Bioactive properties of fermented spreadable product manufactured from pistachio kernels

Abstract

A functional fermented plant-based product was developed by using raw and roasted pistachio kernels. The product was prepared from aqueous pistachio slurries by heating and fermenting with a lactic culture. Antioxidant, ACE-inhibitory and α-amylase inhibitory activities along with soluble protein and phenolic contents of the products were measured during storage. Raw pistachios exhibited significant antioxidant and ACE-inhibitory activity along with a low level of α-amylase inhibitory activity which were further enhanced by fermentation with the lactic culture. On the other hand, roasting pretreatment resulted in lower soluble protein content, antioxidant activity, phenolic content and consequently lower bioactivity in the end product. Plant-based pistachio products can be manufactured from raw pistachios in order to obtain high antioxidant and ACE-inhibitory activities.

Highlights

Unfermented raw pistachios had high levels of antioxidant and ACE-inhibitory activities.

The bioactive properties of the fermented pistachio product were reduced by roasting pretreatment.

Fermentation with lactic culture slightly enhanced antioxidant and ACE-inhibitory activities of the pistachio product.

Introduction

Consumers around the world have increased interest towards plant-based foods due to concerns for health and environment in recent years; therefore, the food industry has begun to focus on development of new plant-based food products. Plant-based dairy analogues provide an alternative to dairy products for consumers with lactose intolerance or milk protein allergy and vegan population who prefer a plant-based diet. Nuts are commonly used in plant-based food production due to the presence of structural components, proteins, carbohydrates, fat and dietary fiber, as well as nutritional and functional components including vitamins, minerals and bioactive compounds.

Pistachios (Pistacia vera L.), one of the most popular nuts in the world, were mostly produced in the USA, Iran, and Türkiye in amounts of 400, 242 and 239 thousand tons in 2022, respectively (FAO 2023). Pistachios are rich in nutrients, containing high amounts of protein, fat, carbohydrate, calcium, potassium and magnesium (Reale et al. 2024). The consumption of pistachio has been associated with various health benefits, reduction of oxidative stress and cholesterol and prevention of type-2 diabetes which was explained by the presence of bioactive compounds such as polyphenols, carotenoids specifically lutein, tocopherol, and phytosterols (Terzo et al. 2019). Hence, pistachios are considered as a good raw material option in the production of plant-based dairy analogues and beverages (Marulo et al. 2024; Reale et al. 2024). Moreover, fermentation of plant-based products with lactic acid bacteria (LAB) leads to the release of bioactive compounds such as peptides, phenolics, and flavonoids from the plant matrices which can act as health promoting components (Singh et al. 2020).

Pistachios have technological and functional potential for creating new plant-based products due to their rich composition of structural and bioactive components. Moreover, fermentation can be applied to improve health functionalities as well as to extend shelf life of the products. In this context, the amounts of soluble protein/peptides and phenolic compounds with antioxidant activity can be augmented by fermentation of plant materials. These components have been found to be associated with antihypertensive and antidiabetic bioactivities that can alleviate metabolic disorders such as diabetes and hypertension (Dwivedi et al. 2024). This study was undertaken to develop a fermented plant-based product from pistachios and to determine its health-promoting bioactivities including antioxidant, antidiabetic and antihypertensive activity.

Materials and methods

Materials

Pistachio kernels (Pistacia vera L.), collected one month early than regular harvest time for use as an ingredient named as boz without the outer shell, were purchased from Gaziantep, Türkiye. Locust bean gum and xanthan gum were supplied from KMK Lab. Ltd. (Istanbul, Türkiye). Commercial thermophilic lactic starter culture (Yo-Flex CH 1), a mixture of defined single strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, was obtained from Chr. Hansen (Istanbul, Türkiye).

