Valorization of Parmentiera aculeata juice in growth of probiotics in submerged culture and their postbiotic production: a first approach to healthy foods

Nowadays, functional foods are greatly accepted by consumers because they improve health and are new sources for substrates to be explored. In this sense, Parmentiera aculeata, a plant distributed in Mexico with beneficial effects on health, has not been chemically explored. In this work, P. aculeata juice was used as carbon source to promote the growth of two probiotic Lactobacillus strains during submerged fermentation. Taguchi’s methodology with orthogonal array L9 was applied for culture conditions optimization. pH, agitation, and inoculum concentration variables, each with three levels, were evaluated and the best treatment was validated through a kinetic culture monitoring some postbiotics traits. We observed an increase in 1.76-times in cellular concentration of L. plantarum 14917, and the main produced postbiotics were short-chain fatty acids such as succinic, formic, acetic, propionic, and lactic acids, which are associated with the probiotic metabolism and are important for human health. In the best of our knowledge, this study is the first to describe the valorization of P. aculeata juice as substrate for growth of probiotic strains and future studies are required to gain further applications in functional food production.


Introduction
Plant's diversity from Mexico has potential applications in food biotechnology, However, multiple plant species remain still functionally uncharacterized, Parmentiera aculeata (Kunth) Seem (syn. P. edulis DC), which is a plant distributed in the Pacific area from Sinaloa to Chiapas, and in the Gulf region from Tamaulipas and San Luis Potosí to Yucatán (Andrade-Cetto and Heinrich 2005; Morales-Sánchez et al. 2015). P. acuelata tree can reach up to 15 m of height, with fruits from 15 to 20 cm long and 6.5 cm wide. It has longitudinal grooves and is yellow-green in color (Andrade-Cetto and Heinrich 2005). Their fruits, roots and bark are recognized in Mexican traditional medicine as treatment for diabetes and renal diseases, among others such as headaches, gallstones, deafness and diarrhea (Andrade-Cetto and Heinrich 2005;Morales-Sánchez et al. 2015). Some of these ailments have been confirmed in biological models, for example their hypoglycemic effects of lactucin-8-O-methylacrylate present in the chloroform extract from dried fruits. Also, the effect of lowering blood sugar levels was observed in a mice model with alloxan-induced diabetes (Pérez et al. 2000). In addition, the fruit extracts have cytotoxic activity and apoptosis induction in breast cancer cells (Estanislao-Gómez et al., 2016). Also, hypoglucemic (Pérez et al. 2000;Andrade-Cetto and Heinrich 2005) al. 2015). In this sense, the exploration of bioactive compounds present in P. acuelata could be applied for functional food formulation, where functional food and nutraceuticals have an increment demand because of their interesting health improvement effects. The first is defined as food products with appearance of traditional food that contains added ingredients to provide health-related benefits for humans. (Topolska et al. 2021). Whereas nutraceuticals are characterized by its presentation in pills, powders or other medicinal forms not generally associated with food (Penson and Banach, 2020). Probiotics and/or prebiotics are usually associated as the providers of those functionalities. Interestingly, probiotics have been associated to improve health in patient with any of the aforementioned diseases. Probiotics consists of lactic acid bacteria (LAB) of genus Lactobacillus, mainly (Kitazawa et al. 2020 On the other hand, to improve the growth of probiotic strains, culture and nutrimental condition's optimization is mandatory. In this sense the submerged fermentation (SmF) in combination with Taguchi's methodology can be used to improve the growth of different probiotic candidates. Taguchi's methodology searches for the optimal conditions and is used in industries to improve the quality processes (Aguilar-Zarate et al. 2014). The aims of this work were 1) to find alternative sources of prebiotics, by demonstrate the ability of P. aculeata juice, used as carbon source, to promote the growth of two probiotic Lactobacillus strains during SmF, 2) to optimize the culture conditions of SmF to improve the cellular concentration of Lactobacillus strains and, 3) to characterize the postbiotics production during SmF by the probiotic Lactobacillus strains.

Culture medium and reagents
The DeMan-Rogosa-Sharpe (MRS) broth were purchased from BD Bioxon (New Jersey, USA). All reagents used in this study were reactive grade and were purchased at Productos Químicos de Monterrey (Monterrey, Mex) and Jalmek Científica (San Nicolás de los Garza, México), standards were obtained from JT Baker (Phillipsburg, NJ, USA) or Sigma Aldrich Chemical Co. (St. Louis, MO).

