Effect of packing conditions on the acrylamide content in black ripe olives

Abstract

BACKGROUND

Black ripe olives are considered a ‘low‐acid canned food’ and must be sterilized in order to be safe. Acrylamide, a potential carcinogen, is formed in black olives during the thermal treatment. It is known that the formation of acrylamide is dependent on the pH of the olive flesh. The purpose of this study was designed to assess the influence of packing conditions on the acrylamide production in black olives.

RESULTS

A reduction in acrylamide content of up to 20–30% was achieved by raising the pH of the fruit before packing above 7. Lactic acid improved color and addition of calcium chloride to the cover brine enhanced firmness. Sodium bicarbonate decreased acrylamide by up to 35%, although severe softening occurred. Packaging under CO₂ atmosphere increased acrylamide relative to air or nitrogen, indicating its unsuitability for packing this product. Likewise, no effect on acrylamide levels was observed among pitted and sliced olives, unlike in whole fruits, in which a higher concentration of acrylamide was detected, attributable to precursor localization in the seed, which produced significantly more acrylamide than the flesh or woody endocarp during isolated sterilization.

CONCLUSION

The study identifies pH modulation as an effective mitigation strategy for acrylamide in black olives, but highlights trade‐offs between reduction efficacy, texture quality and packaging conditions. The proposal does not solve the problem completely, but it may be viable on an industrial scale and will therefore provide the table olive industry with new tools to mitigate the presence of acrylamide in black olives. © 2026 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

INTRODUCTION

The annual worldwide production of table olives is around 3 million tons, similar to that of olive oil. In particular, this food product is a significant source of economic profit for Mediterranean countries such as Spain, Turkey, Egypt, Greece and Italy. Among the different table olive processing styles, black olives, also known as Californian‐style black olives, are one of the three major trade preparations commercialized worldwide. They are very attractive to consumers due to their shiny black color, so they are used for fast foods such as salads, pizzas, sandwiches and many others.

The elaboration method involves a first stage of fruit preservation in acidic solution for 2–14 months. ¹ When the market requires black olives, they are treated with a dilute NaOH solution followed by two tap water washes under continuous aeration. ² The color changes to black due to the oxidation and polymerization of the phenolic compounds, and it is stabilized by immersing the fruits in a ferrous gluconate solution. Acids (acetic and lactic acids, or CO₂) are currently added to the solutions after the alkaline treatment to obtain a pH of around 7–8 before packing the product.

Black olives commercialized whole, pitted or sliced ³ are packed with fresh brine or even without liquid in tins, jars and aluminium or plastic pouches, and are sold in a wide range of formats, from 50 to 5000 g containers. All of them must be sterilized because of the neutral pH of the product at a temperature between 121 and 125 °C to reach an accumulated lethality of 15 F₀. ³

As occurs in most heated foodstuffs, acrylamide formation in black olives can occur through several mechanisms, and the thermal treatment is a key step, the content depending on the heating intensity (F₀), among other variables. ⁴ , ⁵ , ⁶ , ⁷ , ⁸ , ⁹ , ¹⁰ Unlike the well‐established reaction of asparagine and reducing sugars for acrylamide formation in most heated foods, ¹¹ the mechanism in black olives remains elusive. ¹² , ¹³ , ¹⁴ Small peptides bound to polyphenols ¹⁵ and compounds with low molecular weight (<1000 Da) ¹⁴ have been proposed as precursors of acrylamide production in black olives, and recently the involvement of small phenolic compounds, ¹⁶ even their quinones ¹⁷ or water‐soluble larger components (>10 000 Da), has been considered relevant. ¹⁸ However, none of these mechanisms have been demonstrated in industrially processed black olives.

The influence of the crop year, irrigation and processing conditions on acrylamide formation in black olives has been studied ¹⁹ , ²⁰ , ²¹ along with mitigation strategies to prevent or decrease the generation of this toxic substance in this product, ⁶ , ²² including the addition of certain additives such as amino acids, phenolic compounds and sodium sulphite ²³ or fortification with fresh and lyophilized Cassia grandis. ²⁴ Because of modifications of the physicochemical and sensory characteristics of the olives and legal restrictions, most of these substances have not been tested at an industrial scale. Recently, an alternative processing method of black olives based on the acidification and pasteurization of this product has been proposed. ⁹ , ¹⁰

Overall, there is consensus that acrylamide is produced in packed olives during the sterilization step. Also, many variables may influence acrylamide content in black olives, including cultivar and darkening conditions. However, packing and sterilization are key steps for the formation of acrylamide in this product. Hence, the study reported here was focused on the packing conditions that could affect the quality characteristics of black olives, ²⁵ together with their acrylamide level.

