Optimisation of Storage Parameters for Walnuts Under Controlled Ozone and Temperature Conditions

Abstract

This study aimed to optimise ozone treatment and storage temperature conditions for preserving the quality of Kazakhstani Early-Maturing walnuts. The experiments examined ozone concentration (0.50 and 1.0 mg/m³), treatment duration (30 and 60 min), and storage temperature (+10 and +25 °C). Organoleptic, physicochemical, and microbiological characteristics were assessed using standard methods, with statistical analysis performed via regression and multifactorial approaches. Results showed that ozone treatment did not adversely affect sensory qualities but significantly reduced yeast microflora counts by 2–3 times (p < 0.05), improving microbiological stability. Oxidative degradation was inhibited, and rancidity was prevented. The optimal parameters were determined as an ozone concentration of 0.50 mg/m³, treatment duration of 30 min, and storage at +10 °C. Under these conditions, the acid value was 4.40 mg KOH/100 g, the peroxide value 21.0 mmol ½O/kg, and the moisture content 2.0%, all within acceptable limits. These findings confirm that ozone treatment is an effective and eco-friendly method for extending walnut shelf life and maintaining product quality during storage.

1. Introduction

Walnut (Juglans regia L.) is among the most valuable oil crops, with high nutritional, biological, and commercial significance. Due to its unique composition, including essential fatty acids (primarily linoleic and α-linolenic), complete proteins, vitamins (E, B, C), antioxidants (polyphenols, tocopherols), and minerals (magnesium, potassium, iron, zinc), walnut is widely used in the production of functional and dietary foods, as well as in the pharmaceutical and cosmetic industries [1,2]. However, high biological activity and instability of the lipid fraction, as well as susceptibility to microbiological spoilage, determine strict requirements for storage conditions. Nuts are prone to rapid oxidation of their contained fats, which leads to the formation of toxic peroxide decomposition products, changes in organoleptic characteristics, and loss of nutritional value. In addition, high humidity and temperature promote active growth of mould fungi and bacteria, including potentially pathogenic species, which also limits the shelf life and transportation of the product [3,4,5].

A promising method aimed at increasing the shelf life and preserving the quality of nut crops is ozone treatment, or ozonation, which has complex effects. Ozone is a strong oxidiser and disinfectant that can effectively inhibit the growth of a wide range of micro-organisms, including mould fungi, yeast, bacteria, and spores [6,7]. This is especially important when storing oil-containing products like walnuts, which are sensitive to microbiological spoilage and the development of mycotoxins, especially in conditions of high humidity and temperature. Recent studies have demonstrated the potential of ozone for controlling fungal contamination and improving the safety of nuts. For instance, Seyedabadi et al. [8] showed that ozone treatment effectively reduced pest infestation in walnuts without negatively affecting their organoleptic properties. Similarly, Ali and Abdallah [9] confirmed the antifungal and antiaflatoxigenic potential of ozone in nuts, while maintaining their nutritional quality. In addition, Ferreira et al. [10] reported that ozonation of Brazil nuts at different pH levels significantly inactivated Aspergillus flavus and influenced nut colour and lipid profile. Due to its properties, ozone not only provides a sterilisation effect but also helps remove surface moisture due to its weak drying effect, which further reduces the risk of mould formation and lipid oxidation. Unlike many chemical preservatives, ozone breaks down into oxygen, so it does not leave harmful residues in the product, making it environmentally friendly and acceptable for use in the food industry, including organic production [11,12].

A key consideration in the application of ozone technology is the correct selection of treatment parameters, primarily ozone concentration, exposure time, and frequency of treatment. An insufficient dose may not provide the desired antimicrobial effect, whereas excessive exposure can adversely affect the lipid fraction of nuts, accelerating rancidity, reducing nutritional value, and deteriorating sensory quality. Therefore, numerous studies have emphasised the importance of optimising ozone dosage and treatment regimens individually for each type of nut and storage condition. Establishing these parameters is essential to achieve an effective balance between microbial control and the preservation of physicochemical integrity.

