Microbiological and Physical–Chemical Quality of Pickled Vegetables Produced by Rural Family Agribusinesses ^†

Abstract

The aim of the present study was to assess the microbiological and physical–chemical quality of pickled vegetables produced by rural family agribusinesses in the mountainous region of Espírito Santo State, Brazil. This descriptive, observational and cross-sectional study was carried out on the basis of secondary data provided by the Food Monitoring Program of Espírito Santo State, Brazil. Data were extracted from sample collection terms and from analysis reports on 58 samples of pickled vegetables produced by 37 rural family agribusinesses, which were collected between June and September 2022. The analyses applied to these samples included Salmonella sp. incidence; molds, yeasts, and Enterobacteriaceae counts; and pH, titratable acidity, and chloride content in sodium chloride determination. The microbiological limits for these products are the absence of Salmonella sp., 10² for Enterobacteriaceae, and 10³ for molds and yeasts. For physical–chemical quality, these products must have a pH of 4.5 or lower. All samples were in compliance with the legislation provided for Salmonella and Enterobacteriaceae. In total, 13.79% of the samples (n = 8) presented mold and yeast counts higher than the microbiological limit of 10³. The pH of 12.06% (n = 7) of the samples was higher than 4.5, which exceeded the limit established by the legislation. The titratable acidity ranged from 0.25 to 2.82 g of acetic acid/100 g; its mean value reached 0.89 g of acetic acid/100 g. The chloride values ranged from 0.10 to 5.70 g of NaCl/100 g; its mean value reached 1.85 g of NaCl/100 g. Thus, 25.86% (n = 15) of the samples did not comply with the legislation. These results suggested that microbiological and physical–chemical quality were compromised in some of the analyzed samples. This finding indicated likely flaws in good manufacturing practices and quality controls, and it can pose risks to consumer health.

1. Introduction

Vegetables are sources of vitamins, minerals, dietary fiber, and antioxidants that, in turn, are important components of healthy human diets. Adequate intake of these food types is associated with a lower risk of developing chronic noncommunicable diseases [1]. Vegetables present a short shelf-life, since they remain metabolically active after harvest, in addition to being exposed to physical, chemical, and biological factors and to the influence of environmental factors capable of affecting their appearance, flavor, aroma, and nutritional value, as well as their deterioration [2].

Transforming fresh food into processed food in undertakings, such as rural family agribusinesses, is an alternative to avoid waste and losses, to extend the postharvest consumption period, to use surplus derived from agricultural production, to add value to raw materials, and to increase one’s income [3].

Rural family agribusinesses are companies owned by family farmers who operate in rural areas and occupy spaces exclusively used for food-processing purposes or who use their home kitchen, or a space adjacent to it, as well as family labor [4,5]. These companies preserve traditions and cultural habits. Their recipes are part of family traditions that are passed on from generation to generation. Their production process is carried out at a small scale; it is an art rather than just a technique, and it makes their products unique in terms of flavor and presentation [6] from other products, since their quality is different from that of conventional products; moreover, it generates an artisanal, colonial, and agroecological image for consumers [7].

In Brazil, the sector that manufactures canned vegetables and similar products produced 973,046 tons and sales of 1,015,408 tons in 2021 [3,5]. Pickled vegetables stand out among several food types processed in agrobusinesses. According to the Brazilian National Health Surveillance Agency, this product is prepared with the edible parts of tubers, roots, rhizomes, bulbs, stalks, sprouts, leaves, inflorescences, petioles, fruits, seeds, and cultivated mushrooms, which are packaged virtually raw, rehydrated or precooked, immersed or not in appropriate covering liquid, and exposed to technological processing before or after they are hermetically closed in containers to avoid changes in them [8].

Pickled vegetables are susceptible to contamination at different production, distribution, and consumption chain stages. Therefore, it is essential to comply with good manufacturing practices throughout the entire production chain [9] to prevent these food products from becoming potential carriers of foodborne diseases and from posing risks to consumer health [8]. However, high costs and strict requirements to meet food quality and safety standards, as well as knowledge and financial limitations, make agribusinesses face difficulties in following health regulations and, consequently, in expanding their access to markets to sell their products [7,8].

According to public data from the Ministry of Health, in Brazil, 16,839 outbreaks of foodborne illnesses were reported between 2000 and 2023, totaling 304,147 cases and 262 deaths [10]. Considering this, assessing the quality of pickled vegetables through microbiological and physical–chemical analyses can indicate the safety level of these food products in terms of risks to population health [11]. However, research on pickled vegetables produced by agribusinesses remains scarce. This finding makes it difficult to formulate public policies, as well as to develop projects in the health sector and actions to be taken by public authorities to help family farmers increase the supply of nutritional- and health-quality products, especially agroecological ones [4].

