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Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2023 Feb 6;60(4):1303–1312. doi: 10.1007/s13197-023-05675-6

Milk fermentation with prebiotic flour of Vasconcellea quercifolia A.St.-Hil.

Lilian de Fátima Ferreira da Silva 1,2, Kétlin Fernanda Rodrigues 1,2, Adriano Gennari 2, Eduardo Miranda Ethur 2, Lucélia Hoehne 2, Claucia Fernanda Volken de Souza 2, Elisete Maria de Freitas 1,2,✉
PMCID: PMC10020413  PMID: 36936124

Abstract

Non-conventional food plants have bioactive compounds and a high nutritional value. Among these, Vasconcellea quercifolia has nutritional benefits, but it is also easy to cultivate and has a low production cost. In this study, the flour from the unripe fruit of V. quercifolia was evaluated in terms of its potential as a prebiotic for the probiotic bacteria Lactobacillus acidophilus and Bifidobacterium lactis. To do so, fermented milk samples were prepared with 2%, 3%, and 6% of flour and 8.25 log CFU/mL of each microorganism. Samples were analyzed in terms of the number of viable cells of L. acidophilus and B. lactis, as well as pH level, total solids, titratable acidity, and texture in the course of 21 days of storage at 4ºC. The obtained microbial viability revealed the in vitro symbiotic effect of flour from V. quercifolia on the probiotic strains of L. acidophilus and B. lactis, which reached 10.20 and 11.19 log CFU/mL, respectively, after 21 days of storage, showing a significant difference in cell growth of 1.7 and 2.5 log CFU/mL compared with the control. The pH level decreased from 4.8 to 4.5 after storage time, so it did not alter the conditions for the growth of bacteria. The physical and chemical parameters analyzed did not reveal significant differences (p > 0.05), which indicates product stability. Therefore, flour from the unripe fruit of V. quercifolia has a prebiotic property and can be used as a nutritional supplement for L. acidophilus and B. lactis.

Keywords: Non-conventional food plants, Native species, Probiotics, Lactobacillus acidophilus, Bifidobacterium lactis

Introduction

The growing interest in certain foods and food components that are physiologically active is changing the eating habits of a good portion of the population. This prompts industries to develop products that promote good health in order to attract consumers and expand their market share (Vega-Gálvez et al. 2015; Granato et al. 2020). In this context, vegetable species that are not widely cultivated, native plants in particular, are promising alternatives for new food products, not only for their nutritional properties but also for their economic viability.

Vasconcellea quercifolia A.St.-Hil. (Caricaceae) is a native plant from Brazil with little-known properties and applications that have a high potential for use in human nutrition. It is one of the non-conventional food plants (NCFP) in Brazil. NCFP are vegetable species with high nutritional value that are not normally consumed in conventional diets (Kinupp and Lorenzi 2019).

In addition to its ecological importance, V. quercifolia, tolerates low temperatures. Also, because of its gene resistance to phytopathogens, it has a high nutritional value and a high concentration of total fibers (Kinupp and Lorenzi 2019). Da Silva et al. (2022) determined that the flour obtained from its unripe fruits presents 32.80% of total fibers. This value is higher than those recorded in other foods: from 6 to 15% for green banana flour (Santos et al. 2018); from 18.9 to 23.7% for carioca beans flour (Bento et al. 2022); 16.5% for yellow pea flour (Setia et al. 2019); 12.5% for pequi pulp flour (Pereira et al. 2022); from 7.4 to 10.3% for lentil flour (Liu et al. 2022); from 9.5 to 15.4% for pigeon pea flour (Chinma et al. 2022); from 15.6 to 16.3% for bacupari flour (De Melo et al. 2022). The fibers found in vegetables are carbohydrates, a non-starch polysaccharide that cannot be digested by the human small intestine but can be fermented by colonic bacteria in short-chain fatty acids (SCFA), such as acetate, propionate, and butyrate. For having these characteristics, these fibers are excellent food components to be used as prebiotics (Ribeiro et al. 2022), which are non-digestible food components that selectively promote the growth and activity of beneficial colonic bacteria, named probiotics.

Among these microorganisms, the genera Lactobacillus and Bifidobacterium are the most commonly used in the food industry. These microorganisms are normal inhabitants of the human gastrointestinal tract (GIT) and comprise the dominant microbiota in the small intestine (Iravani et al. 2015). To be added to food products, these microorganisms must show high viability in these products and must remain alive in the intestine to be able to provide their beneficial effects to the human organism (Ozcan et al. 2016).

