Residual moisture and volatile content determination

Residual moisture and volatile content (%, w/w) of saffron samples were determined in duplicate using the gravimetric method recommended in ISO 3632-2:2010 [2]. Briefly, the plant material (2.5 g) was dried till constant weight in an oven set at 103°C ± 2°C for 16 h. The content was calculated by the following equation:

The relative standard deviation (RSD%) of five measurements per sample extract (n = 5) for five samples (n = 5) by the same analyst was calculated to assess the intra-day precision of the method (1.9% to 6.9%).

CS, FS, and AS estimation

Saffron stigmas were ground in a pestle mortar and passed through a sieve of 0.5 mm mesh, following the crushing and sieving method proposed in ISO 3632-2:2010 [2]. Aqueous extracts of ground stigmas were prepared as follows. An amount of 0.1 g stigmas of ground stigmas (±0.001 g) was mixed with deionized water in a 200 mL volumetric flask. Major constituents (crocetin sugar esters and picrocrocin) were extracted by rigorous agitation (1,000 r/min) at ambient temperature (25°C), away from direct sunlight for 1 h. Prior to analysis, an aliquot from the extract was diluted (1:10, v/v) with deionized water. For each sample studied, duplicate extracts were prepared, and each one was diluted twice. The UV-Vis spectra of water extracts were recorded in the region 200–600 nm using a 1 cm pathway quartz cell in a Shimadzu UV 1601 (Kyoto, Japan) spectrophotometer. The data were processed with the aid of UVProbe 2.61 (Shimadzu Co., Kyoto, Japan) software facilities. CS, FS, and AS of the samples, expressed as $E_{max}^{1\%}$ values were calculated on a dry basis, according to the ISO 3632-2:2010 method. The equation:

Where D represents absorbance value; m represents the mass of the test portion (g); λmax = 440 nm (for crocetin sugar esters); λmax = 257 nm (for picrocrocin); and λmax = 330 nm (for safranal). The reported values are the average values of four measurements per sample, all performed by the same analyst.

Geographical area of the cultivation

According to the Cooperative records (personal communication) most saffron farms are located in a region around the village Krokos that extends to the west and along the lake Polifytos (Figure S1). It is worth noting that only a few of the farms are located more than 30 km away from this focal point, where all the annual produce is collected and further processed to form Krokos Kozanis (e.g., 40°04’13”N, 21°51’48”E to 40°15’3”N, 21°48’4”E) (Figure S1).

Climate parameters

Meteorological data that were extracted from the records of the HNMS [10] show the variation in monthly mean, average max, and average min temperatures (°C), and precipitation volumes (mm) throughout the period from December 2021 to December 2022 (Table 1). The sequence of the growth stages of Crocus sativus plant is given in Figure S2 [11]. Moreover, statistical meteorological data for the period 1955–2022 (from the same source) are presented in supplementary Figures S3 and S4 to assist discussion.

In the period from December 2021 to March 2022, the total precipitation volume was almost 50% higher than the average value of respective data recorded for the period 1955–2021 (Figure S3). In contrast, April and May 2022 were far much drier months; there was a nearly 50% reduced precipitation compared to the reported 65-year average and the trend reversed the summer months (June−August), which were exceptionally wet. September 2022 was unusually dry. This period is critical for boosting the flowering season. October with 32 mm and November with 63 mm were not adequate to result in an appreciable yield per acre (Cooperative administration, personal communication). As far as mean temperature is concerned (Figure S4), there was an ascending trend of more than 1°C in the period October to December 2022, compared to the 65-year average recorded across the timeline. It is stressed that both May and October 2022 were quite hot and dry compared to the historical data signifying the need for further research about the changing climate effect on the local saffron production and the quality characteristics of Krokos Kozanis.

Type of farming

Since the beginning of the century, according to the Cooperative records, the majority of farmers are registered or in the process of achieving organic farming certification.

Scale of individual production

After drying and home-processing by each registered member for about 1–2 months, pure saffron in the form of dried filaments is produced. In the 2022 saffron harvest, the majority of growers delivered to the Cooperative installations approximately 0.01 kg to 2 kg (see Figure 2) indicating that the typical scale of individual production is very small. Nevertheless, about 5% of the growers managed to deliver more than 5 kg of saffron. The distributed sites of home production entail diverse practices (e.g., cleaning and drying conditions, sorting of stigmas, number of persons involved) within this small area of cultivation. However, the product of each grower that is delivered to the Cooperative is further processed (sorting, storage, packaging) and finally, the annual produce is homogenized and standardized as Krokos Kozanis. The particular system describes the conditions under which saffron has been domestically produced in the area around the village of Krokos for more than 50 years.