Vegetal material and strains
Fruits from P. acuelata were collected in "El Gavilan" locality (22.5250397,-100.0429407), Ciudad Valles, San Luis Potosi, Mexico. The fruit was selected according to the color scale described by Angón-Galván (2006), focusing on fruits with a 3-4 color scale. Fruits were transported to Food Research Laboratory at FEPH-UASLP and conserved in plastic bags in refrigeration at 4-6 °C until further use. Lactobacillus paracasei subsp. paracasei ATCC 25302 and L. plantarum subsp. plantarum ATCC 14917 were kindly provided by the Food Research Department at Autonomous University of Coahuila, Mexico.

For submerged cultures
The Lactobacillus strains were activated in assay tubes with 10 mL of MRS broth and incubated at 37 °C for 24 h. The inoculum for the fermentation was prepared from re-seeding the overnight culture in new broth and incubated for 16 h at 37 °C. Incubation of the treatments was carried out at 37 °C for 120 in accordance with conditions described in Table 1.
Growth was expressed as cells number per milliliter (cel/ mL) from cells counts using a Neubauer's chamber. The experimental was performed in duplicate.

Experimental data analysis and prediction performance
The results from the nine SmF cultivation runs were processed using the software Statistical version 7.1 (Statsoft, Tulsa, OK, USA).

Validation
The strain with the higher cellular concentration was validated as suggest the experimental data analysis using the optimal culture conditions. In addition, the SmF kinetic were cultured for 120 h and was monitored each 24 h to determine cellular concentration, pH changes, titrable acidity (citric acid as % CA, lactic acid as % LA), total soluble solids (TSS, °Brix), reducing sugars (RS, %) and postbiotic compounds. Cultures were realized in triplicates. All results were compared between statistical groups, using analysis of variance (ANOVA) and Tukey's multiple comparisons.

Chemical profile from free-cell culture medium
Culture medium from each cultivation was centrifuged (Hermle, Z206A, Germany) at 5000 rpm for 5 min and free-cell culture medium (supernatants) was used for the following determinations: pH (Oakton, 700, Vernon Hills, USA), TA was determined according to procedure described in NMX-FF-011-1982 and was expressed as % CA, SS expressed in °Brix using the methodology described in NMX-F-436-SCFI-2011, ART using Lane-

Results
The chemical profile of juice from P. aculeata is presented in Table 2. The value of TSS was 11%, where RS corresponds to 9%. In addition, titrable acid was 0.16 and 0.25% expressed as citric and lactic acids, respectively. After SmF cultivations, experimental data analysis was analyzed for Taguchi methodology. In Fig. 1, the results of nine culture assays are presented, where the treatment 2 exhibited the major cellular concentration for both Lactobacillus probiotic plantarum 14917 was carried out using the optimized conditions. A 1.76-fold increase in cell concentration (1.36 × 10 9 cell/mL) was obtained compared to the expected value predicted by the Taguchi model (Fig. 2). First, the exponential growth of L. plantarum subsp. plantarum 14917 in SmF was observed in the first 24 h followed by a stationary phase in the next hours. A second growth increase was appreciated with the maximum growth at 120 h. In this sense, a decrease of pH to 3.28 (Fig. 2) and a titratable acidity of 0.69% citric acid was observed at 120 h. In addition, the consumption of 1°Brix (TSS) and 1.59% RS was evident at the end of the growth kinetics (Fig. 2).
The maximum substrate consumption was observed at 72 h with a consumption of 13.4 g/L and 9.5 g/L of glucose and xylose, respectively (Fig. 3). In addition, arabinose was not present during the kinetic growth. The main metabolites obtained at the end of the cultivation was lactic acid, with a production of 6.9 g/L in a typical culture from L. plantarum strains, but the maximum production was observed at 96 h with a value of 11.7 g/L. Additionally, the production of ethanol reached a maximum value of 0.2349 g/L at 72 h (Fig. 3). The production of SCFA was not statistically significant (Fig. 4). Initial and final concentrations of succinic acid were 0.3125 and 0.3198 g/L, respectively, indicating a 7.3 mg/L consumption. Formic acid initial and final values were 0.9520-0.6793, respectively, with a 0.2730 g/L consumption. Meanwhile acetic acid concentrations were 0.16636-0.73148 g/L, at the start and end of cultivation