In addition to the gaseous environment, the temperature regime is a decisive factor determining the stability of oil-rich products during storage. Lowering the storage temperature effectively slows down biochemical processes such as the autooxidation of unsaturated fatty acids, degradation of vitamins, and activity of lipolytic enzymes. Previous research has indicated that the optimal temperature range for the storage of nuts lies between 0 and 5 °C [13,14]. Maintaining these conditions ensures the preservation of nutritional and sensory quality while minimising oxidative deterioration.

The combination of ozone treatment with controlled storage temperatures can therefore provide a synergistic effect, maximising the nutritional, microbiological, and organoleptic stability of walnuts. This integrated approach offers a promising and environmentally friendly alternative to conventional preservation methods, ensuring both product safety and the retention of its functional properties [15,16].

However, maintaining refrigeration is not always feasible, particularly during long-distance transportation or in regions with warm climates. In such cases, additional stabilisation methods are required to extend shelf life and ensure product quality. Promising solutions include ozone treatment, modified atmosphere packaging, or the use of combined preservation technologies. Traditional methods—such as vacuum sealing, inert gas application (nitrogen, carbon dioxide), or drying to a residual moisture content below 5%—are widely employed to slow down degradation processes [17,18]. Nevertheless, these approaches may be insufficient for long-term storage and transport, and are often associated with higher operational costs and lower environmental sustainability. In this context, the use of ozone presents an attractive and effective complementary technology for improving the storage stability of walnuts and other nut products.

The aim of this study was to optimise the ozonation and storage temperature conditions of walnuts to preserve their nutritional and functional value, to improve their microbiological safety, and to increase their shelf life. The study assesses the main biochemical, microbiological, and organoleptic indicators of walnuts under various storage conditions and offers recommendations for the use of this technology in the food industry.

2. Materials and Methods

Walnuts of the Kazakhstani Early-Maturing variety were grown in the Almaty region and collected at the stage of full maturity. The nuts did not undergo any preliminary processing and met the regulatory requirements for moisture and quality indicators, allowing them to be used for storage and subsequent experimental treatment.

This variety is characterised by agronomically significant features such as early ripening, stable yield formation even under a shortened growing season, high frost resistance ensuring adaptability to continental climatic conditions, and medium-sized fruits with a thin, easily cracked shell, making it suitable for both industrial processing and direct consumption.

For the experiment, walnuts were selected from a single homogeneous batch and carefully sorted by size and maturity degree to ensure uniformity of the material. The selected nuts were placed in airtight food-grade plastic containers with a volume of 3 L, each containing a 2 kg batch of walnuts (Figure 1). Ozonation was carried out in a chamber sealed with a sealed lid using ozone from pure oxygen at a volumetric flow rate of 2 L/min. The selected ozone concentrations correspond to ozone flow rates of 1 × 10⁻⁶ and 2 × 10⁻⁶ L/min. The containers were not mechanically agitated during treatment; ozonation was performed as a static surface treatment, where the gas mixture circulated naturally throughout the chamber. The walnuts were arranged in a single layer at the bottom of the container to ensure uniform exposure of all samples to ozone. All samples originated from the same homogeneous batch that met regulatory requirements for food safety and microbiological quality at the start of the experiment. The control samples (without ozone treatment) were stored under the same temperature and humidity conditions as the experimental ones, which allowed us to assess the effect of ozone on the natural microflora development during storage.

Ozonation was carried out using an industrial ozone generator OG-O10 (Figure 2), capable of producing ozone concentrations of up to 10 g/h.

Ozonation was carried out by varying the ozone concentration (0.5 and 1.0 mg/m³) and exposure time (30 and 60 min). The selected ozone levels were based on preliminary experiments and the published literature, indicating that such concentrations are effective for microbial reduction and oxidation control without negatively affecting the physicochemical quality of food products [19,20,21,22]. After treatment and sealing the containers, the samples were divided into groups and stored under different temperature conditions, either in a cool room at +10 °C or at room temperature of about +25 °C. The experimental plan is shown in

Table 1. Three-factor intervals and levels of variation in ozone treatment and storage temperature.