NCFP can be consumed fresh and may also be transformed into flour and used as supplements to functional foods. Studies have shown that flour from vegetable species can be used as a fermentable substrate for the growth of probiotics, especially Lactobacillus and Bifidobacterium present in milk and dairy (Boudjou et al. 2014; Staliano et al. 2015; Klajn et al. 2021). Since there has been no research on Vasconcellea quercifolia to investigate its potential as a prebiotic food, this study aimed to evaluate the prebiotic potential of flour from the unripe fruit of V. quercifolia on probiotic bacteria, namely Lactobacillus acidophilus and Bifidobacterium lactis.

Material and methods

Material

Green fruits of mountain papaya (V. quercifolia) were collected from specimens of species found in the municipalities of Arroio do Meio, state of Rio Grande do Sul (RS), Brazil and Cruzeiro do Sul (RS), Brazil, as described in Da Silva et al. (2022). Samplings were conducted from January to March 2018, which corresponds to the species’ fruiting period.

Methods

Analysis of milk fermentation with flour from V. quercifolia

Flour from the unripe fruit of V. quercifolia was added to ultra-high temperature (UHT) whole milk at a concentration of 2%, 4%, and 6% (w/v). The milk was fermented by either a strain of L. acidophilus (La-05®) or B. lactis (Bb-12®), bringing to a total of three treatments per bacteria, in addition to the fermented milk samples that had no flour addition (control samples) (Batista et al. 2017). During the fermentation period (48 h), the pH level and cell viability were analyzed after 0, 4, 12, 24, and 48 h. The fermentation assay was carried out in triplicate.

Inoculum preparation and milk fermentation

The probiotic cultures of L. acidophilus (La-05®) and B. lactis (Bb-12®) were provided in the lyophilized form by Danisco®. They were first diluted into Man, Rogosa, and Sharpe (MRS) liquid medium and stored at 37 ºC for 48 h (Ethik, 403-3D). L. acidophilus (La-05®) and B. lactis (Bb-12®) were kept in Petri plates with MRS agar under refrigeration to be used later (Vinderola and Reinheimer 1999).

The isolated bacteria were resuspended in MRS liquid medium and incubated (at 37 ºC for 48 h). Then, cells were centrifuged (10 min, 5,000 × g) (Universal 320, Hettich), washed in 10 mM of a phosphate-buffered saline (PBS) solution twice, and homogenized in 40 mL of UHT milk.

The UHT milk was divided into four samples of 50 mL, three of which received flour from V. quercifolia at a concentration of 2%, 4%, and 6% (w/v), and one did not receive flour (control). All the samples were individually pasteurized at 65 ºC for 30 min. Then, 10 mL of bacterial inoculum, which was prepared beforehand, was added to the total volume of the milk. The samples with L. acidophilus (La-05®) and B. lactis (Bb-12®) were named 0La, 2La, 4La, 6La, and 0Bb, 2Bb, 4Bb, 6Bb. The numbers correspond to the concentration levels of flour and the letters correspond to the probiotic species used.

The samples were kept in an orbital shaker incubator (Marconi, MA 830) set at 150 rpm and 37 ºC for 48 h. Aliquots of 3 mL were taken after 0, 12, 24, and 48 h to assess the pH level and cell viability. The fermentation assay was carried out in triplicate.

Assessment of cell viability and pH level

For determination of cell viability, an 1.0 mL aliquot of fermented milk with flour from V. quercifolia was submitted to serial dilution in buffered peptone water 0.1% (w/v). Next, aliquots of 20 µL of the diluted sample were transferred to Petri plates with MRS agar medium and then incubated at 37 ºC for 48 h for later counting of the number of colonies (Kim et al. 2008).

Analysis of fermented milk during storage

The fermented milk samples were prepared with a concentration of 2% of flour from V. quercifolia + L. acidophilus (La-05®) and 4% of flour from V. quercifolia + B. lactis (Bb-12®). They fermented for 12 h. A strain of Streptococcus thermophilus (provided by Diagramma®) was used as the starter culture to prepare the fermented milk samples.

