Effect of lactic acid fermentation on the nutritional quality and consumer acceptability of African nightshade

Abstract African nightshade (ANS) is among many underexploited and neglected indigenous vegetables. This study assessed the effect of lactic acid fermentation (LAF) on nutritional and sensory quality in Solanum villosum (Sv) and Solanum scabrum (Ss). Spontaneously fermented (SF) and controlled fermented (CF) conditions using Lactobacillus plantarum LP90 and Leuconostoc mesenteroides LM58 were employed for 15 days and 120 h. From the fermented pickles, relish products were prepared using cooking oil and a variety of spices. The relish products were subjected to a consumer acceptability test. Results show a significant drop in pH to <3.5, increasing titratable acidity (TTA) to around 0.6 after 120 h and 15 days of CF and SF, respectively. LAF resulted in a 2.6–5 and 1.6–4.8‐fold significant rise in β‐carotene in pickles and their relish products. All pickles and relish products exhibited a significant decrease (p < .05) in vitamin C by 88.33%–95.90%. LAF significantly reduced total phenolic (26%– 43%) and Chlorophyll (16.45%–39.25%). On the other hand, LAF showed improvement in minerals content (P, Ca, Fe, and Zn) and reduction of tannin (76.27%–92.88%) and oxalate (77.33%–90%) levels. LAF relish products were highly preferred by the consumers, with S. villosum controlled fermented relish (SvCFR) leading. All fermented relishes were stable at ambient (27°C) and refrigeration (4°C) temperatures after 6 months of storage. Generally, LAF is an effective method for ANS preservation, with improved nutritional quality and safety. LAF can therefore be recommended to small‐scale farmers, processors, and households for ANS preservation. Ultimately, this method enhances the nutrition and sensory quality, safety, and livelihood.

The pH and titratable acidity (TTA) were recorded daily for SF and CF.
Pickle products SsSFP and SvSFP from SF and (SsCFP and SvCFP) from CF were cooked for 10 min mixing with cooking oil, onions, and different spices (garlic, pepper, cardamom, turmeric, cumin, clove, and cinnamon) employed in relish-making. Four relish products of S. scabrum controlled fermented relish pickle (SsCFRP), S. villosum controlled fermented relish pickle (SvCFRP), S. scabrum spontaneous fermented relish pickle (SsSFRP), and S. villosum spontaneous fermented relish pickle (SvSFRP), respectively. The relishes were then cooled, packaged in PET bottles, and stored at refrigeration and ambient temperatures for shelflife study.

| Determination of titratable acidity
Titratable acidity (TTA) was determined, as described by Rajković et al. (2007). About 10 ml of brine solution from spontaneously and controlled fermented Ss and Sv was mixed with 10 ml of distilled water in a conical flask. The mixture was titrated with 0.1N NaOH using two drops of phenolphthalein indicator (LOBA Chemie, India) to a persistent pink end. TTA was calculated in terms of LA anhydrous as follows:

| Determination of vitamin C
The total vitamin C content of the fresh leaves, pickle, and relish products was determined (Kapur et al., 2012). About 10 g of sample was mixed with 50 ml of 3% metaphosphoric acid and 8% acetic acid solution (LOBA Chemie, India) and centrifuged at 4000 rpm (revolutions per minute) (centrifuge 5810, Germany) at ambient temperature for 15 min. Four milliliters (4 ml) of the extract was treated with 0.23 ml of bromine water (3%) (LOBA Chemie, India), followed by the addition of 0.13 ml of 10% thiourea solution (LOBA Chemie, India), and then 1 ml of 2,4 dinitrophenylhydrazine solution (LOBA Chemie, India).
TTA( % ) = 0.09 × Titer (ml) The mixture was incubated in a thermostatic water bath at 37°C for 3 h. The mixture was then cooled for 30 min and then treated with 6 ml of chilled 85% sulfuric acid (LOBA Chemie, India). The absorbance of the resulting red-colored solution was measured using a Spectrophotometer (Heidolph, Germany) at 521 nm. The total ascorbic acid content was estimated, based on the standard curve of ascorbic acid, and the result was expressed as mg/100 g (wet basis).
The supernatant was collected and filtered using Whatman No. 1 filter paper (Whatman International Ltd., India), and then was reextracted two times until the residue was colorless. The supernatant was combined, and the volume was recorded. The supernatant was saponified with 40% potassium hydroxide (LOBA Chemie, India) and left at ambient temperature for 12 h. The mixture was then transferred into the separating funnel and treated with the same volume of 10% w/v sodium chloride (LOBA Chemie, India) for removing moisture. The mixture was then shaken vigorously, and the upper phase was collected and dried over anhydrous sodium sulfate (LOBA Chemie, India). The β-carotene content was estimated based on the standard curve of β-carotene, and the result was expressed as mg/100 g (wet basis).