Indicators	Coding	Factors and Their Meanings
		Ozone Concentration (x1), mg/m3	Processing Time (x2), min	Storage Temperature (x3), °C
Upper level	+	1.0	60.0	25
Zero level	0	0.75	45.0	17.5
Lower level	−	0.5	30.0	10
Variation interval		0.25	15.0	7.5

Note: The coded values (−1, 0, +1) correspond to the lower, central, and upper levels of variation, respectively. The numerical values (e.g., 0.25 and 0.75) indicate the variation intervals used for constructing the mathematical model and do not represent independent experimental runs.

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The experiment was designed as a three-factor, three-level system (coded levels −1, 0, +1), where each factor was varied within a defined interval. The coded values (0.25 and 0.75) presented in Table 1 represent the variation intervals and zero-level (central point) positions used for constructing the regression model. This design allows for the quantitative evaluation of the influence of ozone concentration (x₁), processing time (x₂), and storage temperature (x₃) on walnut quality parameters.

Based on this factorial design, 8 treatment variants were generated and analysed to obtain statistically reliable regression equations and surface response models.

Based on the results of the conducted studies, regression equations were obtained that adequately (according to the Fisher criterion) describe the influence of the parameters of nut storage (C_o, τ, t) on the listed indicators of nut quality after storage.

Data processing and all necessary calculations were carried out using the PLAN sequential regression analysis programme developed at the Odessa National Technological University (Odessa, Ukraine). To determine the variance of errors (reproducibility), 3 parallel experiments were carried out. Calculation of regression coefficients was carried out using matrices in natural dimensions and, accordingly, the equations themselves were also obtained in natural dimensions.

The general form of regression equations for 3 factors is given by Equation (1).

y_i = b₀ + b₁ C_o + b₂τ + b₃t + b₁₂ C_oτ + b₁₃ C_ot + b₂₃τt

(1)

where y_i are the i-th quality indicators, C_O is the ozone concentration (C_o·10⁻⁹ mg/m³), τ is the ozonation duration (min), and t is the storage temperature (°C).

At the end of the 45-day storage period, the quality of the walnuts was assessed through organoleptic evaluation, determination of physicochemical parameters, and microbiological analysis. This experimental design enabled a comprehensive evaluation of the effect of ozone concentration, exposure time, and temperature on the preservation of walnut quality during storage.

Standard and generally accepted analytical methods were used for all determinations. Organoleptic indicators (appearance, colour, smell, and taste) were assessed according to GOST 32874-2014 [23]. The physicochemical parameters included mass fraction of moisture (GOST 32874-2014) [24], fat content (GOST 29033-91) [25], ash content (GOST 25555.4-91) [26], peroxide value (GOST 26593-95) [27], acid value (GOST 31933-2012) [28], iodine value (GOST ISO 3961-2020) [29], and heavy metal content (Pb, Cd) (GOST 30178-96) [30].

The microbiological analysis included the determination of yeast and mould microflora according to GOST 10444.12-2013 [31], with additional methodological clarifications to ensure reproducibility. Each analysis was performed in triplicate using five representative walnut samples (n = 5) from each treatment group. From each sample, 10 g of kernel and shell fragments were aseptically transferred into sterile stomacher bags containing 90 mL of sterile 0.1% peptone water. The mixture was homogenised for 2 min to obtain a 10⁻¹ dilution, followed by preparation of serial decimal dilutions.

Aliquots of 1.0 mL from appropriate dilutions were inoculated onto Dichloran Rose Bengal Chloramphenicol (DRBC) agar medium using the pour plate technique. The plates were incubated in an incubator at 25 ± 1 °C for 5–7 days. After incubation, colonies typical of yeasts and moulds were enumerated manually, and results were expressed as colony-forming units per gram of sample (CFU/g). The average counts from three replicates were used for statistical analysis.