Product preparation

Raw pistachio kernels were soaked in water at a ratio of 1:3 (pistachio: water, w/w) at 4 °C for 1 h to remove the inner peels. Peeled kernels were dried at room temperature for 6 h before use. Half of the pistachios were used raw, and the other half were roasted at 135 °C for 15 min (Hojjati et al. 2013). The raw or roasted pistachios (40 g) were mixed with water at a ratio of 1:3 (pistachio: water, w/w) at 23,000 rpm for 1 min using a blender (AR1061, Arzum, İstanbul, Türkiye). The slurries were enriched with sucrose at a level of 4% (w/w) to increase fermentation rate and then they were homogenized by using a probe-homogenizer (Ultraturrax T18, IKA Werke, Staufen, Germany) at 15,500 rpm for 2 min. The resulting slurries were heat-treated in a water bath at 85 °C for 30 min and cooled immediately to fermentation temperature of 43 °C. After inoculation with the LAB culture (0.05%, w/w), fermentation continued for approximately 5 h in an incubator at 43 °C until the pH of the slurries decreased to 4.5.

The fermented slurries at 43 °C were mixed with a gum solution to obtain 0.4% locust bean gum, 0.4% xanthan gum, and 0.72% salt in the final product and then homogenized with a probe-homogenizer. The gums and salt in the gum solution were dissolved by heating to 90 °C and then the mixture was cooled to 43 °C before mixing. The products were placed in sterile lidded containers and stored at 4 °C for 20 days to be analyzed after 1, 10 and 20 days. Unfermented products were produced in the same way to determine the impact of fermentation on the measured properties.

Physicochemical analyses

The proximate composition of the pistachio kernels was determined according to Tsantili et al. (2010). The pH values of the samples during fermentation and storage were determined with a pH-meter. The titratable acidity of the sample was determined by titrating with 0.01 N NaOH in the presence of phenolphthalein indicator and expressed as %lactic acid.

Bioactivity analyses

Water-soluble extracts of the samples were prepared for bioactivity analyses. The samples were diluted two-fold with distilled water and homogenized (Ultraturrax T18, IKA Werke, Staufen, Germany) at 7000 rpm for 30 s. Then, the mixture was placed in a water bath at 40 °C for 30 min. pH of the unfermented samples was adjusted to 4.5 with 4 M HCl before heating. The mixture was centrifuged at 2500 x g, at 4 °C for 20 min and then the pH of the supernatant was adjusted 7 with 1 M NaOH and re-centrifuged. The obtained supernatant was mixed with hexane at 10:1 ratio (supernatant: hexane, v/v) and re-centrifuged to separate oil residue. The clear lower aqueous phase was filtered through Whatman 4 filter paper and used for the analysis. The soluble protein content of the samples was determined according to the modified Lowry method by using diluted water-soluble extract (Hartree 1972). Soluble protein content was calculated using a standard curve prepared with bovine serum albumin at a concentration range of 0- 0.4 mg/mL.

Folin-Ciocalteu method was used to determine total phenolic content (TPC) expressed as gallic acid equivalent according to Singleton and Rossi (1965) with some modifications. The extract was diluted eight-fold with distilled water before analysis. Standard calibration curve was prepared by using gallic acid in the concentration range of 0- 0.2 mg/mL. Total flavonoid content (TFC) was expressed as rutin equivalent. Rutin was used as a standard in the concentration range of 0- 0.8 mg/mL to draw calibration curve.

The cupric reducing antioxidant capacity (CUPRAC) was measured according to the method of Apak et al. (2004). The free radical scavenging capacity was determined using the ABTS (2,2′-azinobis-3-ethylbenzotiazoline-6-sulfonic acid) method as described by Re et al. (1999). Trolox standard calibration curve in the concentration range of 0- 0.04 mg/mL was used for quantification.

The α-amylase inhibitory activity was determined according to Esfandi et al. (2022) by using an α-amylase from porcine pancreas (Sigma Chemical Co., St. Louis, MO, USA). Angiotensin-I-converting enzyme (ACE) inhibitory activity was measured based on the principle of inhibiting the conversion of the hippuryl-L-histidyl-leucine substrate into hippuric acid in the presence of an ACE inhibitor substance. ACE from rabbit lung (EC 3.4.15.1, Sigma Chemical Co., St. Louis, MO, USA, 100 mU/mL) was used for the analysis. Hippuric acid in the samples was determined by RP-HPLC using a C18 column (Tracer excel 120 ODSA 5 μm, Teknokroma, Barcelona, Spain).

