for Week 6 (Figure 1).

Aim 2, randomized, controlled, cross-over (open label, two-arm) pilot study:

Following the initial clinical evaluation, the first 20 eligible participants will be randomly allocated to one of two treatments in a sequential fashion: the “Control” (usual diet + 28 g cream cheese/day) or the “Bottarga Supplementation” (usual diet + 20 g bottarga/day) for 8 weeks. Telephonic check-ins and supplement supplies will follow the same procedures as described for aim 1. At the end of the 8-week period, participants will attend an in-person follow-up exam. Afterward, participants will undergo a 2-week washout period, during which they will follow their usual diet and refrain from taking any supplements. An in-person clinical assessment will be conducted at the end of the washout period. Following the washout, participants will crossover to the supplementation/treatment they did not receive during the first 8 weeks (i.e., the initial control group will receive bottarga supplementation, and the initial bottarga group will receive cream cheese). This second 8-week supplementation phase will follow the same procedures for telephonic check-ins and supplement supplies. After the second 8-week phase, participants will undergo a final in-person clinical assessment.

During control supplementation participants will be provided cream cheese and instructed to consume 2 tablespoons daily, ensuring a similar caloric intake to that of those receiving bottarga supplementation to prevent any bias in the outcomes. Specific instructions for daily consumption will be provided (Figure 2). The nutritional composition of the cream cheese is presented in

Table 1. Nutrition facts of cream cheese (product label).

Nutrition Facts
	Cream cheese (28 g)
Calories (kcal)	70
Fat (g)	6.5
of which saturated	3.6
of which monounsaturated	1.6
of which polyunsaturated	0.3
Trans fat (g)	0
Carbohydrates (g)	1
of which sugars	1
Dietary fibers (g)	0
Protein (g)	2
Salt (mg)	300
Sodium (mg)	120
Cholesterol (mg)	20

.

2.6. Study Measurements

At baseline and at follow-up visits, the participants will complete the following measures, as summarized in the text below for both aims 1 and 2 (

Table 2. Study measurements.

Measures and Timing	Study Phase
	Phase 1 (Dose-Confirming Study)		Phase 2 (Randomized, Controlled, Cross-Over Pilot Study)
	Baseline	6 Weeks	Baseline	8 weeks	Washout Period	8 Weeks
Dietary questionnaires to evaluate n-3 fatty acids and olive oil intake [Mediterranean Diet Adherence Screener (MEDAS)]	X	X	X	X	X	X
Anthropometrics/Body composition weight, height, waist and hip circumference, bioelectrical impedance analysis (BIA)	X	X	X	X	X	X
Resting blood pressure and heart rate	X	X	X	X	X	X
Biochemical measures (fasting glucose, liver function tests, hemoglobin A1C, hs-CRP, lipid profile)	X	X	X	X	X	X

). Experienced clinic staff performing the evaluations will be blinded to participants’ intervention allocations.

2.6.1. Sociodemographic Data and Mediterranean Diet Adherence Screener Score (MEDAS)

Sociodemographic characteristics (i.e., age, sex, race, and educational level) will be collected using an online questionnaire administered via REDCap. The MEDAS score [29] will also be obtained online to assess self-reported adherence to a diet rich in plant- and marine-derived fats (e.g., fatty fish, nuts, seeds, olive oil). Individuals with high adherence to the Mediterranean diet (MEDAS ≥ 9) will be excluded from this study.

2.6.2. Fasting Blood Draws

Participants will be asked to abstain from all caloric intake after 9 p.m. on nights prior to clinic visits and arrive in fasting condition. Water intake is encouraged and regular medications should be taken. Biochemical measures will include: fasting glucose, liver function tests (total protein, albumin, total bilirubin, direct bilirubin, indirect bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase), hemoglobin A1C, high-sensitivity C-reactive protein (hs-CRP), and a lipid profile.

2.6.3. Blood Pressure and Heart Rate

After at least 5 min resting in a seated position, an average of three readings (each taken in 1 min intervals) will be obtained using automated/calibrated sphygmomanometers (10 series, Omron, Kyoto, Japan or similar) following the joint American College of Cardiology/American Heart Association guidelines.

2.6.4. Anthropometrics/Body Composition

Measurements will be conducted in a private exam room, with participants wearing shorts or underwear and no shoes. Height will be measured using a clinical stadiometer (to the nearest 0.5 inch) (Portable Stadiometer 213, SECA, Hamburg, Germany). Body weight, BMI, lean mass, and percent body fat will be assessed using bioelectrical impedance analysis (BIA) (BC-418 Segmental Body Composition Analyzer, Tanita, Tokyo, Japan, or a comparable device).