MATERIALS AND METHODS

Olives

Fruits of the Hojiblanca cultivar (Olea europaea L.) with surface green‐yellow color were mechanically harvested in the province of Seville (southern Spain). Olives were placed in fiberglass underground tanks and covered with a preservation liquid, acidified water with 20 g L⁻¹ acetic acid, at the Agrosevilla SCA factory (La Roda de Andalucía, Seville, Spain).

Olives were darkened at the Agrosevilla SCA factory or at the Instituto de la Grasa pilot plant. When necessary, the olives were pitted and sliced using an industrial machine (OFM, Dos Hermanas, Spain).

Darkening process at Instituto de la Grasa pilot plant

Fruits of the Hojiblanca cultivar (Olea europaea L.) stored for 5 months in fiberglass tanks at the Agrosevilla SCA factory under acidic conditions were used for the darkening stage at the Instituto de la Grasa pilot plant. ²⁶ Olives (5 kg) were placed in two cylindrical oxidation chambers and immersed in 5 L of a NaOH solution (30 g L⁻¹) until it penetrated to the stone. Subsequently, the lye was removed, and the olives were covered with 5 L of a mixture of preservation liquid–tap water (1/1) for 20 h. After draining, the fruits were washed in fresh tap water for 24 h and CO₂ was bubbled to neutralize the high pH value in the alkaline pulp. On the third day, fruits were immersed in a ferrous gluconate solution (1 g L⁻¹) for 5 h to fix the black color formed. ² Also, in this ferrous solution, CO₂ was bubbled to continue neutralizing the high pH value of the pulp.

Sterilization treatment at Instituto de la Grasa pilot plant

Both jars and pouches with different commercial presentations were sterilized in a computer‐controlled Steriflow retort (Madinox, Barcelona, Spain). The containers were placed in the retort, which, once closed, was kept at 30 °C for 10–15 min. Subsequently, the temperature inside the retort was increased to 121 °C in 21 min, which was maintained for the time required to achieve an accumulated sterility of 15 F₀ in each container. Finally, the cooling step was performed by decreasing the retort temperature to 30 °C in 20 min.

The temperature inside the jars and pouches during sterilization was controlled using programmable thermometers (Thermo buttons, Thermotrack PC, Progres‐Plus, France).

Experimental design

Effect of additives and pH before packing on acrylamide formation

A batch of olives were darkened as described in the previous section in two oxidation chambers at the pilot plant of the Instituto de la Grasa by bubbling CO₂ during the second washing and the ferrous gluconate steps to neutralize the high pH value of the pulp and reach a value of pH 7.5 before packing (control). In another two chambers, the pH was left at 9.2 units (high pH). Whole olives (170 g (140 mL)⁻¹ brine) and pitted olives (145 g (170 mL)⁻¹ brine) at pH 7.5 were packed in A314 glass jars (JUVASA, Dos Hermanas, Spain), and submitted to sterilization. The cover brine composition was 35 g L⁻¹ NaCl and 0.2 g L⁻¹ ferrous gluconate. Olives (whole and pitted) with pH 9.2 were packed similarly to those with pH 7.5, and lactic and citric acids were added in the cover brine to reach a pH of 7.0.

The required amount of acid in each jar was previously determined by titration of the paste olive/brine with lactic or citric acid solutions. Additionally, half of the jars with added acid were spiked with 6 g L⁻¹ CaCl₂ to assess the influence of the presence of calcium ions on fruit firmness.

In another assay with darkened fruits from the Instituto de la Grasa pilot plant, whole olives (with pH 9.5) were packed with brine containing 0.2 g L⁻¹ ferrous gluconate (control) and supplemented with 1 g L⁻¹ lactic acid or varying concentrations of ascorbic acid (1, 2 and 4 g L⁻¹).

The effect of NaHCO₃ on acrylamide formation in olives was assessed using four batches of darkened fruits from four table olive factories, which were processed in a similar manner. Sodium bicarbonate was added to the cover brine at concentrations of 16 and 32 g L⁻¹.

After sterilization, all jars (six for each treatment) were stored at room temperature for 2 months to determine acrylamide content and fruit quality parameters.