Statistical analysis

The sample preparation was replicated three times on different days. Analyses were carried out in at least two parallel samples per batch. Two-way ANOVA analysis was used to determine the effects of roasting and fermentation on measured properties of the samples. Tukey’s multiple comparison test was applied to compare means when a significant effect was determined. A significance level of 0.05 was used in all analyses. Minitab software (Version 21, State College, PA, USA) was used as a statistical analysis program.

Results and discussion

Physicochemical properties of pistachio product

Pistachio kernels have a rich nutrient content reflected on the composition of the product (Table 1). The composition of pistachio kernels was comparable to those reported in earlier studies (Tsantili et al. 2010). Dilution of pistachios with water allowed a composition similar to that of yogurt in the product (Craig and Brothers 2021). The final consistencies of the fermented spreadable pistachio products are shown in Fig. 1.

Table 1.

Composition of pistachio kernel and the product obtained

Component	Pistachio kernel (%)	Pistachio product (%)
Fat	43.9	8.1
Protein	25.4	4.7
Carbohydrate	22.0	7.7
Moisture	6.2	78.2
Ash	2.5	1.3
Calories (kcal/100 g)	584.7	122.5

Fig. 1.

Consistency of fermented spreadable pistachio products from raw and roasted pistachios

The pH of pistachio slurries decreased from 6.5 to 4.5 approximately after 5 h of fermentation as a result of sucrose enrichment. Carbon source available in the plant materials is vital for fermentation. Sucrose has been commonly used as an ingredient for enrichment of plant materials such as soy or quinoa in production of yogurt analogues (Huang et al. 2022). Fermentation increased the acidity significantly in pistachio-based products produced from raw and roasted kernels with no significant change during storage (Table 2).

Table 2.

Acidity of fermented pistachio products from raw and roasted kernels during storage

Property	Day	Raw	Roasted
Titratable Acidity (lactic acid, %)	0	0.06 ± 0.01 bB	0.08 ± 0.01 aB
	1	0.31 ± 0.03 aA	0.29 ± 0.04 bA
	10	0.30 ± 0.02 aA	0.29 ± 0.03 aA
	20	0.32 ± 0.03 aA	0.30 ± 0.02 aA
pH	0	6.27 ± 0.06 aA	6.22 ± 0.09 aA
	1	4.36 ± 0.05 aB	4.33 ± 0.04 aB
	10	4.33 ± 0.09 aB	4.31 ± 0.08 aB
	20	4.29 ± 0.04 aB	4.29 ± 0.05 aB

Values are mean ± standard deviation (n = 3). Means marked with different letters are different depending on heat treatment (lower case) and day (upper case) (p < 0.05)

Bioactivity of the pistachio products

Soluble protein content

Soluble protein content shows the presence of small protein aggregates or fractions in aqueous phase of food products. The products with raw and roasted pistachios had similar soluble protein content before fermentation (Table 3). While soluble protein content of the sample with raw pistachios did not change by fermentation, that of the sample with roasted ones decreased. Proteins in oil seeds can undergo changes by roasting such as denaturation, aggregation and cross-linking with other compounds such as carbohydrates via Maillard reaction. These changes cause formation of protein aggregates with reduced solubility. Acidification of the sample by fermentation can cause interactions between these aggregates resulting in lower amount of soluble proteins. On the other hand, proteolysis by LAB in the samples can produce smaller soluble protein fractions. This was clearly observed in both samples over the storage period. Similarly, different lupin varieties fermented with LAB showed an increase in the soluble protein content (Lampart-Szczapa et al. 2006).

Table 3.