The strains of L. acidophilus (La-05®) and B. lactis (Bb-12®) were prepared as described in item 2.3.1. Then, the microorganisms were grown in plates under the following conditions: L. acidophilus in MRS agar medium with bile salts (0.15%) and B. lactis in MRS agar medium with sodium propionate (0.3%) and lithium chloride (0.2%), both incubated in anaerobiosis at 37 ºC for 72 h. S. thermophilus grew in M17 agar medium, incubated in aerobiosis at 37 ºC for 48 h (Vinderola and Reinheimer 1999). The isolated colonies were resuspended to grow, then centrifuged (10 min, 5,000 × g), washed in 10 mM of PBS, and homogenized in 10 mL of UHT milk, forming the inoculum.

In isolated experiments, 10 mL of the inoculum of each probiotic was added to 100 mL of UHT milk, both containing 10 mL of S. thermophilus. Then, 2% of flour was added to the milk sample with L. acidophilus and 4% to the milk sample with B. lactis, both fermented at 37 ºC for 12 h. The fermented milk samples were stored at 4 ºC for 21 days, in glass bottles with screw caps, and analyzed at 0, 7, 14, and 21 days (Batista et al. 2017) in terms of the viability of the probiotic and S. thermophilus strains, as well as pH level, texture, titratable acidity (TA), and total solids.

Assessment of cell viability of microorganisms in fermented milk

An aliquot of 1.0 mL of the fermented milk sample with flour was used for the serial dilutions in buffered peptone water (0.1%) (w/v). Then, aliquots of 20 µL of the serial dilutions were transferred to Petri plates under specific growth conditions for each bacteria (item 2.2), and both were incubated in anaerobiosis at 37 ºC for 72 h. S. thermophilus was quantified in M17 agar medium after incubation in aerobiosis at 37 ºC for 48 h (Vinderola and Reinheimer 1999).

Assessment of titratable acidity and pH level

TA was assessed via titration with a sodium hydroxide solution (0.1 M) in 20 mL of fermented milk with flour and 40 mL of water and two drops of a phenolphthalein solution (1%). The pH level was assessed through the potentiometric method using the electronic pH meter Digimed DM-22 (São Paulo, state of São Paulo), in which an aliquot of 2.0 mL was used for assessment. Both methodologies followed the analytical guidelines proposed by Instituto Adolfo Lutz (2008).

Assessment of total solids and texture

To assess the total solids, 5.0 g of fermented milk was weighted on a porcelain capsule previously dried on a stove (at 105 ºC for one hour). Then, the capsule containing the sample remained on the stove at 105 ºC for 4 h until the first weighting. Then, it was repeatedly weighted every hour until the weight was constant (Instituto Adolfo Lutz 2008). The texture of the fermented milk was assessed in terms of the firmness parameter using a Texture Analyzer (Brookfield, model CT3-10 K) with the following specifications: target at 20%, hold time at 0 s, trigger force at 10 g, speed at 3 mm/s, cycle count at 2, cylinder probe TA3/100, fixture TA-RT, and load cell at 10,000 g.

Statistical analysis

To compare the groups, a one-way analysis of variance (ANOVA) was used followed by a Tukey test for multiple comparisons using the software InfoStat. The differences were considered significant when p < 0.05. All of the analyses were carried out in triplicate of samples and analyses.

Results and discussion

Evaluation of the flour's prebiotic potential

Figure 1A and 1B show the pH level values of the fermented milk samples containing 0%, 2%, 4%, and 6% (w/v) of flour from the unripe fruit of V. quercifolia that were fermented by the probiotics strains for 48 h. The addition of flour to the fermented milk samples resulted in a significant decrease (p < 0.05) in the pH level for both bacteria in comparison with the control sample. After 12 h, the fermented milk samples with L. acidophilus and B. lactis reached the ideal pH level (− 4.4) in all flour concentration levels.

Fig. 1.

Fig. 1

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pH control and cell growth during 48 h of milk fermentation with Lactobacillus acidophilus and Bifidobacterium lactis with 0%, 2%, 4%, and 6% of flour from the unripe fruit of Vasconcellea quercifolia A.St.-Hil. a pH control during fermentation with L. acidophilus; b pH control during fermentation with B. latis; c cell growth during fermentation with L. acidophilus; d cell growth during fermentation with B. latis. ( Inline graphic) 0%, ( Inline graphic) 2%, ( Inline graphic) 4% e ( Inline graphic) 6% flour. Different lowercase letters represent differences between concentrations at the same time. Uppercase letters represent differences between times at the same concentration level. Different letters represent statistical difference ( p < 0.05)

The presence of flour directly and significantly decreased the pH level for both bacteria after 4 and 12 h. However, there was no significant difference (p > 0.05) between samples at a concentration of 4% and 6%. The addition of flour did not influence the pH level at any concentration level after 24 and 48 h. Both probiotic cultures survive in pH levels above 4.0. Acidity levels below that mark may be detrimental to the bacteria's development (Paquin 2009).