Influence of type of atmosphere on acrylamide content in olives packed without cover brine

Two batches of whole olives from the Agrosevilla SCA factory (120 g) were packed in plastic complex pouches of polypropylene and polyester (PET PVDC PAST+PP G) with an O₂ permeability lower than 10 cm³ m⁻² day⁻¹ (SPgroup, Villarrubia, Spain) without liquid. A Tecnotrip sealer (EVT‐7‐G‐TDSD, Terrasa, Spain) was used to pack the samples with three different internal atmospheres: air, nitrogen or carbon dioxide. Subsequently, the pouches were sealed using an Audion Elektro sealer equipment (Weesp, The Netherlands) and submitted to sterilization. After 2 months of storage at ambient temperature, acrylamide formation was measured.

Effect of type of commercial presentation and flesh/cover brine ratio on acrylamide content in olives

Pitted and sliced olives from an industrial batch were packed in two different container sizes: 314 mL bottles (A314) and 1890 mL bottles (0.5 gal). Both were sterilized to reach 15 F₀ sterility units at the cold point. This experiment assessed how presentation and packaging size affected acrylamide formation.

In another experiment, whole olives (120 ± 2 g) and pitted olives (97 ± 2 g, equivalent in flesh weight to 120 g of whole olives) from two different industrial batches were packed in plastic bags with 120 or 140 mL of cover brine. Both were sterilized to reach 15 F₀ sterility units at the cold point. The study evaluated how the ratio of olive flesh to brine volume influenced acrylamide levels. After 2 months of storage at ambient temperature, acrylamide was analyzed in both the brine and olive pulp.

Contribution of different parts of olive to acrylamide formation

Darkened olives at the Instituto de la Grasa pilot plant were used for these experiments.

Whole darkened olives (145 g) and olive stones (145 g) were covered with 170 mL of tap water in A314 jars, sterilized at 121 °C and stored for 2 months at ambient temperature before analysis of acrylamide (experiment 1).

For a second experiment, 7 g of whole stone, wood shell (outer stone layer) and seed (inner kernel) from darkened olives were placed in 25 mL glass tubes with 8.5 mL of tap water, sealed and sterilized at 121 °C. The acrylamide content in the liquid was analyzed after 2 months of storage at ambient temperature.

Physicochemical analysis

The pH of olive flesh was measured by puncturing the pulp of 10 fruits with a pH Spear instrument (Eutech Instruments, Thermo Scientific, Waltham, MA, USA).

Moisture levels of olives, stone and seed were measured by oven drying at 105 °C to constant weight.

Olive color was measured using a ColorFlex EZ spectrophotometer (HunterLab, Reston, VA, USA). Interference by stray light was minimized by covering samples with a box featuring a matte black interior. The surface color of the fruits was expressed as reflectance at 700 nm (R ₇₀₀), lower reflectance values indicating darker fruit. Each measurement represents the average value of 10 olives.

For firmness determination, the shear compression force in newtons (N) to break three pitted olives was measured using a Kramer shear compression cell coupled to a TA.TXplus texture analyzer (Stable Micro System, Surrey, UK). The crosshead speed was set to 200 mm min⁻¹. The value (N (100 g)⁻¹ pitted fruit) reflects the mean of eight replicate measurements.

Analysis of acrylamide

The acrylamide content in the olive pulp was analyzed according to a method described elsewhere. ²⁷ Briefly, olives were pitted and homogenized in a blender. A portion of 5 g was mixed with 10 mL of ultrapure water and spiked with 100 μL of the internal standard (¹³C₃)acrylamide (10 mg L⁻¹). The mixture was homogenized for 2 min with an Ultra Turrax homogenizer and then filtered through filter paper (Whatman No. 1440‐110). All extracts were finally filtered through a 0.22 μm pore size nylon filter, and an aliquot (20 μL) was injected into a high‐performance liquid chromatography (HPLC) instrument. The quantification of acrylamide was performed using an HPLC–MS system (QDA, Waters, USA).

For the analysis of acrylamide in liquid samples, 5 mL was mixed with 10 mL of ultrapure water and 100 μL of internal standard (10 mg L⁻¹ (¹³C₃)acrylamide). The mixture was filtered through a 0.22 μm pore size nylon filter, and 20 μL was injected into the HPLC system. Analysis was run in triplicate.