| Determination of Chlorophyll
Chlorophyll content of the raw leaves and pickle products was determined as described by Su et al. (2010), with some modifications. About 1.0 g of sample was taken and ground into a fine pulp using mortar and pestle with about 10 ml of 80% acetone (LOBA Chemie, India). The pulp was centrifuged at 4000 rpm at ambient temperature for 5 min, and the green supernatant was then transferred to a 50 ml volumetric flask for Chlorophyll determination. The sediment in the centrifuge tube was scrapped and ground with the same mortar and pestle with a small amount of 80% acetone (LOBA Chemie, India) to extract the residual Chlorophyll. The mixture was centrifuged at 4000 rpm at ambient temperature for 5 min; the supernatant was then mixed with the previous supernatant into the volumetric flask. Re-extraction was done until no perceptible green color was left in the residue. The supernatant was made to a 50 ml volumetric flask with 80% acetone (LOBA Chemie, India). The extract was placed into the refrigerator for 10 min to lower the temperature. The absorbance of the extract was read at 663 and 645 nm using a Spectrophotometer (Heidolph, Germany), and 80% acetone was used as the blank. The amount of Chlorophyll was calculated using the empirical formula: Where A = absorbance at specific wavelengths; V = final volume of chlorophyll extract; W = fresh weight of the tissue extracted.

| Determination of the total phenolic content
The total phenolic content of raw Ss and Sv, pickle, and relish products was determined by the Folin-Ciocalteu method (Mahdavi et el., 2011). Homogenized samples (10 g) were mixed with 30 ml of 80% methanol (LOBA Chemie, India) and centrifuged at 3000 rpm at ambient temperature for 15 min. The residue was re-extracted twice. Precisely, 1 ml of the methanol extract was diluted 10 times with the extraction solvent. An aliquot (0.5 ml) of the diluted sample was mixed with 2.4 ml of deionized water, 2 ml of sodium carbonate (2%) (LOBA Chemie, India), and 0.1 ml of Folin-Ciocalteu reagent (FCR) (LOBA Chemie, India). The mixture was incubated in a dark place at ambient temperature for 60 min, and the absorbance was determined at 750 nm using a Spectrophotometer (Heidolph, Germany).

| Determination of the mineral content
Minerals (P, S, Ca, Fe, Zn, Cu, Mn) were analyzed according to the method of (Croffie et al., 2020). The samples (raw leaves, pickle, and relish products) were dried at 60°C in the conventional oven (UN30/Germany). The samples were ground using mortar and pestle and sieved in a sieve of 60-micron size (Shanghai sieves/ China). Four grams of the sample was mixed with 0.9 g of binder (Hoechstwax) (Cereox fluxana-BM-0002-1/Germany) using a pulverizer (PulverisetteTM/Germany) at the speed of 150 revolutions per sec for 30 min. The mixture was then compressed to form a pellet using a hydraulic pressing machine (Vaneox Fluxana PP25, Germany) at 15 psi (pounds per square inch). The Energy-Dispersive X-ray Fluorescence (EDXRF) (Xlab pro-Spectro xepos Spectrometer/ Germany) was used to quantify the mineral content. The minerals reading was corrected using spinach leaves standard/Standard

| Oxalates' determination
The oxalate content in raw leaves, pickles, and relish products was determined by the titration method, as described by Agbaire (2011).
In one (1 g) sample, 75 ml of 3 M sulfuric acid was added while stirring using a magnetic stirrer for 60 min. The mixture was filtered using Whatman filter paper No. 1 (Whatman International Ltd, India).