When analyzing the fermentation of barley and malt by L. acidophilus, Rathore et al. (2012) reached a pH level of 3.5 in 24 h, similar to the values found in this study for the same fermentation time. This data corroborate the findings of Matejčeková et al. (2017), who obtained a pH level of 4.42 using 9% of buckwheat flour with chocolate flavor in lactic acid fermentation by L. rhamnosus for 8 h. Barat and Ozcan (2018) claim that B. lactis is more tolerant to lower pH levels when compared with other species of Bifidobacterium. These values also corroborate the findings of Ozcan and Kurtuldu (2014), who analyzed the fermentation of yogurt by B. lactis using barley fibers and oats as prebiotics keeping the fermentation acidity above a pH level of 4.6, ideal for yogurt and a stable bacteria growth. Acidity is an important parameter for the perception of flavor in dairy beverages. It is desirable that the pH level is close to 4.4 (Batista et al. 2017). This value was observed in the milk samples with flour from the unripe fruit of V. quercifolia and fermented by commercial probiotic strains (Fig. 1 A and B).

Figure 1 C and D show the number of probiotic bacteria throughout the 48-h fermentation process of the fermented milk samples with 0%, 2%, 4%, and 6% (w/v) of flour from V. quercifolia. In the control sample, the cell growth of L. acidophilus differed (p < 0.05) at all fermentation times (Fig. 1C). There was no significant difference in the fermented milk samples with flour from the unripe fruit of V. quercifolia that were fermented for 12 and 24 h, regardless of flour concentration (p > 0.05). One can opt for the lowest concentration as an optimal condition for both times.

After 12 h of fermentation under the different assessed conditions, L. acidophilus (La-05®) and B. lactis (Bb-12®) will be at the end of the exponential growth phase. During the following hours of fermentation, the microbial cultivation will have reached the stationary phase. The viable cells in this phase have continued to produce lactic acid, therefore, reducing slightly the pH values of fermented milk (Liang et al. 2022).

Cell growth decreased in the sample with 6% of flour between 12 and 24 h, with a significant difference between both times (p < 0.05). The highest cell density (10.21 log CFU/mL) was found under the same conditions (6% of flour in 24 h), but with no statistical difference compared with the other samples in 24 h (p > 0.05). Therefore, the best conditions for L. acidophilus were 2% of flour in 12 h of fermentation, as it reached the stationary phase of cell growth at 9.51 log CFU/mL. In the control sample, the stationary phase was reached in the final hours of fermentation. In 48 h, a cell decline occurred in the samples with flour.

Salmerón et al. (2015) analyzed the fermentation of barley and malt (1%) by L. acidophilus and observed a maximum growth of 8.11 and 8.07 log CFU/mL in 10 h, which represents a growth of 11%. Sah et al. (2016a) analyzed the fermentation of pineapple peel and pulp (1%) (w/v) by the same bacteria and obtained a cell growth of 1 log CFU/mL in 24 h of fermentation. The growth of L. acidophilus with flour from V. quercifolia was similar: 1 log CFU/mL. In the sample fermented with 2% of flour, the growth increased from 8 to 9 log CFU/mL in 12 h, increasing the number of colonies by 10% compared with time zero.

The cell growth of B. lactis in the fermented milk sample with flour from the unripe fruit of V. quercifolia showed a significant difference (p < 0.05) compared with the control at the stipulated times (except for time zero). After 4 h of fermentation, the growth of B. lactis was significantly higher in the samples with 4% and 6% of flour. In 12 h, the sample with 4% of flour showed a higher growth of probiotic bacteria, reaching 10.10 log CFU/mL. In 24 h of fermentation, the samples with 4% and 6% of flour showed a higher cell concentration, reaching 10.08 and 9.99 log CFU/mL, respectively. At the end of the fermentation process (48 h), the fermented milk sample with 2% of flour had reached the stationary phase of cell growth (10.65 log CFU/mL) after 24 h of fermentation.

The statistical comparison between the mean number of B. lactis under the conditions of 4% of flour in 12 h (σ = 0.04) and 2% of flour in 48 h (σ = 1.01) revealed that there was no significant difference (p = 0.3998) in the growth of the probiotic strain.