Discussion
The chemical profile of fruit juice from P. acuelata is related with the fruit maturity according to the color scale constructed, considering the fruit firmness, TSS and titrable acidity as reported in previous studies (Angón-Galván 2006). Our results were similar in humidity and TSS levels, but high in titrable acid expressed as citric acid and lower in ash content in comparison with previous reports (Angón-Galván 2006). These differences can be attributed to geographical conditions and the season of the year when the fruits were harvested, as referred by other authors (Angón-Galván 2006). The chemical profile of P. aculeata fruit juice contains sugars that can be used as substrate for the stimulation of probiotic growth, similar to previously reported fruit juices, such as apple, orange, pomegranate, among others (Jaiswal and Abu-Ghannam 2013;Londoño et al. 2015;Mousavi et al. 2011;Pérez-Leonard and Hernández-Monzón 2015;Perricone et al. 2014). However, few studies are focused in application of Taguchi's methodology for probiotics growth optimization, but has been applied to describe the relationship with indole-3-acetic acid production in a symbiotic and nonsymbiotic nitrogen-fixing bacteria (genera Agrobacterium, Paenibacillus, Rhizobium, Klebsiella oxytoca, and Azotobacter) or to optimize the immobilization conditions for Lactobacillus penntosus cells (Shokri and Emtiazi 2010;Wang et al. 2020). In the best of our knowledge, our study is the first in describing the utilization of juice from Parmentiera aculeata as substrate for probiotic growth and its optimization using submerged cultures. Different sources of fruit and vegetables have been explored as substrates for growth of probiotics, for example, the juice from Aloe vera has been used for growth of L. plantarum and L. casei (González, Domínguez-Espinosa, and Alcocer, 2008;Pérez-Leonard and Hernández-Monzón 2015). While white cabbage (Brassica oleracea var. capitata) was used for the growth of other probiotics as well as L. plantarum ATCC 8014; L. rhamnosus ATCC 9595 and Lactobacillus brevis ATCC 8287 (Jaiswal and Abu-Ghannam 2013). Other authors have assayed pomegranate juice for growth of L. plantarum DSMZ 20174, L. delbruekii DSMZ 20006, L. paracasei DSMZ 15996 and L. acidophilus DSMZ 20079 (Mousavi et al. 2011), and sweet lemon juice was fermented with L. plantarum LS5 (Hashemi et al. 2017).
Particularly, most of the studies reported an efficiency for probiotic effect (expressed as colony-forming units, CFU) of 1 × 10 6 -1 × 10 12 /dosage (Guarner et al. 2017;Jurado-Gámez et al. 2013). In this study, we explored the efficiency by means of cellular growth expressed as cel/mL during the utilization of P. aculeata juice, since this plant is used as livestock feed and for traditional Mexican medicine (Andrade-Cetto and Heinrich 2005;Morales-Sánchez et al. 2015;Pérez et al. 2000). Results describing the growth of probiotic strains using MRS and Mueller-Hinton media have been reported previously, where cellular concentrations at 24-48 h were 2.5-4.5 × 10 9 cel/mL for microorganisms from gut microbiota such as L. brevis, L. casei and Lactobacillus delbrueckii/Streptococcus thermophiles (Niño Herrera et al. 2020).
Concerning to the growth conditions, particularly pH, it changed during the SmF fermentation at similar values as those previously reported. After 48 h of fermentation using Aloe vera juice with L. plantarum NCIMB 11718 and L. casei NRRL-1445 a final pH of 4.6 and 5.6, respectively, were observed (González et al. 2008). In another report, after 72 h of incubation at 37 °C, an increase of pH from 3.2 to 3.4-3.6 was observed in pure and mixed cultures of three L. plantarum strains (L. plantarum subsp. plantarum PTCC 1896, L. plantarum AF1 and L. plantarum LP3) using fermented bergamont juice (Hashemi and Jafarpour 2020). These results are similar with our results, where a pH of 3.35 and 3.28 at 72 and 120 h were obtained, respectively. Fermentation of apple juice with L. plantarum subsp. plantarum ATCC 14917 revealed a change in the initial pH 6.2 to a final pH of 3.68 in 72 h (Li et al. 2019). The same strains have been assayed in pomegranate fermentation for 24 h and pH of 3.5 (Mantzourani et al. 2019). Additionally, a probiotic beverage of pineapple juice was fermented with L. plantarum 299 V during 24 h with an observed pH value of 3.8 (Nguyen et al. 2019). The increase in titrable acidity was due to the carbohydrate metabolism of sugars present in the juice, causing a decrease of pH, this behavior was reported for other fruit juices (Vivek et al. 2019). The titrable acid values obtained for L. plantarum subsp. plantarum ATCC 14917 are lower in comparison to previous reports, values of 1.6 to 1.9% citric acid after 6 h in fermented sweet lemon juice with L. plantarum LS5 (Hashemi et al. 2017)were obtained. Similar values were reported for L. acidophilus DSMZ 20079, L. plantarum DSMZ 20174, L. delbrueckii DSMZ 20006, L. paracasei DSMZ 15996 in pomegranate juice fermentation (Mousavi et al. 2011).