Considering that most studies in the literature focus on large-scale production chains or standardized industrial environments, this work directs its attention to locally based businesses. The aim of the present study was to assess the microbiological and physical–chemical quality of pickled vegetables produced by rural family agribusinesses operating in the mountainous region of Espírito Santo State, Brazil. The research sought to identify potential risks to food safety and to verify whether the products meet the quality standards required for consumption, considering the specific production and processing conditions adopted by these local businesses.

2. Materials and Methods

2.1. Study Type and Sample Collection Data

This descriptive, observational, and cross-sectional study was carried out with secondary data derived from the monitoring process applied to pickled vegetables produced in Espírito Santo State and collected between June and September 2022 as part of a State Food-Monitoring Program (PROALI) developed by the State’s Health Surveillance Agency in Vitória City/ES, Brazil.

Data extracted from sample collection terms, such as sample collection date, product name, brand, producer, manufacturing date, expiration date, batch, packaging content, and information about the manufacturer (name and full address), as well as data from microbiological (Salmonella sp., Enterobacteriaceae, and Molds and Yeasts) and physical–chemical (pH, titratable acidity and chlorides in sodium chloride) analysis reports, were used to assess the microbiological and physical–chemical quality of the pickled vegetables.

The inclusion criteria adopted to form the investigated sample comprised all data on pickled vegetables immersed in covering liquid, acidified either artificially or by fermentation, are naturally acidic, or marinated, which were produced by rural family agribusinesses operating in the mountainous region of Espírito Santo State and collected between June and September 2022. This region was selected at the beginning of agritourism and family agribusiness development [12]. It comprises 11 municipalities, namely Afonso Cláudio, Alfredo Chaves, Castelo, Conceição do Castelo, Domingos Martins, Marechal Floriano, Santa Leopoldina, Santa Maria de Jetibá, Santa Teresa, Vargem Alta, and Venda Nova do Imigrante. Samples whose analysis results were inconclusive were excluded from the study.

Pickled vegetables that gave rise to the analysis reports and sample collection terms were randomly collected by health authorities from the state’s Health Surveillance Agency in the retail trade operating in the mountainous region of Espírito Santo State, as well as at sale points located in the agribusiness companies themselves, without previous notice, as these food products were put for sale and offered to consumers on the basis of their availability at collection time. Each product was collected in a single sample, at a minimum amount of 100 g. The collected products were packaged in sealed and labeled packages; a sample collection form was issued for each product, which included sample data, such as sales description, brand, producer, manufacturing date, expiration date, batch, packaging content, packaging type, and information about the manufacturer (name and full address). Furthermore, the aforementioned form comprises information about the sample collection itself (date, time, collection location, food temperature at collection time, and collection purpose) and about all the requested analytical tests. The products were subsequently sent to the Central Public Health Laboratory of Espírito Santo (LACEN/ES), where they were subjected to laboratory analyses, whose final reports were issued and sent to the State Food Monitoring Program.

2.2. Microbiological Analyses

Microbiological analyses focused on investigating the incidence of Salmonella spp. Enterobacteriaceae, as well as molds and yeasts, were applied to pickled vegetables by LACEN/ES. All analyses were applied to each sample in triplicate.

The Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) method, Chapter 5: Salmonella, online version [13], was used to investigate Salmonella. The results are expressed as the presence or absence of 25 g [14,15]. Enterobacteriaceae counting was based on the deep-sowing plate-count method, according to the methodology described by the American Public Health Association (APHA) [16]. The results are expressed in colony-forming units (CFUs)/g, and the adopted microbiological limit was 10² CFU/g [17].

Mold and yeast counts were performed via the surface-sowing plate-count method, according to the FDA/BAM methodology, Chapter 18: Yeasts, Molds and Mycotoxins [18]. The results are expressed in CFU/g, and the adopted microbiological limit was 10³ CFU/g [14,15].

The results of the microbiological analyses were compared with the microbiological standards set by Brazilian sanitation legislation for food products for compliance-checking purposes [14,15,17,19].

2.3. Physicochemical Analyses

The physical–chemical analyses of the pickled vegetables were carried out by LACEN/ES to determine the pH, titratable acidity, and chloride content in the sodium chloride. All analyses were applied to each sample in triplicate.

The pH determination process was based on analytical standards set by the Adolfo Lutz Institute (IAL) [20], procedure n.017/IV. The results were assessed on the basis of sanitary laws, which establish a pH value for pickled vegetables equal to or lower than 4.5 [8,21].