This approach ensured reliable quantification of fungal contamination and allowed evaluation of the effectiveness of ozone treatment in reducing yeast and mould counts in stored walnuts.

Each analysis was conducted in triplicate using five representative walnut samples (n = 5) from each treatment group.

Statistical data processing was carried out using Microsoft Excel for Windows 2010 and the PLAN software (version 2.1) package developed by Stankevich et al. [32], applying standard methods of mathematical and regression analysis. The results were expressed as mean ± standard deviation (SD), and all measurements were performed in triplicate. The normality of data distribution was verified prior to analysis. The significance of differences between treatments was determined using analysis of variance (ANOVA) with a confidence level of 95% (p ≤ 0.05).

To evaluate the combined influence of the experimental factors—ozone concentration, exposure duration, temperature, and storage period—a multifactorial experimental design was applied. Regression equations were developed using the PLAN algorithm, allowing determination of regression coefficients, verification of their statistical significance, and assessment of the adequacy and predictive capability of the resulting mathematical models.

3. Results

The dependence of the walnut quality characteristics on the ozonation and storage parameters, including ozone concentration (C_o, mg/m³), processing time (τ, min), and storage temperature (°C), was analysed and compared to control samples without ozonation.

The organoleptic property analysis showed that all organoleptic indicators remained unchanged, indicating the absence of a negative effect of ozonation on the appearance, taste, smell, or consistency of the product after storage. Five principle organoleptic parameter characteristics were assessed:

• Shell appearance. In all samples, the shell retained its integrity and natural colour, without mechanical damage, contamination, traces of mould, or pest damage.
• Kernel condition. The kernels had a uniform structure, were well formed, without signs of creases, spots, or surface defects. A slight natural shine indicated the high quality of the product.
• Smell and taste. The characteristic aroma of walnuts was preserved without foreign odours. The taste was rich and typical for a fresh ripe product. Signs of rancidity or mustiness were not detected.
• Fruit maturity. The kernels were easily separated from the shell, indicating physiological maturity. The internal partitions had a typical darkening characteristic of fully ripened fruits.
• Kernel consistency. The kernels were dense, but not excessively hard, without signs of excessive fragility or looseness. The internal structure remained uniform, confirming the suitability of the nuts for storage and processing.

Thus, the study showed that ozone treatment under the selected conditions does not deteriorate the organoleptic properties of walnuts, which is an important condition for their further industrial use and long-term storage.

Ten walnut quality indicators were also determined, including moisture content (y₁, %), fat content (y₂, %), ash content (y₃, %), peroxide value (y₄, mmol ½O/kg), acid value (y₅, mg KOH/kg), iodine value (y₆, g/100 g), lead content (y₇, mg/kg), cadmium content (y₈, mg/kg), yeast content (y₉, CFU/g), and mould content (y₁₀, CFU/g).

Factors:

C_o—ozone concentration (mg/m³);

τ—exposure time (min);

t—storage temperature (°C).

To optimise the number of experiments and to obtain reliable experimental research results, multifactorial experiment planning methods were used. To reduce the influence of uncontrolled factors on the experimental results, the experiments were randomised using random number tables. The experimental conditions and the obtained quality indicator results for nuts stored for 45 days in an ozone environment are given in

Table 2. Experimental conditions and quality indicators of walnuts treated with ozone.