2.6.5. Waist (WC) and Hip Circumference (HC)

Measurements will be taken using a non-stretchable clinical measuring tape. Waist circumference (WC) will be measured at the midpoint between the lowest rib and the top of the iliac crest, following a normal expiration. Hip circumference (HC) will be measured around the widest portion of the buttocks, with the tape held parallel to the floor. Each measurement will be taken twice; if the two values are within 1 cm of each other, their average will be used. If not, the measurements will be repeated.

2.7. Safety of the Study Supplements

To maintain the safety of the study supplements (bottarga and cream cheese), the following measures will be implemented:

• Food storage at study site: the bottarga and cream cheese will be stored in a dedicated refrigerator at a temperature of 4 °C (39 °F) or below, in sealed, air-tight containers to maintain freshness and quality, in compliance with food safety guidelines.
• Packaging for distribution:

  -

  Both bottarga and cream cheese will be provided in pre-packaged, sealed portions to minimize handling and ensure product safety. They will be distributed to participants in insulated cooler bags containing ice packs to maintain a temperature of 4 °C (39 °F) or below during transport.

• Transport and Storage by Participants:

  -

  Participants will be instructed to refrigerate both the bottarga and cream cheese immediately upon arriving home, maintaining storage at or below 4 °C (39 °F).

  -

  Participants will also receive instructions to consume the products within the specified use-by period and to report any issues with product condition promptly.

• Additional Food Safety Considerations:

  -

  Given the short travel time expected for most participants and the use of insulated cooler bags with icepacks, there is minimal concern about food safety during transport.

  -

  Participants will receive written guidelines on proper handling and storage of both Bottarga and cream cheese, in alignment with food safety protocols.

2.8. Study Incentives

For aim 1, the dose-confirming (open-label, single-arm) study, each participant completing the baseline clinical eligibility assessment will receive a USD 50 gift card. Each participant completing the 6-week clinical protocol will also receive a USD 100 gift card. For aim 2, the randomized, controlled, cross-over (open-label, two-arm) pilot study, each participant completing the baseline clinical eligibility assessment will receive a USD 50 gift card. Participants will also receive a USD 100 gift card if they complete the 8-week follow-up and another USD 100 gift card after the second 8-week supplementation follow-up. The gift cards will be provided upon completion of each respective visit.

2.9. Statistical Analysis

All questionnaires and consent forms will be collected online using a RedCAP platform via participant smartphones, tablets, or laptops. Anthropometric and physical fitness data will be electronically filed by clinic staff at baseline and at each time point. All data will be collected using study codes to maintain de-identified datasets. Initial data cleaning/management will be performed using Excel. The data will be imported into R, Stata, and SAS, as needed, for more advanced statistical analyses. Continuous variables will be assessed for normality using the Shapiro–Wilk test. Continuous characteristics that are normally distributed will be presented as mean ± SD, whereas quantitative characteristics with skewed distributions will be presented as median (q1, q3). Qualitative characteristics will be described as frequency (%). Differences at baseline in the mean values of a quantitative variable between two groups will be assessed using the independent t-test or the non-parametric Wilcoxon test, as appropriate. The paired t-test or the non-parametric signed-rank test will be used for within-group comparisons. Differences in mean values between the control and intervention groups will be examined using the t-test or the non-parametric Wilcoxon test, as appropriate, when one time point is analyzed independently. Differences in qualitative characteristics will be compared using the chi-square, Fisher’s exact, or McNemar’s tests (for paired comparisons), as appropriate. Highly correlated variables will be checked for collinearity and treated in separate models as needed. The two groups will be compared on an intention to treat basis, adjusting for covariates as needed. Statistical significance for all analyses will be p < 0.05 and two-tailed. No formal power calculation will be performed because, to our knowledge, no prior human studies have evaluated the health effects of bottarga consumption, and therefore no reliable estimates for effect size or variability exist to inform such a calculation. As this is an exploratory pilot study, our primary objective is to assess feasibility, acceptability, and preliminary indications of potential effect rather than to achieve statistical power for hypothesis testing. A crossover design is selected to enhance efficiency by allowing each participant to serve as their own control, thus reducing inter-individual variability. Findings from this pilot will provide critical data on effect size, variability, and adherence, which will serve as the basis for parameter estimates and power calculations in the design of a future full-scale randomized controlled trial.

3. Results

3.1. Nutritional Profile of a Commercial Bottarga Sample

The nutritional composition per 100 and 20 g of a commercial bottarga product, as stated on the product label, are presented in

Table 3. Nutrition facts of bottarga (product label of Trikalinos grey mullet bottarga).