Determination of tannin
The tannins were determined by the Folin-Ciocalteu method according to Chandran and Indra (2016). A 10 g of homogenized sample (raw ANS, pickle, and relish) was mixed with 30 ml of 80% methanol (LOBA Chemie, India) and centrifuged at 3000 rpm at ambient temperature for 15 min. The residue was re-extracted twice. Precisely, 1 ml of the methanol extract was diluted 10 times with the extraction solvent.
An aliquot (0.5 ml) of the diluted sample was mixed with 7.5 ml of distilled water, 1 ml sodium carbonate (35%) (LOBA Chemie, India), and 0.5 ml of Folin-Ciocalteu reagent (FCR) and diluted to 10 ml with distilled water (LOBA Chemie, India). The mixture was shaken well and kept at room temperature for 30 min. The tannic acid standard was used to prepare the standard curve. Absorbance for test and standard solutions was measured against the blank at 700 nm with a Spectrophotometer (Heidolph, Germany). The tannin content was expressed in mg of tannic acid equivalents/100 g of wet basis.

| Microbial determination of relish products
Total bacteria, fungal counts, coliform, and Lactobacillus were analyzed separately by the pour plate technique as described by Karami et al. (2017), Rahimi et al. (2019), and Singh et al. (2013) with slight modification. Briefly, 1 g of homogenized relish products was mixed with 9 ml of sterile peptone salt (LOBA Chemie, India) and vortexed for 5 min.

| Shelflife test of the relish products
The shelflife study of relishes was conducted at refrigeration (4°C) and ambient temperatures according to Pala and Agnihotria (1994), with some modifications. Relishes in transparent polyethylene (PET) plastic-capped bottles were kept at refrigeration (4°C) and ambient temperatures for 6 months consecutively. Monthly monitoring of TTA, pH, total bacteria, LAB, yeast, and mold was performed.

| Consumer acceptability test
The consumer acceptability test was conducted in the Moshi District A 9-scale Hedonic test was used (Yang & Lee, 2018), with 1 = dislike extremely, 5 = neither like nor dislike, and 9 = like extremely.
Panelists were served with all four coded relish products and filled out an evaluation form after tasting.

| Statistical analysis
Experiments were conducted in triplicate, and data were expressed as mean values ± SD. SPSS software version 25 was used for data processing. The one-way analysis of variance (ANOVA) and post hoc test (least significant difference) were used to compare the mean between the species of Solanum for the fermented pickles and relish products, and the differences were considered significant when p < .05.

| Effect of LAB on African nightshades
African nightshades (ANSs) (S. scabrum and S. villosum) at an initial pH of 7.4 were fermented spontaneously and under controlled conditions for 15 days and 120 h, respectively. A sharp drop in pH to 4.7 and pH to 3.5 was observed within 24 h for both spontaneous fermentation (SF) and controlled fermentation (CF), respectively (Table 1). Furthermore, a slight pH drop in SvCFP and SsCFP has observed a similar trend. A sharp pH drop indicates the ability of acidification by LAB during fermentation (Stoll et al., 2021). LAB produces various organic acids, with predominant lactic acid (LA), hence the preservation effect (Degrain et al., 2020). A combination of L. plantarum LP90 and L. mesenteroides LM58 in 3% brine solution resulted in a sharp pH drop. Therefore, faster, more profound, stable, and more controlled fermentation can be achieved using starter cultures (Stoll et al., 2021).  (Degrain et al., 2020;Stoll et al., 2021;Wafula, 2017). Heterofermenters, mainly Leuconostoc mesenteroides and L. brevis, can initiate the fermentation process, while L. plantarum occur later (Belitz et al., 2009;Hutkins, 2006;Sangija, 2021).
On the other hand, a steady pH drop was observed in SF, resulting in a prolonged fermentation time with a pH of 3.5 attained in 72 h for SvSFP and 192 h for SsSFP, respectively (Table 1). Naturally occurring bacteria grow over 1-2 weeks to produce lactic acid (LA), whereas added salt controls the type and rate of the fermentation (Behera et al., 2020). In this study, the addition of brine solution (4% salt and 2% sugar) facilitated the attainment of an optimal pH of 3.5 within 3-8 days of spontaneous fermentation (Table 1).
Similarly, a sharp increase in TTA to 0.4 within 24 h was observed in CF, instead of SF (Table 1). Wafula (2017) reported an increase in TTA due to ANS fermentation. Lactic acid (LA) production prevents the growth of food poisoning bacteria and other spoilage microorganisms, thus enhancing product safety (Behera et al., 2020) and sensory quality.