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Figure 2. 

Box-plot showing the variation in the size of each grower’s annual produce during the 2022 harvest period in the Kozani region, among 611 registered members of the Cooperative. Mean (line) and median (symbol) values are highlighted in the box

Demographic profile of saffron growers

Based on the responses of 172 individual growers (almost 1 out of 3 registered members of the Cooperative) about the location of their farms, the geographical distribution of the respondents covered the whole saffron cultivation area (see Figure S1). Only 11% of those saffron growers are under 35 years old; these young farmers are balanced in gender and with a very high level of literacy (technical schools, university degree, Figure S5A) but also with experience in saffron production, due to their enrolment in past family activities. Their annual produce ranged from 220 g to 1.6 kg with only one of them reaching 3 kg of dry saffron. As far as concerns the rest of the older growers, half of them have not completed high school (secondary education) but there is still a significant proportion (22%) that holds a higher-level diploma (e.g., vocational or academic degree). Approximately 75% of the growers are women (Figure S5B), a figure that deserves special attention considering that saffron production represents mainly a secondary agricultural activity in the local community in Kozani. The available data imply that for 2 out of 3 respondents, saffron cultivation offers a complementary income to their family. The registered growers are adequately educated (Figure S5C and D) for adopting good agricultural and hygiene practices. It is not clear whether the motivation is only economically driven or/and emotionally linked with the local tradition. It is worth noting that a small proportion of the > 35-year-old growers (almost 14%) seem to have been engaged in the cultivation within the last 5 years after being trained by other family members. Over 75% of the respondents are sole members, representing their families in the Cooperative. Given that almost half of the registered members are small-scale producers (e.g., < 1 kg dry saffron on an annual basis, see Figure 2), they claimed much lower demand for external workers in comparison to those who produce up to 5 kg or even 10 kg of saffron. As expected, there is a higher demand during the short, labor-intensive period of harvesting flowers than in the later stages of domestic production [9].

Residual moisture and volatile content data

In the region of Kozani, saffron collection period during which each registered grower may deliver their product to the Cooperative installations lasts about 4 months (until the 31st of March). In the course of the internal QC system of the Cooperative, each product presented in dried, whole filament form is sampled and rapidly inspected (a) with the use of infrared moisture balance, to assess whether its residual moisture falls below an internal maximum limit (< 10%, w/w); and (b) visually, to track and remove residual extraneous matter (inorganic or organic) or floral waste (e.g., pollen). Each delivered quantity is recorded facilitating, thus, the transparency of the supply chain from producers to the Cooperative.

In this study, the standard ISO 3632-2 method was used to assess the moisture and volatile content (%) of 547 test samples. The results verified that almost 90% of the samples that were analysed in a period between 1 month and 3 months after processing had the moisture and volatile content < 10%, in line with the internal specification of the Cooperative’s QC system. The frequency distribution plot, shown in Figure 3, indicates that the majority of the moisture values fluctuated asymmetrically in the range of 6.5−9.5%, with a positive skewness, towards the lower moisture and volatile content values.

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Figure 3. 

Frequency distribution histogram of residual moisture and volatile content values for saffron samples (n = 547) representing each grower’s annual produce during the 2022 harvest period in Kozani region

Among the remaining 10% of the test samples, a few outliers were detected falling below 5.5% (w/w) and quite a larger number surpassed the moisture and volatile content value of 9.5% (w/w). The latter observation could signify individual products that are more vulnerable to spoilage or quality deterioration if stored improperly in the course of processing (e.g., water activity ≥ 0.53 or relative humidity > 23%) [9]. It is worth noting that only a few samples did not comply with the ISO 3632-1 specification for the maximum allowed residual moisture content (i.e., 12%, w/w). They represent, in total, about 8 kg of saffron that were delivered to the Cooperative shortly after drying and cleaning. The relative amount of this type of sample is minor compared to the annual production in the Kozani region during 2022 (1,261 kg). Even so, it is emphasized that the specific internal QC tool of the Cooperative needs to be regularly validated against reference methods for moisture determination. A proper validation plan will help to safeguard the desirable high quality of the PDO Krokos Kozanis.

Metrological issues

The UV-Vis spectral intensities of saffron aqueous extracts at 440 nm, 257 nm, and 330 nm are of major importance for the classification of traded saffron into quality categories. They are used to estimate the ISO-based main quality characteristics of the spice, that is, CS, FS, and AS, respectively. The results are expressed only on a dry basis that presumes the determination of residual moisture content (see Materials and methods). In practice, absorption at 440 nm has the greatest impact on economic transactions because, when a sample fulfills the requirements of any category regarding CS ($E_{440 nm}^{1\%}$, it also fulfills the other spectrophotometric parameters $E_{257 nm}^{1\%}$ and $E_{330 nm}^{1\%}$) for the same category [12]. CS is strongly associated with the actual content of crocins, the dominant pigments in aqueous extracts. These compounds are esters of crocetin mainly with gentiobiose and/or glucose units with a maximum absorption at 440 nm. Their structural characteristics are shown in Figure 4.