Nutrition Facts
	Per 100 g	Per 20 g
Energy	338/1411	68/282.2	kcal/Kj
Protein	34.8	6.96	g
Lipids	20.9	4.18	g
Saturated fatty acids	6.5	1.3	g
Monounsaturated fatty acids	9.7	1.94	g
Polyunsaturated fatty acids	4.5	0.9	g
Trans fatty acids	0.1	0.02	g
Carbohydrates	2.6	0.52	g
of which sugars	<0.5	<0.1	g
Dietary fibers	<0.5	<0.1	g
Cholesterol	484.9	96.98	mg
Ash	2.8	0.56	g
Macrominerals
Sodium	725	145	mg
Phosphorus	327	65.4	mg
Potassium	234.2	46.84	mg
Calcium	65.3	13.06	mg
Trace minerals
Iodine	256	51.2	μg
Chromium	20	4	μg
Iron	10.5	2.1	mg
Zinc	4.05	0.81	mg
Lipid-soluble vitamins
Vitamin A	1040	208	μg
Vitamin Κ	21.9	4.38	μg
Water-soluble vitamins
Vitamin Β5
(Pantothenic acid)	78.4	15.68	mg
Vitamin Β7 (biotin)	21.9	4.38	μg
Vitamin Β12	6.4	1.28	μg

, demonstrating that the product is nutritionally dense with protein and lipid contents, as well as rich in various micronutrients.

3.2. Lipid Analysis of Bottarga

The lipid class composition of bottarga, determined through spectroscopy, reveals a predominance of wax esters (72.9 mol%), indicating their major role in the lipid profile of the product. Minor but significant contributions come from triacylglycerols (8.8 mol%) and free fatty acids (8.2 mol%), with phosphatidylcholine accounting for 6.2 mol% and cholesterol present at 3.8 mol%. Cholesterol esters were nearly absent (0.1 mol%). Notably, n-3 fatty acid chains represent 12.6 mol% of total lipids, with docosahexaenoic acid (DHA) alone contributing 4.9 mol% (

Table 4. Percentage of total lipids ^a.

Type of Compound	mol%
Wax esters (WEs)	72.9 a
n-3 fatty chains	12.6 b
DHA	4.9 b
EPA	3.1 b
Triacylglycerols (TAGs)	8.8 a
Free fatty acids (FFAs)	8.2 a
Phosphatidyl choline (PC)	6.2 a
Cholesterol (Ch)	3.8 a
Cholesterol esters (CEs)	0.1 a

^a Relative contents were measured from normalized areas of the following peaks of the ¹³C NMR spectrum: 64.46 ppm for WE, 61.98 ppm for TAG, range 180–177 ppm for FFA, 71.56 ppm for Ch, 73.57 ppm for CE, 54.38 ppm for PC based on a previously described method [30]. The NMR measurements have an accuracy of approximately ±5%. The values are presented as mean of two replicate measurements. ^b Measured by ¹H-NMR based on a previously described method [31].

).

3.3. Anticipated Outcomes of the Pilot Study

This pilot study aims to investigate the potential health effects of bottarga consumption in humans, with primary outcomes determined by the most clinically relevant findings observed during Phase 1. Based on preliminary evidence, we hypothesize that bottarga supplementation will be associated with improvements in lipid profiles as well as reductions in inflammatory markers and other cardiometabolic markers compared to baseline measures. To date, no controlled human trials have been published. However, clinical use in Greece—where physicians have recommended bottarga for patients with vascular stents or dyslipidemia—has been anecdotally reported as associated with favorable outcomes. Should this study yield positive results, it would justify further clinical investigation and also support the broader promotion of bottarga as a sustainable and functional blue food/nutritional supplement.

4. Discussion

The current study will investigate the potential health benefits of bottarga, a traditional Mediterranean delicacy made from cured fish roe, focusing on its effects on inflammatory and cardiometabolic risk markers in individuals with overweight and prediabetes. Following a preliminary 6-week dose-confirmation phase, the main study will assess the impact of bottarga consumption in a randomized, crossover clinical trial over 18 weeks. Although bottarga is rich in protective macro- and micronutrients, its health effects have not yet been studied in humans, with existing evidence limited to animal studies. This study aims to address that gap by evaluating whether bottarga confers similar benefits in humans. The focus on adults with overweight and prediabetes is particularly relevant, as this group is metabolically susceptible and may benefit more substantially from lipid-based dietary interventions aimed at prevention and early risk modification.