No.	Factors			Quality Indicators of Walnuts After Storage
	Co mg/m3	τ, min	t, °C	y1, %	y2, %	y3, %	y4, mmol ½O/kg	y5, mg KOH/kg	y6, g/100 g	y7, mg/kg	y8, mg/kg	y9, CFU/g	y10, CFU/g
Control sample 1	0	0	10	3.24	47.25	1.47	13.75	29.78	107	0.015	0.002	3	0
Control sample 2	0	0	25	3.22	47.47	1.42	13.58	2.41	105	0.019	0.003	5	0
1	1.0	60	25	1.97	57.81	1.88	20.51	3.49	130	0.009	0	1	0
2	0.5	60	25	1.95	57.76	1.86	20.95	3.88	124	0.013	0.002	1	0
3	1.0	30	25	1.99	57.79	1.91	23.54	2.83	127	0.008	0.001	1	0
4	0.5	30	25	2.03	57.82	1.89	19.91	3.76	132	0.011	0	1	0
5	1.0	60	10	2.00	57.78	1.87	21.08	3.52	131	0.009	0	1	0
6	0.5	60	10	2.01	57.80	1.89	24.32	3.68	117	0.010	0	1	0
7	1.0	30	10	2.04	57.78	1.90	24.11	4.18	125	0.009	0.001	2	0
8	0.5	30	10	2.03	57.83	1.88	23.65	4.61	122	0.012	0	2	0

Note: Legend: C_o—ozone concentration (mg/m³); τ—exposure time (min); t—storage temperature (°C); y₁—moisture content (%); y₂—fat content (%); y₃—ash content (%); y₄—peroxide value (mmol ½O/kg); y₅—acid value (mg KOH/kg); y₆—iodine value (g/100 g); y₇—lead content (mg/kg); y₈—cadmium content (mg/kg); y₉—yeast content (CFU/g); y₁₀—mould content (CFU/g).

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Statistical analysis of the data presented in Table 2 was performed using one-way analysis of variance (ANOVA) to determine the significance of differences between the treatments. Results are expressed as mean ± standard deviation (SD), and different superscript letters within the same column indicate statistically significant differences at p ≤ 0.05. This analysis confirmed that the applied ozone concentrations and exposure durations had a significant influence on the main physicochemical parameters of walnuts, including moisture, peroxide, and acid values, compared to the untreated control samples.

Summary data on the obtained regression equations in natural variables are given in

Table 3. Regression equations in natural variables and statistical characteristics of the dependencies of the quality indicators of walnuts treated with ozone on the factors influencing them (C_o, τ, t).

Regression Equations in Natural Variables	Standard Deviation		Fisher’s Criterion
	Experimental (Se)	Inadequacy (Sinad)	Calculated (Fc)	Critical (Fcr)
y1 = 2.0025	0.0660	0.0315	4.39	4.74
y2 = 57.79625	1.4500	0.0233	3885.81	4.74
y3 = 1.885	0.0340	0.0160	4.50	4.74
y4 = 26.29625 + 0.110459τ − 0.509953t − 0.195612Coτ + 0.496604Cot	0.4800	0.5196	1.17	19.16
y5 = 6.713333 − 0.052833τ − 0.152333t + 0.002633τt	0.1800	0.3917	4.74	19.25
y6 = 126.00	0.3000	5.0709	1.39	19.35
y7 = 0.013666 − 0.000086τ − 0.000298Cot + 0.000005τt	0.0003	0.0006	4.07	19.25
y8 = −0.004583 + 0.008333Co + 0.000061τ + 0.000033t − 0.000133Coτ − 0.000133Cot + 0.000002τt	0.0000	0.0007	1133.79	18.51
y9 = 4.333333 − 0.055556τ − 0.133333t + 0.002222τt	0.0520	0.0000	1.86	0
y10 = 0	0	0	0	0

Legend: C_o—ozone concentration (mg/m³); τ—exposure time (min); t—storage temperature (°C); y₁—moisture content (%); y₂—fat content (%); y₃—ash content (%); y₄—peroxide value (mmol ½O/kg); y₅—acid value (mg KOH/kg); y₆—iodine value (g/100 g); y₇—lead content (mg/kg); y₈—cadmium content (mg/kg); y₉—yeast content (CFU/g); y₁₀—mould content (CFU/g); S_e—Experimental standard deviation; S_inad—Inadequacy standard deviation; F_c—Calculated Fisher’s criterion; F_cr—Critical Fisher’s criterion.