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Figure 4. 

Chemical structures and nomenclature of major all-trans and 13-cis-isomers of crocetin esters (I, II) with sugars (A–D) that can be found in saffron extracts with polar solvents

The analytical protocol that is described in the ISO 3632-2:2010 standard refers to ISO 3632-1:2011 for minimum limits and definitions of saffron quality categories. However, there are no accuracy or maximum tolerance data stemming from the application of the protocols. In this study, the RSD% of the spectral intensity of four working solutions (2 working solutions per replicate extract) per tested saffron sample at each wavelength was plotted to assess their range characteristics. As shown in Figure 5, these values were quite low for the majority of the samples tested, ranging from ±0.1% to ±4.7% when absorbance was expressed as $E_{440 nm}^{1\%}$. The RSD% values were also low when absorbance measurements were expressed as $E_{257 nm}^{1\%}$ (up to 4.7%) and $E_{330 nm}^{1\%}$ (up to 6.5%). The results show that the ISO 3632 standard methodology for the pretreatment and subsequent spectrophotometric measurements gave reproducible results for more than 98% of the samples under study. Thus, only in very few cases, the RSD% exceeded 8%. This finding increased confidence in the use of the experimental data for further evaluating the sample-to-sample differentiation in terms of their organoleptic attributes.

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Figure 5. 

Box-plots showing the variation in RSD% values upon measurement of CS, FS, and AS values (E^1% at 440 nm, 257 nm, and 330 nm, respectively) of every individual grower’s annual produce (n = 547) during the 2022 harvest period in the Kozani region. Mean (line) and median (symbol ×) values are highlighted inside each box

Apart from the variation in measurements, it is necessary to emphasize here other two issues of methodological concern. According to ISO 3632, the UV-Vis extinction coefficient values at 257 nm expressing the FS of the spice, are associated with picrocrocin, a known bitter constituent of saffron that absorbs strongly at 250 nm. Nevertheless, existing scientific evidence shows that the characteristic UV-Vis peak of the band at 240–280 nm (see Figure S6) can be ascribed also to crocins, flavonoids, and other glycosylated molecules that are present in aqueous extracts of saffron [12, 13]. The second issue refers to the expression of extinction coefficient values at 330 nm as the ‘AS’ of the spice, implying association with the content in safranal. The latter compound is the main volatile and strongly aromatic component of the product that presents a maximum absorption at around 310 nm. As it has been stated by several researchers in the past [14], the method proposed in ISO 3632-2 for the preparation of a test extract using water as a solvent is not selective for safranal or any other volatile constituent of saffron, due to their lipophilic nature. Therefore, the particular ISO parameter provides only partially reliable information about the intensity of the aroma of the product. It is most likely that $E_{330 nm}^{1\%}$ values are associated with the presence of cis-double bonds in the carotenoid skeleton of crocetin. Cis-crocetin isomers may occur naturally at relatively low concentrations e.g., < 1% (dry weight) in Krokos Kozanis shortly after processing. Thus, an increase in $E_{330 nm}^{1\%}$ values that is usually evidenced on harsh processing or during storage is unlikely to be due solely to the expected release of safranal [14, 15]. It is also important to stress that the ISO 3632-1 standard defines specific thresholds (20 as the minimum and 50 as the maximum) for $E_{330 nm}^{1\%}$ values in traded saffron, regardless of the quality category of the spice. This means that special attention should be given to samples with extreme values (either too low or too high) of the so-called “AS”.

Exploring patterns in the database for Krokos Kozanis quality characteristics

Variability among freshly produced saffron

The results for each of the tested quality parameters indicated wide ranges of values in the annual produce of Krokos Kozanis, e.g., ±80 units for CS, ±25 units for FS, and ±10 units for AS. As shown in Figure 6A, the CS values presented a normal distribution and ranged from 193 units to 290 units. Noticeably, more than 97% of the test samples that were analysed within 1–3 month period after processing, complied with the ISO 3632-1 specifications for classification to category I based on CS ($E_{440 nm}^{1\%}$ > 200), FS ($E_{257 nm}^{1\%}$ > 70), and AS (20 < $E_{330 nm}^{1\%}$ < 50). Apart from this, the vast majority presented CS > 230 units and FS > 80 units. The latter values correspond to the minimum limits that were recently proposed by CCSCH for the classification of traded saffron to the “extra” quality category. Noticeably, 80% of those “extra” quality samples were quite homogeneous regarding CS; the values varied close to a mean value of 245 units ± 15 units (Figure 6A).