Titratable acidity was determined via potentiometric titration according to the analytical standards set by IAL [20], procedures n. 311/IV and 312/IV. The results are expressed as g of acetic acid/100 g.

The test applied to chlorides in sodium chloride (NaCl) was based on precipitation titration, according to the analytical standards set by the IAL [20], procedure n. 028/IV. The results are expressed as g of NaCl/100 g, mass/mass.

Analyses of both the titratable acidity and the chloride content of sodium chloride were used to feature the sample since it lacks parameters provided by sanitary legislation.

2.4. Data Analysis

The data were tabulated in a Microsoft Excel^® 2019 spreadsheet, and the samples were categorized on the basis of similar pickled vegetable features to compare the results. The data were subjected to descriptive statistical analysis.

2.5. Ethical Aspects

The study was approved by the Research Ethics Committee (CEP-Comitê de Ética em Pesquisa) of the Health Sciences Center of Federal University of Espírito Santo (UFES), in compliance with guidelines set by the National Health Council, under CAEE n. 66175522.5.0000.5060 and Opinion n. 5895.875. The Institute of Education, Research, and Innovation in Health (ICEPi) provided authorization to collect secondary data at the State Health Secretariat of Espírito Santo (Sesa) at the state’s Health Surveillance Agency.

3. Results and Discussion

Analysis reports of 59 samples of pickled vegetables monitored by the State Food Monitoring Program were assessed. One sample was excluded from the study because it was a pickled vegetable that did not meet the inclusion criteria of having been produced by a rural family agribusiness operating in the mountainous region of Espírito Santo State, Brazil. Thus, 58 samples produced by 37 rural family agribusinesses were selected for this study. They were grouped into 5 categories, namely appetizer/caponata, pickled mushrooms, vegetable pickles, pickled peppers, and dried tomatoes/confit (Figure 1).

3.1. Microbiological Quality of Pickled Vegetables

Salmonella sp. was not detected in any of the samples analyzed (

Table 1. Microbial count per category in pickled vegetable samples.

Pickled Vegetables Category	N. of Samples	Salmonella sp. (in 25 g) (1)	Enterobacteriaceae (CFU/g) (1)	Molds and Yeasts (CFU/g) (1)
Appetizer/caponata	12	Not detected	<102	<10 a <103
Pickled mushroom	9	Not detected	<102	<103 a <1.5 × 104
Vegetable pickles	16	Not detected	<102	<10 a <103
Pickled pepper	13	Not detected	<102	<10 a <6.0 × 103
Dried tomato/confit	8	Not detected	<102	<102 a <103

⁽¹⁾ Microbiological limit established by Brazilian sanitation legislation: Salmonella sp. = not detected, Enterobacteriaceae = 10², and molds and yeasts = 10³ for food types analyzed in the current study. a: Connectives indicating numerical ranges, equivalent to “–”.

). Similar results were reported by Rodríguez-Cavallini et al. [22], who assessed ready-to-eat food types, such as pickled samples—small pickles and appetizers—processed by small companies and did not find Salmonella in them. The intestinal tracts of humans and animals, mainly poultry and pigs, are the main Salmonella reservoirs [23]. This bacterium is found in animal-origin products, such as meat, poultry, eggs, dairy products, and seafood [24], although raw vegetables have been listed as significant sources of Salmonella [25]. Food contamination by these microorganisms can take place because of failures in temperature control, improper food handling, or cross-contamination by previously contaminated surfaces, containers, and kitchen utensils [26]. Therefore, the results of the present study demonstrated that the assessed pickled vegetables were appropriately handled and processed and that they were in compliance with Brazilian sanitation legislation for Salmonella sp.

All the samples presented Enterobacteriaceae counts < 10² CFU/g (Table 1); they were in compliance with the legislation. A similar result was reported by Torres et al. [27], who assessed pickled pumpkin and pink pepper and reported Enterobacteriaceae counts < 10² CFU/g (estimated value).

The family Enterobacteriaceae is found in the environment, as well as in the intestines of both humans and animals [11]. It is capable of colonizing several parts of processing plants, but it is easily inactivated by sanitizers and destroyed by heat treatment [28]. Therefore, it is a useful indicator of hygiene in manufacturing processes, process failures, or postprocessing contamination in pasteurized food products [28]. Therefore, the results of the assessment herein applied to pickled vegetables suggested that these food products were manufactured under hygienic conditions and appropriately processed since they were in compliance with the legislation.