Sah et al. (2016b) reduced the time of yogurt fermentation from 7.75 to 5.83 h with 1% of pineapple peel powder, which also increased cell density. Kun et al. (2008) obtained a growth of 107 to 108 CFU/mL after 6 h of fermentation by strains of bifidobacteria (including Bb12) using pure carrot juice. In the present study, the best conditions for fermentation by B. lactis were reached with 4% of flour from V. quercifolia in 12 h, reducing the growth time of the probiotic bacteria by 36 h compared with the control sample. The presence of flour from vegetable origin may represent a stimulating source of nutrition for the microbial growth of probiotic cultures. A decrease in the fermentation time is beneficial for the industrial processing of dairy products since it reduces the costs of production (Lee and Lucey 2010).

Regarding the antimicrobial activity of the same species used in this work, Faccio et al. (2015) observed that the mucilage lyophilized in 5% aqueous solution (w v−1) does not present antimicrobial activity against the microorganisms Staphylococcus aureus and Escherichia coli. On the other hand, the pure lyophilized mucilage presents more expressive activity on S. aureus concerning the effect on E. coli. Another experiment with a species of the same genus (Vasconcellea pubescens), performed by Vega-Galvez et al. (2021), evaluated the antimicrobial potential of the extract obtained from papaya pulp on S. aureus, E. coli and Bacillus cereus and found that the extracts obtained from organic solvents (ethanol, acetone and methanol) exhibited antimicrobial activity against the three microorganisms.

Cell growth of probiotic strains during storage of fermented milk

Figure 2 A and B show the cell growth of the commercial probiotic bacteria L. acidophilus (La05) and B. lactis (Bb12), respectively, in the milk samples fermented with flour from the unripe fruit of V. quercifolia and stored at 4 ºC for 21 days. The fermentation conditions were 2% of flour during 12 h for L. acidophilus and 4% of flour during 12 h for B. lactis.

Fig. 2.

Fig. 2

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Cell growth of a Lactobacillus acidophilus and b Bifidobacterium lactis in the fermented milk samples with 2% and 4% (w/v), respectively, of flour from the unripe fruit of Vasconcellea quercifolia A.St.-Hil. ( Inline graphic) 0%, ( Inline graphic) 2% e ( Inline graphic) 4% flour. Different lowercase letters represent differences between concentrations at the same time. Uppercase letters represent differences between times at the same concentration level. Different letters represent statistical difference ( p < 0.05)

Until the 14th day, the cell growth of 1.5 log CFU/mL of L. acidophilus was significant (p < 0.05). It was constant (p > 0.05) until the 21st day (Fig. 2A). In the control sample, there was no significant increase in cell growth (p < 0.05). After 14 days, there was a significant decrease (p < 0.05) of 1 log CFU/mL, indicating cell death. After 21 days, the sample with flour showed a cell growth of 10.20 log CFU/mL of L. acidophilus, this value was 2 log CFU/mL higher than the control at the end of the fermentation process. Furthermore, the sample with flour did not undergo cell death, as the control sample did, suggesting that the addition of 2% of flour may increase the lifetime of L. acidophilus in milk.

Batista et al. (2017) noted that the concentration of flour directly influenced cell viability. They reported growth of L. acidophilus from 7.11 to 8.77 log CFU/mL in the fermented milk sample with 1% of flour from unripe banana (FUB) in the course of 21 days of storage. These data corroborate the data found in the fermented milk samples with 2% of flour from the unripe fruit of V. quercifolia, since both studies reveal growth of 1.6 log CFU/mL in samples with flour after 21 days of storage.

Furthermore, L. acidophilus experienced a cell growth of 29% in the sample with flour from V. quercifolia when compared with the control sample, while using a percentage of flour that is two times lower than the one used by Boudjou et al. (2014), who reported a cell growth of L. acidophilus of 26% during the production of kefir with 4% (w/v) of bean flour (Vicia faba L.).

In the milk samples fermented by B. lactis with 4% of flour from V. quercifolia, the bacteria underwent a significant cell growth (p < 0.05) of 2.5 log CFU/mL until the 14th day. They continued to grow until the 21st day, but it showed no statistical difference (p > 0.05) compared with the previous time (Fig. 2B). After 21 days, the microbial growth reached maximum cell concentration during fermentation (11.19 log CFU/mL). In the control sample, the bacteria showed no significant cell growth (p > 0.05) and underwent cell death after 14 days, as they decreased by 0.9 log CFU/mL. There was a significant difference between the 14th and the 21st days (p < 0.05). After 21 days, the sample with flour showed 3.5 log CFU/mL more than the control sample, which means that this product has higher probiotic viability. The presence of flour preserved the probiotic strains and the cell increment of the bacteria B. lactis.