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Figure 6. 

Frequency distribution histograms of $E_{max}^{1\%}$ values for the tested saffron samples (n = 547) that represent each grower’s annual produce during the 2022 harvest period in the Kozani region (see Materials and methods). (A) $E_{440 nm}^{1\%}$ (CS); (B) $E_{257 nm}^{1\%}$ (FS); (C) $E_{330 nm}^{1\%}$ (AS)

$E_{257 nm}^{1\%}$ values ranged widely between 70 units and 110 units (Figure 6B) but in half of the tested samples, they varied approximately ±5 units around the mean value (91 units). The frequency distribution analysis implied again, high homogeneity regarding the FS of the annual saffron produced in the Kozani region. It is stressed though that FS exceeded 100 units in a small number of samples that represented 13% of the total annual production. Based on the survey responses of the respective growers, various heating media (e.g., air heater, wood stove, electric oven, etc.) have been used to dry the fresh stigmas in much less than 12 h. This observation may be of importance for future evaluation of the impact of traditional processes on the organoleptic characteristics of Krokos Kozanis. Its chemical composition depends on the drying conditions applied, the intensity of the color and flavor or the aroma notes of the dry stigmas are also expected to vary. For example, the product becomes richer in pigments, picrocrocin, and safranal after short-time drying at relatively high temperatures (70°−90°C) [15–17] as it is also evidenced for color brightness, bitterness, and aroma intensity, assessed by a sensory panel [18].

The $E_{330 nm}^{1\%}$ values signifying the AS ranged between 30 units to 50 units, regardless of whether the samples were classified in the “extra” quality category or not (Figure 6C). In 84% of the samples, AS values ranged between 36 units and 45 units while a minute proportion (approximately 5% of the samples) exhibited $E_{330 nm}^{1\%}$ values that exceeded 45.5 units, very close to the upper ISO threshold. It is stressed that the latter samples displayed high variability in CS, ranging from 164 units to 257 units. This observation verifies that the $E_{330 nm}^{1\%}$ parameter is loosely associated with the quality grading of the spice.

Changes in CS, FS, and AS values upon storage

A second round of analysis was performed for a subset of the 547 stored samples, nearly at the end of the first year after harvest. Sub-sampling was carried out considering: (a) the remaining amount of the stigmas; (b) the representativeness in annual produce; and (c) the initial CS values at the first round of analyses. Thus, we selected 66 samples (whole filaments) that represented nearly 40% of the 2022 production, evenly grouped according to initial CS values, e.g., 200−230 units, 230−250 units, 250−270 units, and > 270 units. The pattern of changes in CS of each stored sample at the end of the first year after its harvest and drying (November 2023) is shown in Figure 7.

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Figure 7. 

Changes in CS values of selected saffron samples (n = 66) that represent approximately 40% of the total 2022 production of Krokos Kozanis, at the end of the first year after harvest. Orange and blue bars correspond to average values from the first round (1 month to 6 months after harvest) and second round of analyses (12 months after harvest), respectively

Based on the CS values (Figure 7), the stored samples presented either a slight increase (4−7%) or decrease (10−11%) relative to the initial values but the changes did not exceed more than ±20 units on a dry weight basis. Despite the fluctuations, CS remained high so that all of the samples were still classified to ISO category I at the end of the first year after harvest. Special attention was paid to a sub-set of potentially extra quality Krokos Kozanis (CS = 230−260) because the majority (33 out of 38) of those samples retained the distinct premium quality characteristic. Moreover, all of the samples with very high initial CS (> 260) could be also classified in the “extra” quality commercial category.

A well-known pattern recognition method that entails PCA [19] of the data helped to get an insight into trends and explore patterns of variation in the compositional data of the 2022 saffron harvest in Kozani. Two principal components (PCs) were extracted from the data explaining 97.4% of the total variance. A closer look into the score and corresponding loading scatterplots of the samples showed a clear pattern of variation across the first PC primarily due to variance in the CS values [R²(X) = 93.3%] (see Figure S7A). Distinct differences in AS and FS [R²(X) = 4.1%] accounted for the sample distribution along the second PC (see Figure S7B and C). It is important to note that the PCA score scatterplot illustrates in a multivariable approach the data shown in Figures 6 and 7. Additionally, the representation of the scattered data reflects multiple sources of product variability in terms of organoleptic quality characteristics that may arise from different production, processing, and storage conditions per grower given the minimal genetic variability of Crocus sativus L. species [20].