| Effect of fermentation on the nutritional quality
The results on β-carotene content in LAF are presented in Figure 1.
LAF significantly increased the β-carotene content in pickle and relish products. The β-carotene content in fresh S. scabrum and S. villosum was 48.7 mg/100 g and 31.1 mg/100 g, respectively ( Figure 1).
In relish-making, heat treatments and powdered spices used as ingredients may also have contributed to the reduction of βcarotene. Likewise, cooking sweet potato leaves above 5 min and cilantro above 10 min decreased β-carotene (Kao et al., 2014). The β-carotene level reported in this study is lower than the decrease of β-carotene content by 24.2% of cooked, fermented tomatoes (Bartkiene et al., 2015). On the other hand, LAF significantly decreases vitamin C content in pickle and relish products ( Figure 2  and Fe 3+ ), oxygen, alkaline pH, and high temperature (Lee & Kader, 2000). Oxidized AA, dehydroascorbic acid can be reduced to AA and irreversibly oxidized to form diketogulonic acid, with no vitamin C activity (Lee & Kader, 2000). Blanching prevents AA oxidase, phenolase, cytochrome oxidase, and peroxidase action, indirectly responsible for AA loss (Lee & Kader, 2000). However, blanching has been reported to decrease vitamin C content by 28%-82.4% due to dissolution and oxidation (Lee & Kader, 2000;Oboh, 2005).
Furthermore, cooking has been reported to reduce vitamin C content by 30% and maintaining potatoes hot for 1 h further decreased vitamin C by 10% (Hägg et al., 1998;Lee & Kader, 2000). During fermentation, sucrose is converted to fructose and glucose, carbonyl groups of fructose react with vitamin C, reducing vitamin C content (Lee & Kader, 2000). Filannino et al. (2016) reported a reduction of vitamin C content after fermentation with L. plantarum and L. brevis. The remaining amount of vitamin C after fermentation is less to meet the recommended daily allowance of 100-200 mg (Lee & Kader, 2000). Likewise, vitamin C content in pickle and relish products was very low compared to the recommended dietary intake.
Therefore, dietary diversification or fortification of the relishes is highly recommended.
Fermentation significantly increased the mineral content of the S. scabrum and S. villosum pickles and relish products compared to fresh leaves ( to be lower than the values observed in this study. LAF improved the mineral content in pickles and relishes. Iron, Zn, Ca, S, P, Cu, and Ni content was improved by 0.58-2.01-fold, respectively. Fermentation of amaranth leaves, pumpkin leaves, and capwood leaves showed increases in Ca, Mg, Zn, Fe, Se, and Cu (Ifesan et al., 2014). LAB improves nutritional quality and micronutrients' bioavailability in food through antinutrients' degradation by microbial enzymes. Therefore, LAF can be considered a strategy to enhance nutritional and functional quality by activating endogenous enzymes (Nkhata et al., 2018;Rollán et al., 2019) due to low pH.
Also, LAB can produce different enzymes responsible for the hydrolysis of the food matrix into desirable nutritional and sensory quality. Despite sparsely available information, this study provides a finding for the mineral content of fermented S. villosum and S. scabrum pickles and relishes.

| Effect of fermentation on Chlorophyll and polyphenols
Fermentation significantly reduced Chlorophyll a content (p < .05) in SsSFP and Chlorophyll b in SsSFP, SsCFP, SvSFP, and SvCFP, respectively (Table 3). Also, fermentation reduced total Chlorophyll in all pickles (Table 3). During fermentation, acetic and lactic acid (LA) production degraded Chlorophyll a and b (Degrain et al., 2020). During fermentation, pH changes can produce new compounds such as Mgfree Chlorophyll derivatives, a carotenoid with 5,8-epoxide groups, brown pigments (0-quinones), or phenolic compounds' chemical oxidation masking the original green color of the leaves and reducing the Chlorophyll content (Ramírez et al., 2015).
During fermentation, when pH decreases below 6 it contributes to the reduction of deprotonated divalent oxalate (C 2 O 4 2− ) ion power to bind with divalent minerals such as Ca 2+ ion to form insoluble oxalates (Simpson et al., 2009;Wadamori et al., 2014).   enzymes phytase and tannase that degrade the phytate and tannins and improve the availability of calcium, iron, and zinc (Samtiya et al., 2021). Similarly, eliminating toxins such as tannins, oxalates, and phytates through LAF ensures food safety. Therefore, reducing tannins and oxalates can ensure the safety and bioavailability of some mineral elements in pickles and relish products.