The addition of 3% of FUB to the fermented milk also significantly increased the cell viability of B. lactis (p < 0.05). It went from 6.56 to 8.85 log CFU/mL in the course of 21 days (Batista et al. 2017). Although the cell concentration in milk fermented with FUB was lower than that of milk fermented with flour from V. quercifolia by the end of the storage time, the cell growth during this time was higher than 2 log CFU/mL in both studies. Moreover, a preparation of kefir containing B. lactis and supplemented with 4% of bean flour increased the number of viable cells by 11% during the first 7 days (Boudjou et al. 2014). Already in the present study, the cell growth of B. lactis was superior in the medium supplemented with flour from V. quercifolia until the end of the storage time.

The lactobacilli generally have a narrow profile of carbohydrate fermentation when compared with bifidobacteria (McLaughlin et al. 2015), which might explain the greater effect of flour from V. quercifolia on the viability of B. lactis. Furthermore, to provide probiotic effects, the microorganisms must be viable and in high concentrations during the entire shelf life of the product, which occurred in the fermented milk samples with flour from V. quercifolia but did not occur in the samples without flour (control).

The starter culture of lactic acid bacteria Streptococcus thermophilus was also analyzed in terms of cell growth (Table 1) in the fermented milk samples with flour from the unripe fruit of V. quercifolia. In the milk samples fermented by the probiotic bacteria L. acidophilus, there was a significant increase (p < 0.05) in the cell concentration of S. thermophilus of approximately 1 log CFU/mL after 14 days and approximately 0.06 log CFU/mL after 21 days of storage compared with the control sample. In the milk sample fermented by B. lactis, there was no significant cell growth (p > 0.05) with the starter culture of S. thermophilus. However, the sample with flour showed a cell concentration of S. thermophilus 2.5 log CFU/mL higher than the sample without flour after 7 days of storage. The results remained the same within 21 days, when the concentration was above 1 log CFU/mL in the sample with flour compared with the control sample (p < 0.05). Although the cell concentration of S. thermophilus decreased after 14 days of storage at 4 ºC, it was significantly higher (p < 0.05) in the samples that were co-fermented by L. acidophilus and B. lactis compared with the samples without flour.

Table 1.

Cell growth (log CFU/mL) of the bacteria Streptococcus thermophilus in co-fermentation with probiotic strains

Day Streptococcus thermophilus (co-fermented with L. acidophilus) log CFU/Ml Streptococcus thermophilus (co-fermented with B. lactis) log CFU/mL
0% flour 2% flour 0% flour 4% flour
1 8.29 8.61 8.73 8.91
7 8.35 9.60* 9.90* 9.15
14 8.75 9.81* 8.45 10.99
21 8.36 9.06** 8.26 9.76
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* Significant growth (p < 0.05) ** Significant decrease (p < 0.05)

Do Espirito Santo et al. (2012) reported a similar cell growth when they observed superior quantities of S. thermophilus in yogurt co-fermented by L. acidophilus and B. lactis, demonstrating a positive correlation between the bacterial strains. The same authors argued that the decrease in the number of S. thermophilus after 14 days is frequent in commercial cultures of lactic acid bacteria associated with other strains under different growth conditions because of the competition for nutrients and specific conditions, leading to an early cell decline. Therefore, flour may influence cell growth of the starter culture of lactic acid bacteria, even in the presence of the probiotic strains of L. acidophilus and B. lactis.

pH level, titratable acidity, total solids, and texture during storage

The pH values decreased significantly (from 4.8 to 4.5) (p < 0.05) over the course of 21 days in the milk samples fermented by L. acidophilus and B. lactis with 2% and 4% (w/v), respectively, of flour from the unripe fruit of V. quercifolia. The fermented milk samples with flour had a lower pH than the control sample (without flour), which indicates the acid property of flour and the cell proliferation of the lactic acid bacteria. The decrease in pH occurred due to the metabolization of the milk's lactose by the lactic acid bacteria, producing lactic acid in the medium and acidifying the sample. Other authors have reported this effect in the production of fermented dairy products (Januário et al. 2017; Batista et al. 2017).

Table 2 shows the results of the analyses of titratable acidity, total solids, and firmness of the milk samples fermented by L. acidophilus and B. lactis with 2% and 4% (w/v), respectively, of flour from the unripe fruit of V. quercifolia in the course of 21 days of storage at 4 ºC.