Soluble protein and phenolic contents of pistachio products

Property	Day	Raw	Roasted
Soluble protein content (mg/g)	0	16.5 ±1.88 aB	16.3 ± 1.4 aB
	1	16.4 ± 1.07 aB	14.1 ± 1.7 bC
	10	17.5 ± 1.35 aB	15.3 ± 2.0 bBC
	20	21.1 ± 2.34 aA	18.9 ± 1.3 bA
Total phenolic content (mg GAE/100 g)	0	63.6 ± 3.51 aB	62.9 ± 3.9 aB
	1	70.3 ± 3.31 aA	67.0 ± 4.4 bAB
	10	68.7 ± 3.07 aA	67.7 ± 3.5 aAB
	20	69.2 ± 5.14 aA	67.1 ± 2.0 aA
Total flavonoid content (mg RE/100 g)	0	4.5 ± 0.98 aB	4.5 ± 0.8 aB
	1	6.4 ± 1.71 aA	6.1 ± 0.7 aA
	10	5.7 ± 0.37 aAB	5.1 ± 0.4 bB
	20	5.7 ± 0.78 aAB	4.9 ± 0.7 bB

Values are mean ± standard deviation (n = 3). Means were compared according to heat treatment (lower case) and day (upper case) (p < 0.05). GAE: Gallic acid equivalent, RE: Rutin equivalent

Total phenolic and flavonoid contents

Unfermented pistachio slurries from raw and roasted kernels were found to contain similar amounts of phenolic compounds comparable to the levels reported in the literature (Tomaino et al. 2010). Various phenolic compounds including gallic acid, catechin, eriodictyol-7-O-glucoside, genistein-7-O-glucoside, naringenin-7-O-neohesperidoside, quercetin-3-O-rutinoside, genistein, eriodictyol, daidzein and apigenin were reported to be present in pistachio kernels. Flavanols, flavonols, flavan-3-ols, flavanones, isoflavones, proanthocyanidins and anthocyanins were reported as the major flavonoids identified in pistachios (Tsantili et al. 2010).

Roasting pretreatment of pistachio kernels resulted in slightly lower TPC in the fermented product. A similar trend was reported for pistachio flour (Ling et al. 2016). Degradation of phenolic compounds by roasting can be due to oxidation, phenolic-protein interactions and possible interactions between phenolic compounds with Maillard reaction products such as melanoidins (Mehaya and Mohammad 2020). On the other hand, fermentation enhanced the TPC and TFC of the samples significantly. Similar findings were reported for yoghurt analogue produced from fermented soybean and quinoa milks (Huang et al. 2022). Acidic medium and enzymes of LAB capable of hydrolyzing cell wall or the bonds between polyphenols and the cell wall can facilitate the release of phenolic compounds (Liu et al. 2023). Possible depolymerization by acidic medium and bioconversion of polyphenols by LAB during fermentation could also increase the amounts of phenolic compounds and improve their solubility (Thai Huynh et al. 2014). There was a significant decline in TFC with storage time. This can be due to oxidation of flavonoids or their metabolism by LAB (Gaur and Gänzle 2023).

Antioxidant activity

Pistachio kernel is rich in antioxidant compounds including resveratrol, cyanidin-3-galactoside, β-carotene, chlorophylls, lutein, γ-tocopherol, δ-tocopherol and vitamin C (Saitta et al. 2011). Roasting pretreatment caused a slight reduction in antioxidant activity against ABTS in the unfermented pistachio slurry and against copper in the fermented one (Fig. 2a and b). Açar et al. (2009) reported significant reductions in antioxidant activity of pistachio and hazelnut upon roasting at 150 °C for 30 min. Since a mild roasting was applied in this study, the reduction was not as high as those reported in the literature. On the other hand, antioxidant activity of the samples improved by fermentation significantly in accordance with the increases in TPC and TFC. This could be caused by the release of antioxidant components from the kernel matrix to the water-soluble phase by fermentation. Liang et al. (2022) also found an increase in DPPH radical scavenging ability of mung bean milk after fermentation with Lactococcus lactis.

Fig. 2.