Limited animal and in vitro studies (using cell lines and cultures) suggest several potential health benefits associated with bottarga consumption. These include increased plasma and tissue levels of omega-3 polyunsaturated fatty acids (PUFAs), potential anti-cancer activity, and antithrombotic properties. Specifically, a study in rats fed a diet enriched with 10% bottarga for five days demonstrated increased levels of eicosatetraenoic acid (EPA) and a reduction in arachidonic acid (20:4 n-6) in plasma, liver, and kidney tissues [19]. Moreover, three additional studies reported that bottarga lipids exerted in vitro cytotoxic effects on an undifferentiated colon cancer cell line (Caco-2 cells) [20,21] and reduced in vitro viability in human cervical carcinoma cells (HeLa) and murine melanoma cells (B16F10 cells) [22]. Lastly, two studies demonstrated that lipids of mullet roe could inhibit platelet-activating factor and thrombin-induced platelet aggregation, leading to prolonged blood clotting time and potentially exerting cardioprotective effects in humans [16,32].

Our analysis demonstrated that wax esters represent the major lipid class in bottarga, accounting for 72.9 mol% of total lipids. This is followed by triacylglycerols (8.8 mol%) and free fatty acids (8.2 mol%), with smaller contributions from phosphatidylcholine (6.2 mol%) and cholesterol (3.8 mol%). Cholesterol esters were nearly absent (0.1 mol%). Importantly, n-3 fatty acids comprise 12.6 mol% of the lipid content, with docosahexaenoic acid (DHA) alone accounting for 4.9 mol%. These results are similar, though not identical, to those of previously analyzed samples of bottarga. For example, Scano et al. examined commercial products of grated mullet roe from two different companies in Sardinia (Italy) and identified wax esters as the major lipid class, exceeding 50 mol%. In contrast, triacylglycerols and phospholipids constituted minor fractions, while n-3 polyunsaturated fatty acids averaged 40 mg/g of the edible portion [33]. Additionally, an extensive nutritional analysis by Kalogeropoulos et al. on Greek bottarga samples reported that wax and steryl esters accounted for 63.7% of roe lipids, followed by phosphatidylcholine at 20.3% [16]. However, the latter results were based on percentage optical density measurements obtained through thin-layer chromatography (TLC) and are not directly comparable to our findings. These differences underscore the variability in bottarga’s composition, which may be influenced by seasonal variation, preparation techniques, and storage conditions [33,34,35]. Furthermore, reported wax ester content in the literature can vary significantly depending on the analytical methods employed.

Bottarga is recognized as a rich source of n-3 polyunsaturated fatty acids, and it appears to be more stable than other sources, such as fish oils. This enhanced stability is mainly attributed to the fact that a significant proportion of n-3 PUFAs in mullet roe are present in the form of wax esters [36]. Notably, wax esters enriched with n-3 fatty acids have been reported to be less susceptible to oxidation, which offers greater resistance to oxidative degradation compared to ethyl ester or triglyceride forms [37]. Although the effects of wax esters specifically from bottarga have not yet been studied in humans, research on wax esters derived from other marine sources has yielded promising results [38]. A relevant example is the oil obtained from Calanus finmarchicus, a copepod found in the northern Atlantic Ocean, in which over 80% of the fatty acids are present in the form of wax esters. One preclinical study demonstrated that purified wax ester supplementation exerted stronger anti-inflammatory and anti-obesogenic effects in obese mice compared to ethyl esters of EPA and DHA, suggesting superior biological activity of wax ester-bound n-3 PUFAs [39]. In a clinical study, the intake of 2 g of Calanus oil over a 12-week period significantly improved the omega-3 index (O3I)—defined as the relative content of EPA and DHA in erythrocytes—in older adult participants engaged in a moderate exercise program. These findings indicate that wax ester-bound n-3 PUFAs are not only bioavailable but also capable of exerting physiologically meaningful effects in humans [40]. In addition to wax esters, bottarga is a source of phosphatidylcholine, a major phospholipid component of cell membranes that plays a vital role in lipid metabolism, membrane fluidity, and cell signaling. Evidence suggests that phosphatidylcholine supplementation may contribute to the regression of liver steatosis [41].

In conclusion, if the results of the current study on bottarga prove promising, the next step will be to expand the investigation through a larger-scale randomized controlled trial to further validate its potential health benefits—particularly in relation to cardiovascular health and related indices. This future study will compare the effects of bottarga supplementation to those of commercially available n-3 fatty acid supplements on lipid profiles, metabolic and inflammatory markers, and overall health outcomes. Interestingly, the high wax ester content in bottarga—unlike the triglyceride-rich profile of most fish oil supplements—may confer distinct metabolic properties, although their bioavailability in humans remains less well-established. A comparative evaluation is therefore warranted to determine whether bottarga’s specific fatty acid composition and lipid matrix offer competitive or even superior effects relative to conventional n-3 formulations.