Statistical analyses

Statistical analyses were performed using Statistica 8.0 software (StatSoft Inc., Tulsa, OK, USA). One‐way analysis of variance (Duncan's test) was used to compare the mean values with a significance level of 95%.

RESULTS AND DISCUSSION

Effect of pH and additives on acrylamide level

The pH of sterilized whole olives was close to 7.5 regardless of the pH prior to packaging (Table 1). Olives packed at pH 7.5 reached a final pH of 7.36, and those packed at pH 9.25 had a final pH of 7.64. Despite the small difference in the final pH of the olives, significant effects on color, firmness and acrylamide content were observed. As previously reported, ²⁸ olives packed above pH 7 resulted in a less dark color and less firmness than those packed at a pH close to neutral for both whole and pitted olives (Table 1). When fruits were packed at a pH above 7, a favorable impact on the level of acrylamide in the product was also observed. Hence, a reduction of more than 25% of the toxic substance was achieved for whole and pitted olives. This pH dependence of the acrylamide level in black olives has been previously reported, ²¹ , ²³ with maximum formation occurring at pH close to 6 and decreasing as the pH increases. ²³ Although the mechanism of acrylamide formation in olives is still unknown, the relationship between acrylamide formation and pH in olives has shown a similar behavior to that described for potatoes and many other foods following the Maillard reaction. ²⁹ These results underscore the relevance of pH control before packaging, though product physicochemical characteristics must also be considered.

Table 1.

Acrylamide content, pH and quality parameters in olives packed with lactic/citric acids and CaCl₂

	Additive	pH before packing	pH in final product	Acrylamide (μg kg−1)	Acrylamide reduction (%)	Color (R 700)	Firmness (N (100 g)−1 fruit)
Whole olives
Control	None	7.50	7.36	630 ± 41a	—	4.32 ± 0.03d	1316 ± 55c
High pH	None	9.25	7.64	468 ± 14b	25.7	5.07 ± 0.10b	1014 ± 33d
High pH	Lactic acid	9.25	7.01	493 ± 18b	21.8	3.96 ± 0.03e	1206 ± 45c
High pH	Lactic acid + CaCl2	9.25	7.02	483 ± 31b	23.3	4.13 ± 0.10de	1693 ± 55b
High pH	Citric acid	9.25	7.27	495 ± 42b	21.4	4.55 ± 0.07c	1049 ± 48d
High pH	Citric acid + CaCl2	9.25	7.17	487 ± 7b	22.7	5.21 ± 0.03ab	1646 ± 66b
Pitted olives
Control	None	7.50	7.18	498 ± 45a	—	4.15 ± 0.05de	1255 ± 89c
High pH	None	9.25	8.04	347 ± 19c	30.4	5.01 ± 0.04b	998 ± 63d
High pH	Lactic acid	9.25	7.11	454 ± 18ab	8.8	3.98 ± 0.04e	1063 ± 55c
High pH	Lactic acid + CaCl2	9.25	7.06	440 ± 28b	11.5	4.34 ± 0.04d	1795 ± 89a
High pH	Citric acid	9.25	7.33	459 ± 14ab	7.9	4.65 ± 0.05c	1104 ± 68c
High pH	Citric acid + CaCl2	9.25	7.34	444 ± 10b	10.8	5.40 ± 0.10a	1871 ± 67a

Value ± standard deviation of color, firmness and acrylamide content for each olive presentation (whole or pitted) followed by the same letter do not differ at the 5% level of significance according to Duncan's multiple‐range test.

To maintain olive quality while reducing acrylamide, lactic and citric acids were added to the cover brine of olives packed at a pH above 7. While lactic acid but not citric acid improved color in olives packed at pH 9.5, they did not enhance firmness, regardless of the olive presentation. Because CaCl₂ is a frequently used firming agent during the processing of table olives, ⁹ the combined effect of lactic and citric acids with CaCl₂ was tested for improving the firmness, although fruit discoloration was observed when citric acid was employed. It should be noted that CaCl₂ has promoted acrylamide formation in olive model systems, ²³ and in Californian‐style green ripe olives ³⁰ but this negative effect was not observed when it was added to the cover brine of packed black ripe olives (Table 1). Likewise, the reduction of acrylamide production was not affected by the use of these additives in the cover brine of whole olives packed at a high pH, but this reduction was lower in the case of pitted fruits (Table 1).