Antioxidant (a and b), antidiabetic (c) and antihypertensive (d) activities of pistachio products. ABTS: 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), CUPRAC: Cupric reducing antioxidant capacity. Means marked with different letters are different according to heat treatment (lower case) and storage day (upper case) (p < 0.05)

Antidiabetic activity

Pistachio products exhibited a low level of α-amylase inhibition less than 10% (Fig. 2c). The water-soluble extracts of the samples contained phenolics, flavonoids, proteins and carbohydrates. Bioactive compounds including polyphenols, flavonoids and peptides present in the products can exhibit α-amylase inhibition. In addition, soluble dietary fibers can inhibit α-amylase due to interactions between fibers and active site of the enzyme via hydrogen and hydrophobic bonds (Wang et al. 2023). The roasting applied to pistachios slightly increased α-amylase inhibition of the pistachio products.

α-Amylase inhibition levels of the products from raw and roasted kernels increased after fermentation from 8.15±0.50% and 9.29±0.56 to 9.52±0.51% and 9.83±1.27%, respectively. Effect of fermentation was significant only in the product prepared from raw pistachios. Ayyash et al. (2019) also reported nearly 2-fold and 1.3-fold increase in α-amylase inhibition of lupine after fermentation with Lb. reuteri or Lb. plantarum K779, respectively. On the other hand, they did not find any change in the level of α-amylase inhibition by quinoa and wheat. These findings indicate that the composition of plant material has a major impact on α-amylase inhibition and fermentation with LAB can have an additional effect. A reduction in the α-amylase inhibition was observed during storage. Degradation of inhibitory components by the acidic medium and/or hydrolysis of them by LAB can cause this reduction.

Antihypertensive activity

ACE inhibition levels of the products prepared with raw and roasted kernels enhanced after fermentation from 84.22±0.50% and 46.49±1.73% to 87.19±0.71% and 85.51±0.81%, respectively (Fig. 2d). The level of inhibition was in agreement with that reported for bioactive peptides derived from chymotrypsin digestion of pistachio kernels by Dumandan et al. (2014). Soluble peptides and possibly phenolic compounds released by fermentation and/or acidic medium are responsible for ACE inhibition (Marulo et al. 2024).

Roasting of pistachios negatively affected ACE inhibition resulting in a reduction by nearly 50% while following fermentation reversed this effect. This can be due to breaking down of the aggregates formed through e.g. disulfide bridges by roasting in the acidic medium (Akillioǧlu and Karakaya 2009). ACE-inhibitory activity slightly improved during storage possibly due to proteolysis by LAB. Singh et al. (2020) also found that ACE inhibitory activity of soy milk was enhanced by fermentation with various Lb. plantarum and Lb. rhamnosus strains from ∼ 20% to ∼ 50–70%, which was attributed to the release of ACE inhibitory peptides by bacterial proteases during fermentation. Fermentation of plant materials seems to affect ACE inhibitory activity depending on the composition of plant, starter culture and fermentation conditions.

Conclusions

A plant-based fermented spreadable product was developed from raw and roasted pistachio kernels. Developed pistachio product was found to have a strong antioxidant and ACE inhibitory activities and a low level of α-amylase inhibitory activity. Bioactivities of pistachios mainly originated from natural components while fermentation had a slight positive effect. Roasting generally slightly reduced the bioactivities; however, it had a large adverse effect on ACE inhibition which was eliminated by fermentation. Future studies can focus on determination of bioactivities especially ACE inhibitory activity of pistachios by in vivo analysis for potential health applications. In addition, other possible bioactive properties of pistachios and fermentation with other LAB species can be explored.

Abbreviations

ACE

Angiotensin-I-converting enzyme

CUPRAC

The cupric reducing antioxidant capacity

LAB

Lactic acid bacteria

TFC

Total flavonoid content

TPC

Total phenolic content

Author contributions

EE: Methodology, investigation, formal analysis, data curation, writing– original draft. MKA: Conceptualization, methodology, funding acquisition, project administration, resources, supervision, writing, review and editing.

Funding

This work was funded by the Scientific Research Projects Department of Istanbul Technical University. Project Number: MYL-2022-44247.

Data availability

Data available on request due to privacy restrictions.

Code availability

Not applicable.

Declarations

Ethical approval

Ethical approval was not required for this study.

Consent to participate

All authors have read and approved the MS and are aware of its submission to JFST.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no conflict of interests.