Table 2.

Titratable acidity, total solids, and texture of the fermented milk samples with flour from the unripe fruit of Vasconcellea quercifolia A.St.-Hil. during storage under refrigeration

Parameter Day Fermented milk (L. acidophilus) Control (L. acidophilus) Fermented milk (B. lactis) Control (B. lactis)
Titratable acidity (°D) 1 1.13 ± 0.02c 0.20 ± 0.01a 1.50 ± 0.14b 0.20 ± 0.02a
7 1.38 ± 0.23d 0.29 ± 0.001b 1.48 ± 0.13b 0.27 ± 0.03b
14 1.42 ± 0.02d 0.31 ± 0.004b 1.55 ± 0.06b 0.29 ± 0.005b
21 1.45 ± 0.04d 0.31 ± 0.01b 1.54 ± 0.02b 0.31 ± 0.03b
Total solids (%) 1 12.40 ± 0.11b 11.88 ± 0.06a 13.35 ± 0.10b 11.81 ± 0.05a
7 12.02 ± 0.08b 11.78 ± 0.04a 13.45 ± 0.05b 11.66 ± 0.02a
14 12.09 ± 0.05b 11.78 ± 0.07a 13.46 ± 0.04b 12.50 ± 0.06a
21 12.02 ± 0.12b 11.72 ± 0.08a 13.44 ± 0.05b 11.82 ± 0.05a
Firmness (N) 1 0.21 ± 0.001a 0.17 ± 0.001a 0.13 ± 0.002a 0.13 ± 0.001a
7 0.11 ± 0.001b 0.12 ± 0.001b 0.12 ± 0.001a 0.13 ± 0.001a
14 0.13 ± 0.001b 0.11 ± 0.001b 0.12 ± 0.002a 0.12 ± 0.001a
21 0.12 ± 0.002b 0.11 ± 0.001b 0.13 ± 0.002a 0.14 ± 0.001a
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Different letters (a and b) represent statistical differences within the analyzed timeframe (p < 0.05)

The milk samples without flour fermented by both probiotic strains showed a significant difference (p < 0.05) in terms of titratable acidity until the 7th day of storage. After 7 days, the values remained stable, reaching a maximum of 0.31% of acidity (Table 2). The samples with flour showed a significant difference (p < 0.05) compared with the control samples, reaching values above 1% for both probiotic strains (Table 2). The maximum acidity found in the fermented milk samples with flour was 1.45% and 1.55% for L. acidophilus and B. lactis, respectively. The presence of flour from the unripe fruit of V. quercifolia may have been responsible for the final acidity in the milk samples fermented by the probiotic strains.

Juliana et al. (2015), using milk co-fermented by L. acidophilus and Bifidobacterium with 3% of quinoa flour, obtained 1.34% of acidity after 22 days of storage, similar to the findings of this study. The increase in acidity is common in milk fermented by S. thermophilus, L. acidophilus, and B. lactis due to the high production of organic acids by the metabolic processes of the microorganisms during storage (Batista et al. 2017). S. thermophilus is a homofermentative bacteria and has a quick metabolism, being responsible for a great portion (approximately 84%) of the lactic acid produced in fermentation processes. The bacteria of the genus Bifidobacterium also produce acetic acid, because they contain fructose-6-phosphate phosphoketolase, which allows a greater production of adenosine triphosphate (ATP) from carbohydrates than the conventional heterofermentative and homofermentative bacteria, yielding 2.54 mol of acetic acid and 2 mol of lactic acid for every 2 mol of ingested glucose (Oliveira et al. 2012).

The milk samples fermented by L. acidophilus and B. lactis showed 12.40% and 13.46% of total solids, respectively, values significantly higher than those found in the control samples (Table 2). Data found by Batista et al. (2017) corroborate the results of the present study. As they analyzed the addition of 1%, 3%, and 5% of FUB to milk fermented by L. acidophilus and B. lactis, Batista et al. (2017) reported 16.02%, 17.13%, and 19.89% of total solids, respectively. Furthermore, they did not find significant variations (p > 0.05) during 21 days of storage. Products with high levels of solids have longer shelf lives because they inhibit the degradation of lactose and increase the buffering capacity (Padua et al. 2017).