This behavior suggests that acids rapidly penetrated the pulp of pitted olives during the sterilization step, resulting in less of an effect of pH on acrylamide reduction. It is important to clarify that acrylamide mitigation strategies based on the addition of organic acids to many foods are associated with a drop in pH below 6, ⁴ , ³¹ but this is not the case for black olives at pH 9.5. In our study, the addition of acids aimed to maintain the black color of the product.

On the other hand, the recently proposed mechanism for acrylamide formation in black olives, based on the oxidation of phenolic compounds via quinones, ¹⁶ , ¹⁷ prompted us to study the effect of ascorbic acid, a common antioxidant in the table olive industry. Unfortunately, the acrylamide level was not significantly reduced with the addition of ascorbic acid up to 4 g L⁻¹, even though the pH of the olives was reduced from 8 (control) to 6.9 for the highest concentration of ascorbic acid tested (Table 2). The influence of ascorbic acid on acrylamide‐forming Maillard reactions remains controversial. Some authors ²⁹ found a reduction in the level of this toxic compound in potatoes, which could be attributed to the decrease in pH, while other researchers ³² detected a promotion effect, probably associated with the thermal decomposition of ascorbic acid. Indeed, Table 2 evidences a trend towards higher acrylamide level in black olives with increasing ascorbic acid concentration, which casts doubts on the involvement of the oxidation of phenolic compounds in acrylamide generation in black olives.

Table 2.

Effect of ascorbic acid on acrylamide content, color and final pH of black olives packed at pH 9.5

	Acrylamide (μg kg−1)	Color (R 700)	pH
Control	566 ± 30a	4.5 ± 0.1a	8.0 ± 0.1a
Lactic acid (1 g L−1)	506 ± 50a	4.1 ± 0.1b	7.4 ± 0.1c
Ascorbic acid (1 g L−1)	527 ± 103a	4.0 ± 0.1b	7.8 ± 0.0b
Ascorbic acid (2 g L−1)	582 ± 52a	4.2 ± 0.2b	7.1 ± 0.1d
Ascorbic acid (4 g L−1)	608 ± 13a	4.5 ± 0.1a	6.9 ± 0.0e

For each column, value ± standard deviation followed by the same letter do not differ at the 5% level of significance according to Duncan's multiple‐range test.

Effect of sodium bicarbonate and CO₂ on acrylamide level

Among the leavening agents employed in the bakery industry, ammonium bicarbonate (NH₄HCO₃) appears to promote acrylamide production through sugar degradation pathways. ⁴ In contrast, sodium bicarbonate (NaHCO₃) reduces acrylamide levels in bakery products, which has not been attributed to a simple pH effect. ³³ Based on these observations, sodium bicarbonate was incorporated into the cover brine of black olives prior to sterilization. As presented in Table 3, the addition of this compound resulted in a reduction of acrylamide content ranging from 0% to 30%, depending on the batch tested. Obviously, the pH of the olives increased with the addition of sodium bicarbonate, suggesting that the observed acrylamide reduction may be directly related to this pH shift. Furthermore, the color of the olives was not affected by sodium bicarbonate. This suggests a viable option to reduce the acrylamide level in black olives. However, the addition of sodium bicarbonate resulted in a significant decrease in olive firmness of approximately 25% for all batches tested, which is consistent with the well‐documented softening effect of this compound on plant tissues due to degradation of cell wall components. ³⁴ These assays were conducted by adding 16 g L⁻¹ sodium bicarbonate in the cover brine. Increasing the level of this substance to 32 g L⁻¹ reduced the concentration of acrylamide by up to 35% but, again, firmness was greatly affected (Fig. 1). A linear decrease in acrylamide content was observed, paralleling a linear decrease in olive firmness.

Table 3.

Effect of sodium bicarbonate (16 g L⁻¹) on pH, acrylamide and quality characteristics of black olives packed in jars