| Consumer's acceptability of fermented relish
The majority of the consumers preferred SsCFR with a mean score of 7.96. Interestingly, all the three SvCFR, SsSFR, and SvSFR were equally preferred (Table 4) Furthermore, low pH is associated with color destruction in fermented vegetables due to the production of acids, concurrently with findings (Table 3). In a study by Wafula (2017), a similar color mean score (7.9) was reported for fermented ANS. In addition, other parameters, such as texture, appearance, taste, saltiness, sourness, bitterness, and spiciness, were also ranked higher by consumers (Table 4). This suggests that, other than LAF, relish-making further improved the sensory quality of the pickles. Likewise, LAF improved sensory attributes of color, taste, smell, appearance, and general acceptability (Wafula, 2017).

| Shelf life of relish products during storage
Results of the shelflife study of the LAF fermented relish products are presented in Table 5 and Table 6. After six months of room and refrigeration storage of the fermented relish products, no change in pH and TTA was recorded. Also, no coliforms, total bacteria, Lactobacillus, yeast, and mold were detected (Tables 5   and 6). Likewise, Wafula (2017) reported that due to the absence of fermented ANS in a starter culture, some microbes were detected in the spontaneously fermented product after storage (10°C and 25°C). Microbiological results indicated that the relish product is safe for consumption. Low pH (3.2-3.3) inhibited the growth of pathogens (Wafula, 2017). Heat treatment of the pickle to make relishes killed microorganisms in the fermented pickles, including Lactobacillus, which hinders their growth in the relish product (Sangija et al., 2021). Fermentation of S. scabrum with L. plantarum and Leuconostoc mesenteroides inhibited Listeria monocytogenes and Salmonella enterica serovar. Enteritidis .
Therefore, from the microbiological quality point of view, all relishes were shown to be safe after six months of storage at 4°C and 27°C.
Results on a gradual loss in vitamin C in relishes stored at ambient (27°C) and refrigeration (4°C) temperatures are presented in Tables 5 and 6. Vitamin C losses were high at ambient temperature than the refrigeration temperature. Ambient storage at 27°C and the transparent PET packaging allow light intensity, facilitating the denaturation of vitamin C (Lee & Kader, 2000). A low temperature of 4°C with no light intensity effect supports the low losses in vitamin C in refrigeration storage.
On the other hand, β-carotene losses amount to 46%-62% and 20%-29% for ambient and refrigeration storage for the relishes (Tables 5 and 6). Despite the reduction of β-carotene, still the remaining quantity is sufficient to contribute to the recommended daily allowance (adult 75-180 mg and children 30-150 mg) (IBM Watson Health, 2022). This agrees with Singh et al. (2013) that, a high loss of β-carotene in room storage was 85% for glass bottles and 81% for plastic bottles. Oxidation is the leading cause of carotenoid degradation in foods. However, in processed foods, the oxidation mechanism is complex but is facilitated by moisture, temperature, presence of prooxidants, antioxidants, and lipids (Singh et al., 2013).

| CON CLUS ION
Lactic acid fermentation (LAF) enhanced ANS pickle and relish products' nutritional, sensory, and safety quality. Specifically, LAF improved β-carotene and minerals contents but reduced vitamin C, total phenols, and Chlorophyll levels. LAF also reduced tannins and oxalate. The SsCFRP product was the most preferred over other relish products. All relish products were stable at ambient and refrigeration temperatures after six months of storage, hence contributing to product safety. Therefore, LAF can be recommended as the best preservation method for African nightshades (ANSs). From the study, LAF has been shown to retain nutrients, improve safety and shelf life, offer diverse products, subsequently contributing to minimize postharvest losses.

ACK N OWLED G M ENTS
The authors acknowledge the financial support from the Federal Ministry of Food and Agriculture (BMEL) based on a decision of the Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE). Grant/Award Number: 2816PROC04.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.