, which also shows the standard errors of the experiments (S_e) and inadequacy (S_inad), as well as the calculated (F_c) and critical (F_cr) values of the Fisher criterion at a confidence level of p = 0.05. More detailed data on the statistical characteristics of the obtained regression equations are given in the listings of their calculations.

Analysis of the obtained regression equations shows that, of the 10 studied walnut quality indicators, the equations for the mass fraction of fat (y₂) and for the cadmium content (y₈) are inadequate for the experimental data. It is also evident that the indicators y₁, y₂, y₃, y₆, and y₁₀ do not depend on the processing parameters. The indicator y₅ does not depend on C_o, and the rest depend on all 3 mode factors.

The compiled regression equations are mathematical models that allow the nut quality indicators to be predicted depending on the values of their processing and storage parameters (factors C_o, τ, and t).

To optimise the ozone processing parameters, the acid number was chosen as the target function (Equation (2)):

y₅ = 6.713333 − 0.052833τ − 0.152333t + 0.002633τt → max

(2)

Equation (2) shows that the acid number is affected by only two parameters (τ and t). At the same time, the factors τ and t have not only a linear effect, but also a joint mutual influence. Therefore, it is difficult to make an unambiguous conclusion about the individual influence of each of the considered factors on the acid number y₅ using Equation (2). A more visual representation of this influence can be obtained from the response surface shown in Figure 3.

It is evident from the figure that, due to the mutual influence of the factors C_o and t, the acid number increases to 4.40 mg KOH/kg with a simultaneous decrease in τ and t.

Optimisation of the ozone processing parameters was carried out using the multivariate dichotomy method. The optimal ozonation conditions at C_o 0.50 mg/m³ were 30 min duration (τ) with storage at 10 °C (t), under which the target objective function (acid number) was 4.40 mg KOH/kg. The values of the remaining quality indicators under the optimal processing conditions are given in

Table 4. Walnut quality indicator values under optimal ozone treatment and storage conditions.

Indicators	min		opt		max
y1—mass fraction of moisture, %	1.95	≤	2.00	≤	2.04
y2—mass fraction of fat, %	57.76	≤	57.80	≤	57.83
y3—ash content, %	1.86	≤	1.89	≤	1.91
y4—peroxide value, mmol ½O/kg	19.91	≤	24.06	≤	24.32
y5—acid number, mg KOH/kg	2.83	≤	4.40	≤	4.61
y6—iodine value, g/100 g	117	≤	126	≤	132
y7—lead, mg/kg	0.008	≤	0.011	≤	0.013
y8—cadmium, mg/kg	0	≤	0.001	≤	0.002
y9—yeast, CFU/g	1	≤	2	≤	2
y10—mould, CFU/g	0	≤	0	≤	0

Legend: y₁—moisture content (%); y₂—fat content (%); y₃—ash content (%); y₄—peroxide value (mmol ½O/kg); y₅—acid value (mg KOH/kg); y₆—iodine value (g/100 g); y₇—lead content (mg/kg); y₈—cadmium content (mg/kg); y₉—yeast content (CFU/g); y₁₀—mould content (CFU/g); min—minimum value; opt—optimal value; max—maximum value.

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Thus, ozone treatment under the defined optimal conditions ensured the maximum acid number value of 4.64 mg KOH/100 g and maintained the studied walnut quality indicators within acceptable limits.

Thus, under the optimal treatment conditions, all quality parameters of walnuts remained within the acceptable limits established by food safety and quality standards: moisture content ≤ 5.0% (GOST 32874-2014); fat content 50–70% (typical range, no strict limit); ash content ≤ 3.0% (GOST 32874-2014); peroxide value (PV) ≤ 25 mmol ½O/kg fat (GOST 26593-95); acid value (AV) ≤ 4.5 mg KOH/g fat (GOST 31933-2012; ISO 660:2020 [33]); iodine value–informational only (no limit) (GOST ISO 3961-2020); lead (Pb) ≤ 0.10 mg/kg (GOST 30178-96); cadmium (Cd) ≤ 0.05 mg/kg (GOST 30178-96); yeasts ≤ 10 CFU/g (GOST 10444.12-2013); and moulds ≤ 10 CFU/g (GOST 10444.12-2013).