In terms of firmness (the individual appearance of a product that changes according to the profiles of consumers), the fermented milk samples containing L. acidophilus varied between 0.11 N and 0.21 N. Both the sample with 2% of flour and the control sample showed a significant difference (p < 0.05) in firmness between the first day of storage and the following days (Table 2). The firmness of the fermented milk samples after 7, 14, and 21 days of storage did not reveal a significant difference (p > 0.05) compared with the control sample. There was no significant difference between the milk samples fermented by B. lactis and the control sample in terms of firmness, which varied between 0.12 N and 0.14 N during storage time. Batista et al. (2017) obtained 1.43 N and 1.92 N of firmness in fermented milk with 3% and 5% of FUB, respectively. These values are higher than the ones found in the present study using the same probiotic species. The dietary fibers present in foods of vegetable origin can form a complex with protein aggregates via H-bonds, becoming a part of the structural network formed during fermentation (Crispín-Isidro et al. 2015). The low concentrations (2% and 4%) of flour from the unripe fruit of V. quercifolia used in the present study were probably insufficient to form these aggregates and to increase the firmness of the fermented milk samples, resulting in a dairy product with a thinner consistency.

Conclusion

The flour from the unripe fruit of V. quercifolia preserved the viability and allowed the cell growth of both probiotic strains in the fermented milk samples analyzed during cold storage. These results confirm the in vitro prebiotic effect of flour from the unripe fruit of V. quercifolia at a concentration of 2% and 4% on the probiotic bacteria L. acidophilus and B. lactis, respectively. It has a nutritional, protective, and prebiotic effect on the probiotic cultures analyzed, since it was available as a substrate for the metabolism of these microorganisms, serving as a carbon source for cell maintenance. The characteristics of the fermented product, such as pH level, titratable acidity, and total solids, were not altered, in accordance with the legislation for fermented milk in Brazil. Both products had more than 6 log CFU/mL of probiotic bacteria by the end of 21 days of cold storage, allowing them to be classified as probiotic products.

Commercially available prebiotics have a simpler nutritional composition than prebiotic flour obtained from fruits since these can have soluble and insoluble fibers, carbohydrates, phenolic compounds, proteins, minerals, and vitamins. Fruits can raise the nutritional value of dairy products since they can increase the daily ingestion of fibers and increase the viability of probiotic bacteria. V. quercifolia is an underutilized native species with potential for human nutrition and easy propagation. This study points to the applicability of this native fruit in the development of new functional dairy products of high nutritional value.

Acknowledgements

This study was supported by grants from the Coordination for the Improvement of Higher Education Personnel (Capes), Brazil. Funding code: 001.l.

Abbreviations

ANOVA

One-way analysis of variance

ATP

Adenosine triphosphate

B. lactis

Bifidobacterium lactis

CFU

Colony forming unit

E. coli

Escherichia coli

FUB

Flour from unripe banana

GIT

Gastrointestinal tract

L. rhamnosus

Lactobacillus rhamnosus

MRS

Man, rogosa, and sharpe

NCFP

Non-conventional food plants

PBS

Phosphate-buffered saline

pH

Hydrogenionic potential

S. aureus

Staphylococcus aureus

S. thermophilus

Streptococcus thermophilus

SCFA

Short-chain fatty acids

TA

Titratable acidity

UHT

Ultra-high temperature

V. quercifolia

Vasconcellea quercifolia

w/v

Weight per volume

L. acidophilus

Lactobacillus acidophilus

Authors' contributions

The Authors declare that the LFFS, KF R and AG carried out the work, analyzed the results, prepared the figures and tables and wrote the manuscript. EME, LH, CFVS and EMF were responsible for conceiving the idea, carried out the work and revised and supplemented the manuscript.

Funding

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Brazil. Funding code: 001.l

Availability of data and material

Not applicable.

Code availability

Not applicable'.

Declarations

Conflict of interest

The authors declare they do not have any conflicts of interest.

Consent to participate

All authors consent for publication in the Journal of Food Science and Technology.

Consent for publication

Not applicable.

Ethics approval

Not applicable.

Footnotes

Publisher's Note

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Contributor Information

Lilian de Fátima Ferreira da Silva, Email: lilian.silva@universo.univates.br.

Kétlin Fernanda Rodrigues, Email: ketlin.rodrigues@universo.univates.br.

Adriano Gennari, Email: agennari@universo.univates.br.

Eduardo Miranda Ethur, Email: eduardome@univates.br.

Lucélia Hoehne, Email: luceliah@univates.br.

Claucia Fernanda Volken de Souza, Email: claucia@univates.br.

Elisete Maria de Freitas, Email: elicauf@univates.br.

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