	pH	Acrylamide (μg kg−1)	Color (R 700)	Firmness (N (100 g)−1 fruit)
Batch1, control	7.9 ± 0.2b	297 ± 35a	2.9 ± 0.1a	1909 ± 67a
Batch 1, bicarbonate	8.4 ± 0.2a	230 ± 35a	2.9 ± 0.0a	1474 ± 27b
Batch 2, control	6.2 ± 0.1b	673 ± 10a	3.6 ± 0.1a	1765 ± 138a
Batch 2, bicarbonate	7.7 ± 0.1a	592 ± 59a	3.8 ± 0.2a	1390 ± 66a
Batch 3, control	7.4 ± 0.1b	462 ± 16a	3.5 ± 0.0a	1578 ± 152a
Batch 3, bicarbonate	8.2 ± 0.2a	324 ± 7b	3.5 ± 0.2a	1133 ± 30a
Batch 4, control	6.9 ± 0.0b	374 ± 3a	3.1 ± 0.1a	1803 ± 91a
Batch 4, bicarbonate	8.4 ± 0.2a	387 ± 35a	3.1 ± 0.1a	1392 ± 53b
Mean, control	7.1 ± 0.7b	432 ± 138a	3.3 ± 0.3a	1764 ± 155a
Mean, bicarbonate	8.2 ± 0.4a	364 ± 131a	3.3 ± 0.4a	1347 ± 142b

For each column and batch, value ± standard deviation followed by the same letter do not differ at the 5% level of significance according to Duncan's multiple‐range test.

Figure 1.

Influence of sodium bicarbonate addition in the cover brine on acrylamide formation and firmness of black olives. Bars represent standard deviation of duplicates.

Because the pH of black olives is currently around 7–8 and sodium bicarbonate is the predominant carbonate species at this pH, olives were also packed without liquid under CO₂‐modified atmosphere (Table 4). Surprisingly, the acrylamide production was negative for the two batches tested. The content of this toxic substance was significantly higher in olives packed in pouches under a CO₂ atmosphere than in those packed with air or nitrogen. Contrary to the reduction reported in bakery products through the use of CO₂, ³⁵ , ³⁶ this gas must be avoided for the packing of black olives in view of the results found. It should be noticed that there is a current tendency to pack black olives in pouches without cover brine, and the main recommendations emphasize minimizing oxygen within the package. ³⁷ The present results indicate that CO₂ should also be excluded in such packaging systems.

Table 4.

Effect of type of atmosphere on acrylamide formation (μg kg⁻¹) in black olives packed in pouches

Atmosphere	Batch 1	Batch 2
Air	917 ± 77b	1004 ± 44b
Nitrogen	964 ± 38b	991 ± 127b
CO2	1282 ± 48a	1496 ± 193a

For each column, value ± standard deviation followed by the same letter do not differ at the 5% level of significance according to Duncan's multiple‐range test.

Influence of commercial presentation and packing conditions on acrylamide level

Table 5 presents the effect of olive presentation format on acrylamide formation in two different containers (A314 and 0.5 gal jars). Data revealed that the total acrylamide formed in the container, expressed as μg kg⁻¹, was statistically similar for pitted and sliced olives, regardless of container type. These results contrast with those previously published. ²⁰ , ²² One possible explanation could be that the sliced olives studied by previous researchers were washed more intensively than pitted olives, a situation that did not occur in our experiments. Some authors ⁶ indicated that extending the washing phase of pitted olives resulted in acrylamide reduction in the fruits. Furthermore, since acrylamide formation depends on thermal intensity, a specific sterilization program was developed for each container and presentation format (supporting information, Fig. S1). Therefore, both pitted and sliced olives were subjected to the same accumulated sterility at the cold point of the container (F₀ = 15), which could explain the similar levels of acrylamide in pitted and sliced olives.

Table 5.

Effect of type of container and commercial presentation of olives on acrylamide level

				Acrylamide
Container	Commercial presentation	Olive (g)	Brine (mL)	Olive (μg kg−1)	Brine (μg L−1)	Total in container a (μg)	Total in container b (μg kg−1)
A314	Pitted	140	160	430 ± 62	605 ± 61	153 ± 18	1011 ± 108a
A314	Sliced	155	145	463 ± 18	579 ± 17	154 ± 3	973 ± 25a
0.5 gal	Pitted	940	970	496 ± 55	640 ± 18	1048 ± 69	1059 ± 71a
0.5 gal	Sliced	940	960	492 ± 00	662 ± 0	1044 ± 0	1082 ± 0a

^a Sum from the olive flesh and the cover brine.

^b Value ± standard deviation of total acrylamide in the container (μg kg⁻¹) followed by the same letter do not differ at the 5% level of significance according to Duncan's multiple‐range test.