Mold and yeast counts higher than the microbiological limit of 10³ were observed in 13.79% (n = 8) of the samples; 05 of them belonged to the pickled mushroom category, and 03 belonged to the pickled pepper category. Mold and yeast counts in these pickled vegetable categories ranged from <10³ to 1.5 × 10⁴ and from <10 to 6.0 × 10³, respectively (Table 1). These results differed from those reported by Aljahani [11], who assessed 90 samples of pickled pickles purchased from retail stores in Saudi Arabia and recorded mold and yeast counts ranging from 0.0 to 1.50 ± 0.09 log₁₀ CFU/g. The aforementioned author concluded that the analyzed samples were within the acceptable limits (<10³) established by the International Commission on Microbiological Specifications for Foods (ICMSF), which is the same limit provided by Brazilian legislation.

Molds and yeasts are mostly found in the soil and air [29]; although they are mostly aerobic, they are also capable of developing in anaerobic environments [30]. These microorganisms are indicators of unsanitary process control or conditions [31], as well as useful tools to assess general contamination, reduced shelf-life, and deterioration onset [28]. Overall, fungal-associated deterioration takes place under certain conditions, according to which bacterial deterioration is controlled by low water activity, pH, temperature, and/or the presence of inhibitory agents [32]. Very acidic or acidified food products are more prone to contamination with molds and yeasts, mainly spore-formers [33].

Fungal multiplication can be controlled through the adoption of high temperatures and preservatives supplemented with weak acids and antibiotics [34]. Therefore, presumably, pickled vegetables may have been contaminated because of failures that may have taken place during pickled vegetable processing, such as inadequate heat treatment, lack of fast cooling immediately after heat treatment application, and/or not using preservatives supplemented with weak acids or inappropriate amounts of them; alternatively, contamination may take place after processing, such as using poorly sanitized, improperly sealed packaging or incorrect handling at the packaging stage. Furthermore, the presence of heat-resistant spores and environmental contamination may have contributed to contamination with molds and yeasts in the analyzed pickled samples. However, it is necessary to investigate rural family agribusiness processing plants to assess their good manufacturing practices, as are the criteria adopted by them to determine product shelf-life. These complex criteria require specific technical knowledge and affect production costs, which can be a limitation for food producers in the agribusiness field since these companies use family labor and operate in rural areas.

3.2. Physicochemical Quality of Pickled Vegetables

Table 2. Minimum and maximum values recorded for pH, titratable acidity (TA), and chloride content in NaCl in pickled vegetables per category.

Pickled Vegetables Category	N. of Samples	pH	TA (g of Acetic Acid/100 g)	Chlorides in NaCl (g of NaCl/100 g)
Appetizer/caponata	12	3.90–6.10	0.25–0.79	0.50–3.70
Pickled mushroom	9	3.20–4.20	0.30–0.53	0.10–0.40
Vegetable pickles	16	3.60–4.90	0.27–2.47	0.20–3.50
Pickled pepper	13	3.30–4.50	0.51–2.82	0.60–5.70
Dried tomato/confit	8	4.00–4.90	0.79–1.88	0.80–5.30

shows the minimum and maximum values recorded for pH, titratable acidity (TA), and chloride content in NaCl in pickled vegetables per category.

Overall, the pH values ranged from 3.20 to 6.10 (Table 2), and the mean pH reached 4.15. In total, 12.06% (n = 7) of the 58 analyzed samples presented pH values higher than 4.5, i.e., higher than the limit provided by the Brazilian sanitation legislation, and can represent a risk of contamination by Clostridium botulinum [8,21]. Five of these samples belonged to the appetizer/caponata category, with one belonging to the vegetable pickle category, and one belonging to the dried tomato/confit category. Different results were reported by Santana et al. [35], who reported pH values ranging from 3.93 to 4.43 in 33 samples of pickled cucumber obtained from stores in Itaqui County, Rio Grande do Sul State; all the samples had pH values ≤ 4.5, and they were in compliance with Brazilian sanitation legislation. Ding et al. [36] recorded pH values ranging from 1.9 to 4.1, i.e., values ≤ 4.5, in 131 samples of pickled fruits and vegetables purchased at retail stores in Raleigh County, NC, USA, and online suppliers.

pH affects food product shelf-life and protection against invading microorganisms, such as spoilage microorganisms and foodborne pathogens [11]. This variable plays a key role in the quality of pickled vegetables, since it helps control Clostridium botulinum development [35], whose spores are not destroyed by pasteurization and can germinate at pH values higher than 4.6 [37]. Moreover, pH affects the thermal processing intensity, since reducing the pH decreases the resistance of microorganisms to heat [38]. Therefore, pH is the main factor in pickled vegetable conservation processes [39], since adequate pH values guarantee that the acidification procedure is properly carried out [40]. C. botulinum can be a serious contamination problem in canned foods, such as canned vegetables. The presence of this pathogen is characterized by high gas production, leading to swelling of packaging, a strong odor, and loss of color and texture [41]. C. botulinum is an anaerobic, spore-forming bacterium that causes botulism, a disease caused by neurotoxins. It is considered a serious food poisoning that causes paralysis of the respiratory muscles and respiratory failure and can be highly fatal [41,42,43].