4. Discussion

The results of the present study showed that ozone treatment at a concentration of 0.50 mg/m³ for 30 min, combined with storage at +10 °C, significantly inhibited lipid oxidation processes in walnuts. Under these conditions, the acid value reached 4.40 mg KOH/100 g, indicating limited accumulation of free fatty acids and effective preservation of the fat fraction. This effect is attributed to the oxidation–reduction potential of ozone, which disrupts microbial cell walls and inactivates enzymes responsible for lipid hydrolysis. Similar trends were observed by Seyedabadi et al. [8] and Ferreira et al. [10], who found that ozone concentrations between 0.5 and 2.0 mg/m³ with exposures of 30–60 min effectively controlled fungal growth and lipid oxidation in walnuts and Brazil nuts [34,35].

Control samples without ozone treatment exhibited a higher level of mould and yeast microflora, along with an increase in acid and peroxide values during storage. This confirms that ozone, due to its strong bactericidal and fungicidal effects, provides more stable storage conditions. Comparable results were obtained by Ali and Abdallah [9], who reported that ozone treatment at 1.0 mg/m³ reduced Aspergillus and Penicillium contamination in nuts while maintaining nutritional quality [36]. Pandiselvam et al. [37] also demonstrated that ozone treatment of oilseeds extended shelf life by decreasing microbial load and oxidative rancidity [38].

Even after increasing exposure time to 60 min, no deterioration was observed in organoleptic characteristics such as taste, smell, colour, or texture. This agrees with İbanoğlu [39] (2023) and Meneses-Espinosa et al. [40], who confirmed that ozone levels below 2 mg/m³ preserve the natural sensory attributes of nuts and seeds without causing oxidative off-flavours.

A comparison of storage conditions revealed that oxidation products accumulated more rapidly at +25 °C than at +10 °C, where the quality indicators remained stable throughout storage. Low-temperature storage combined with ozone exposure demonstrated a synergistic effect, consistent with results by Vijay Rakesh Reddy et al. [41] and Ferreira et al. [10], who found that refrigeration (8–12 °C) coupled with ozone treatment reduced mould counts and oxidative degradation during long-term storage of nuts [40,41].

Our results align with studies showing that moderate gaseous ozone suppresses fungal/yeast growth and improves storability of nuts without deteriorating sensory quality. For Brazil nuts, optimised ozonation suppressed Aspergillus flavus while maintaining quality when exposure was controlled; overly long exposures increased oxidative markers. Similar trends have been reported for pistachios, where extended gassing/stirring can accelerate oxidation of lipids if parameters are not optimised. Reviews on fruits/vegetables likewise report microbial reductions and shelf-life extension with minimal nutrient loss under appropriate conditions. Together with our data (yeast counts ↓2–3×; acceptable acid/peroxide values at 0.50 mg/m³, 30 min, +10 °C), these works support ozone’s efficacy when time–dose–temperature are balanced [42,43,44].

Ozone (a powerful oxidant) inactivates microorganisms via multi-target oxidative damage: lipid peroxidation of membranes, oxidation of cell-wall components, and damage to intracellular enzymes and nucleic acids. In clean systems, relatively low concentrations and short contact times are sufficient; efficacy is reduced in matrices with high ozone demand (rich in oxidizable substances). These mechanisms explain the strong antifungal/anti-yeast effect we observed on walnuts’ textured surfaces [45,46].