In contrast, acrylamide levels were significantly higher in whole than in pitted olives in both batches studied (Table 6). This difference was evident in the olive flesh, in the cover brine and in the total acrylamide formed in the jar. As previously discussed, the accumulated lethality was the same for whole and pitted olives (F₀ = 15). This finding has previously been reported for commercial olives, ²⁰ , ²² , ³⁰ which could be attributed to the washing step of pitted olives as opposed to whole olives. However, both whole and pitted olives underwent a similar number of washing steps in our assays. Furthermore, a clear, although not statistically significant, trend towards lower acrylamide level was detected with decreasing ratios of olive flesh (g) to cover brine (mL), related to the high water solubility of the toxic component.

Table 6.

Effect of ratio of olive flesh/cover brine on acrylamide content in whole olives (97 g of flesh) and pitted olives (97 g)

		Acrylamide
Commercial type	Cover brine volume (mL)	Flesh (μg kg−1)	Cover brine (μg L−1)	Total acrylamide in jar (μg) a
Batch 1
Whole	120	508 ± 6a	664 ± 93a	129 ± 17a
Whole	140	478 ± 36a	604 ± 35a	131 ± 12a
Pitted	120	358 ± 22b	455 ± 4b	89 ± 4b
Pitted	140	290 ± 23b	409 ± 53b	85 ± 14b
Batch 2
Whole	120	492 ± 14a	298 ± 21a	83 ± 5a
Whole	140	488 ± 44a	250 ± 40a	82 ± 14a
Pitted	120	307 ± 1b	261 ± 22a	61 ± 4b
Pitted	140	296 ± 12b	207 ± 7b	58 ± 3b

^a Sum of acrylamide in flesh and brine. For each batch, value ± standard deviation in column followed by the same letter do not differ at the 5% level of significance according to Duncan's multiple‐range test.

To elucidate the reason for the higher level of acrylamide in whole than in pitted olives, different parts of the black olives were sterilized separately (Table 7). The amount of acrylamide formed from the stones was significantly higher than that from the olive flesh: 150 and 66 μg, respectively. In fact, data from experiment 2 revealed a much higher production of acrylamide from the seed than from the wood shell: 31 and 9 μg, respectively. Overall, these results indicate that the precursors of acrylamide formation in black olives are more concentrated in the seed than in the olive flesh. Recent studies further support this conclusion by demonstrating that these precursors are mainly low‐molecular‐weight compounds (<1000 Da). ¹⁴

Table 7.

Acrylamide content in different parts of olive

	Humidity (%)	Acrylamide in liquid (μg L−1)	Acrylamide in liquid (μg)	Estimated acrylamide in solid a (μg)	Total acrylamide in jar (μg)
Experiment 1
Olive	80.0 ± 0.0	251 ± 25	43 ± 4b	29 ± 1b	66 ± 5b
Stone	37.4 ± 0.5	671 ± 34	114 ± 6a	36 ± 1a	150 ± 7a
Experiment 2
Stone	44.8 ± 2.2	754 ± 154	6 ± 1b	2 ± 0b	9 ± 2b
Wood shell	53.0 ± 7.5	760 ± 84	6 ± 1b	3 ± 1b	9 ± 1b
Seed	79.0 ± 0.4	2181 ± 780	19 ± 7a	12 ± 4a	31 ± 11a

^a Calculated taking into account the acrylamide in the liquid and the humidity of the solid. For each experiment, value ± standard deviation in column followed by the same letter do not differ at the 5% level of significance according to Duncan's multiple‐range test.

CONCLUSIONS

The results of the present study indicated that packing conditions of black olives influence the production of acrylamide during the sterilization stage. Acrylamide formation in sterilized black olives results from the combined influence of pH, additive behavior and the intrinsic distribution of precursors within the fruit, with elevated packing pH emerging as the most effective and technologically viable mitigation strategy despite its impact on certain quality attributes. The addition of lactic acid and CaCl₂ to the cover brine was advisable to maintain color quality and firmness of the olives. Concerning the type of olive presentation, acrylamide production in pitted and sliced olives was statistically the same when they were sterilized with similar accumulated sterility value, F₀ = 15 units. In contrast, whole olives resulted in higher acrylamide production than the former, which was due to the high contribution of the seed part of the fruit to the formation of the toxic substance. Overall, these findings indicate that effective acrylamide reduction in black olives must integrate pH control with a careful selection of additives and an understanding of tissue‐specific precursor distribution, while further mechanistic studies remain essential to develop processing strategies that balance chemical safety with product quality.

CONFLICT OF INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supporting information

Figure S1. Evolution of temperature inside the glass jars during sterilization of pitted (a) and sliced (b) black olives to reach (c) an accumulated sterility of 15 F₀.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.