Furthermore, pH values above the limits provided by legislation can indicate failure in the manufacturing process of pickled vegetables, since acidic or acidic food must be added to the covering liquid during the processing of these food products, or acidification must be performed through fermentation to obtain a final product with a pH equal to or lower than 4.5 [8].

Therefore, acidification was not carried out, or it was not carried out properly, during the pickled vegetable preparation process by considering the type, initial pH, and weight of the raw materials used, the covering liquid volume and the amount of acid or acidic food to be added to the product, as well as the quality control applied to the finished product, on the basis of pH analysis. The main agents of fruit and vegetable spoilage include Gram-positive and Gram-negative bacteria, as well as molds and yeasts [44].

Pickled vegetable samples presented titratable acidity values ranging from 0.25 to 2.82 g of acetic acid/100 g (Table 2), and the mean titratable acidity value reached 0.89 g of acetic acid/100 g. The lowest acidity values were found in the pickled mushroom category, whereas the highest values were observed in the pickled pepper category. Ding et al. [36] reported acidity values ranging from 0.13 to 3.01 g of acetic acid/100 g in 131 samples of commercially purchased pickled fruits and vegetables; these values were close to those reported in the present study. Aljahani [11] reported smaller variations, with values ranging from 0.14 to 0.55 g of acetic acid/100 g, in the titratable acidity results recorded for pickled pickles.

Acidity is an important parameter for preventing microbial spoilage in pickled vegetables, in addition to contributing to typical sensory traits [45], such as their sour taste, which depends on organic acid composition and amounts [46]. Brazilian sanitation legislation does not establish limit values for titratable acidity. However, acidity plays an important role in product stability; it is the main preservative factor for most pickled vegetables [47], as well as an important quality trait, since overall reactions involved in food decomposition processes (fermentation, hydrolysis, oxidation) generate acidic compounds that change medium acidity [48].

The chloride values, expressed in sodium chloride, ranged from 0.10 to 5.70 g of NaCl/100 g (Table 2); its mean value reached 1.85 g of NaCl/100 g. Santana et al. [35] reported chloride levels ranging from 1.70 to 3.47 g of NaCl/100 g in 33 pickled cucumber samples; these values were close to those recommended in the literature, ranging from 2% to 3% [49]. The samples analyzed in the present study presented mean chloride values in sodium chloride lower than those recommended in the literature; pickled mushroom samples presented the lowest values, which may have contributed to the growth of mold and yeasts in this product category. According to Montaño et al. [47], NaCl reduces oxygen solubility in water, which is a relevant parameter used to control aerobic microorganisms and medium water activity to enable conditions less favorable for microbial growth.

Accordingly, 25.86% (n = 15) of the 58 analyzed samples presented unsatisfactory results in comparison with the Brazilian sanitation legislation. In total, 13.79% (n = 8) of these results were associated with microbiological quality, and 12.06% (n = 7) were associated with physical–chemical quality.

These results can also be explained by the difficulty faced by agribusinesses in following sanitary regulations, since a study carried out with family agribusiness producers in Espírito Santo State revealed that 37.4% of them did not have sanitary registration, only 15.4% used a public water supply, 45.2% had a good manufacturing practices manual (GMP), 26.5% adopted standard operating procedures and/or standard operating hygiene procedures, and 67.5% were trained in GMP [3]. This finding is worrisome since these requirements are part of good manufacturing practices for health-safe food production.

4. Conclusions

Pickled vegetables produced by rural family agribusinesses operating in the mountainous region of Espírito Santo State have compromised the microbiological and physical–chemical quality in a small number of samples. However, despite the small number of samples that did not comply with the legislation, the results have indicated failures in good manufacturing practices and quality control procedures adopted in some establishments, which may compromise consumer health. Therefore, it is necessary to encourage inspection actions, as well as developing programs and projects in the sanitation sector, to both guide and help these establishments produce pickled vegetables in compliance with parameters provided by sanitary legislation and to provide quality and food safety for public consumption. Furthermore, considering that many rural agroindustries sell only their products on their own property, which may have limited access to all products, it is suggested that future work involve partnerships with local organizations and cooperatives to allow for better contact with these producers.