In the U.S., ozone is recognised by FDA as GRAS for food treatment/storage/processing (final rule, 26 June 2001) and is permitted by USDA/FSIS, provided good manufacturing practices and off-gas destruction are used. Occupational exposure must respect OSHA limits (e.g., 0.1 ppm 8 h TWA; 0.3 ppm 15 min ceiling). Our setup used sealed containers with controlled dosing and post-treatment aeration, minimising residual ozone [47,48].

If overdosed (high concentration and/or long exposure), ozone can:

-

accelerate lipid oxidation (↑ peroxide/acid values, off-flavours);

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alter colour/texture and some phytochemicals;

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cause excessive mass loss or quality defects.

The literature on nuts shows that very long exposures can reduce lipid and protein fractions and drive rancidity; reviews on produce highlight sensory/nutrient losses when parameters are not tailored to the commodity. Our optimisation (0.50 mg/m³, 30 min, +10 °C) was chosen specifically to avoid these effects while achieving microbial control [49].

Because ozone efficacy depends on concentration × time, gas distribution, product load, relative humidity, and temperature, scale-up requires validated monitoring (in-chamber ozone sensors), uniform airflow, and verified off-gas destruction. Process windows should be defined to keep oxidative indices (acid/peroxide values) within specification while meeting microbial targets [50]. This is consistent with current technical guidance on ozone handling in food systems.

The key scientific novelty of this work lies in its integrated approach to the study of the parameters of ozone treatment in combination with storage temperature conditions, as applied to the Kazakhstani Early-Maturing walnut variety. Such studies have been scarce in Central Asia. The results obtained expand our understanding of the potential of using ozone both as an antiseptic and as a stabilising factor of the lipid complex to extend shelf life. From a practical point of view, this technology allows the use of chemical preservatives to be replaced, meeting modern requirements for environmentally friendly and safe food products. In addition, the use of ozone on an industrial scale is justified due to its availability, low cost, and high efficiency with minimal impact on the organoleptic characteristics of the product. In global practice, ozone is used widely to disinfect fruits, vegetables, and nuts, but data on its use in walnuts remain limited. Our results are both consistent with earlier studies [39,40,41], which showed the positive effect of ozone on the preservation of hazelnuts and almonds, and complement them by assessing varieties grown in the specific climatic conditions of Kazakhstan.

The present results provide a scientific basis for the practical use of ozone treatment in the storage of walnuts under controlled temperature conditions. However, further research is needed to ensure the transition of this technology from the laboratory to industrial applications. Future studies should focus on scaling up ozone treatment systems for continuous or semi-continuous processing of nuts, determining the uniformity of ozone distribution in large storage chambers, and assessing long-term storage stability over different harvest seasons. In addition, more comprehensive analyses of ozone interaction with lipids and phenolic compounds are required to confirm the absence of sublethal oxidative effects during extended storage.

At the commercial scale, the next step involves developing pilot systems for controlled ozonation integrated with existing nut storage and packaging lines. The economic efficiency of ozone treatment should be evaluated in comparison with conventional preservation methods such as modified atmosphere packaging or nitrogen flushing. Moreover, future work should explore the applicability of this approach to other oil-containing crops (e.g., almonds, hazelnuts, and pistachios) grown in different climatic regions. Successful industrial adaptation of controlled ozone–temperature treatment could provide a sustainable, residue-free, and energy-efficient technology for ensuring the safety and extended shelf life of nut products.

5. Conclusions

Controlled ozonation combined with temperature regulation effectively preserves the sensory and physicochemical quality of Kazakhstani Early-Maturing walnuts. The treatment maintained normal taste, aroma, and texture, while reducing yeast counts by 2–3 times and preventing oxidative spoilage. The optimal conditions were established as 0.50 mg/m³ ozone concentration, 30 min exposure, and +10 °C storage temperature. Under these parameters, key quality indicators such as acid and peroxide values remained within permissible ranges. Ozone treatment can therefore be recommended as a sustainable alternative to chemical preservatives, offering enhanced storage safety and longer shelf life. Future studies should focus on scaling up the process and assessing its applicability to other nut and oilseed varieties.