The previously reported sugar consumption by the probiotic strains is similar to the values obtained in our study. L. plantarum MCC 2974 consumed ~ 1° Brix in 72 h during sohiong juice fermentation (Vivek et al. 2019). Additionally, L. plantarum consumed < 1°Brix during tomato juice fermentation. Particularly, glucose consumption was variable during juice fermentation by probiotic strains. While L. plantarum LS5 consumed ~ 2 g/L of glucose during sweet lemon fermentation (Hashemi et al., 2017), three L. plantarum strains (L. plantarum subsp. plantarum PTCC 1896, L. plantarum AF1 and L. plantarum LP3) consumed ~ 4 g/L of glucose during fermentation of bergamot juice (Hashemi and Jafarpour, 2020). These glucose consumption values are lower than those reported for L. plantarum ATCC 14917 in P. aculeata juice.
Other sugar monitoring in fermented P. aculeata was xylose, which is other important source carbon for growth of probiotic strains (Ucar et al. 2020;Zhao et al. 2015). Xylose consumption in a range of 2.26-7.75 g/L have been reported for different Lactobacillus strains, such as L. pentosus, L. brevis and L. buchneri when were growth in cucumber juice supplemented with trehalose, xylose and L-citronelle. We observed a xylose consumption higher for L. plantarum in P. aculeata juice, a value of 8.6-9.5 g/L at 120 and 72 h, respectively.
In fermentation with LAB, the main product was lactic acid, with others organic acids such as formic and propionic (Hashemi and Jafarpour 2020). Analyzing the results of posbiotics production during the fermentation of sweet lemon juice with L. plantarum EM ~ 7.5 g/L of lactic acid was obtained at 48 h and a similar value was reported for fermented bergamot juice at 72 h with L. plantarum strains (Hashemi and Jafarpour 2020;Hashemi et al. 2017). Values of 5.71 g/L lactic acid were reported for fermented papaya juice with L. plantarum GIM1.140 at 48 h (Chen et al. 2018). In our study the higher lactic acid concentration was reached at 24 h, producing 1.5-times more acid (11.3 g/L) than those previously reported. The value of formic acid obtained in our study was similar that the reported for fermented bergamot juice with L. plantarum PTCC 1896, which exhibited a production of ~ 0.8 g/L formic acid (Hashemi and Jafarpour 2020), which was higher that the value obtained in fermented papaya juice with L. plantarum GIM1.140 at 48 h (0.1842 g/L formic acid) (Chen et al. 2018). The production of propionic acid during fermentation, has been associated with anti-obesity properties in experiments with animals treated with fermented juices, including also acetic acid (Park et al. 2020).The production of these acids have been reported (0.2315 g/L) in mixed fermentation with L. rhamnosus GG and L. plantarum A6 using whole teff at 15 h (Alemneh et al. 2021). Succinic acid is associated with the reduction of intestinal pain symptoms (Moradi et al. 2021) and presents a similar inhibitory effect than acetic and propionic acid, for a strong inhibitory effect controlling the growth of molds and yeasts (Lucumi-Banguero et al. 2021).
In addition, during the metabolite profile monitoring of fermented P. aculeata juice ethanol was observed. The obtained value in this study at 120 h was 0.235 g/L, which is higher than the value reported for mixed fermentation with L. rhamnosus GG and L. plantarum A6 during the fermentation of whole teff at 9-12 h with values of 0.044-0.037 g/L, respectively (Alemneh et al. 2021), but are lower in comparison with the produced values during bhaati jaanr production (a rice-based fermentation beverage) with L. plantarum L7, 3.8 g/L at 120 h (Giri et al. 2018).
Several studies have been focused in demonstrate the benefic bioactivities of fermented juices, for example cabbage-apple juice and citrus juice fermented with Lactobacillus strains were evaluated for their positive effect on obesity and allergic rhinitis, among others (Harima-Mizusawa et al. 2016;Park et al. 2020). The results reported in the present work constitute a first approach to a future utilization for functional foods production, but numerous studies are required to confirm the potential of the fermented P. acuelata juice with probiotics as an interesting option for functional beverages.