In vitro resistance of Lactobacillus plantarum LP299v or Lactobacillus rhamnosus GG carried by vegetable appetizer

LWT - Food Science and Technology 116 (2019) 108512

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In vitro resistance of Lactobacillus plantarum LP299v or Lactobacillus rhamnosus GG carried by vegetable appetizer

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Priscilla Araújo Camposa, Eliane Maurício Furtado Martinsa,∗, Maurílio Lopes Martinsa, Aurélia Dornelas de Oliveira Martinsa, Bruno Ricardo de Castro Leite Júniorb, Roselir Ribeiro da Silvaa, Larissa Mattos Trevizanoa a Federal Institute of Southeast of Minas Gerais, Food Science and Technology Department (DCTA/IF Sudeste MG), Laboratory of Food Microbiology, Rio Pomba, MG CEP 36180-000, Brazil b Federal University of Viçosa, Food Technology Department, Viçosa, MG CEP 36570-000, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Fermented vegetables Probiotic Gastrointestinal resistance Functional food

This study evaluated the viability of Lactobacillus plantarum LP299v or Lactobacillus rhamnosus GG in vegetable appetizer, as well as the resistance of the strains to the gastrointestinal tract (GIT) simulated in vitro. Control appetizer and added of probiotic strains were prepared and remained at 8 °C for 90 days. There was no difference in the L* and b* between the treatments and throughout the storage time. The control appetizer presented higher pH and lower acidity compared to probiotic appetizer. Vegetable appetizer showed counts of L. plantarum or L. rhamnosus higher than 7.42 Log CFU/g and 8.84 Log CFU/g respectively, along the refrigerated storage, being verified greater viability for L. rhamnosus (p < 0.05) with no reduction in the counts of both microorganisms over time (p > 0.05). Mean scores above 6.0 (“slightly appreciated”) were attributed to sensory analysis. The appetizer containing L. rhamnosus had a higher preference among consumers. In the in vitro GIT test, considering the consumption of a 100 g portion, in the time 90 days, approximately 8.67 Log CFU/g and 9.53 Log CFU/g of L. plantarum or of L. rhamnosus respectively, would be available to promote consumer benefits, which makes the appetizer apt to be considered probiotics.

1. Introduction In the last decades, there have been great changes in the diet being characterized by processed foods, sugars, fats and oils. This change has contributed to the high incidence of chronic diseases (Tilman & Clark, 2014), which have been modifying the behavior of the population, who started to seek healthier living habits (Saad, Cruz, & Faria, 2011). Thus, consumers are becoming increasingly aware of the relationship between diet and health (Carrillo Prado-Gascó, Fiszman, & Varela 2013), boosting markets for functional food development. Probiotics are among the functional ingredients and, according to Hill et al., 2014, are living microorganisms which, when administered in suitable amounts, confer a health benefit to the host. Dairy products are more suitable for the development of probiotic foods, however, there are individuals with intolerance and allergy to dairy products (Roberts et al., 2018; Suri et al., 2019), vegetarians, cholesterol-restricted diets or those who do not consume dairy products because of habits (Vasudha & Mishra, 2013). Thus, there is a growing search for

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new food matrices as a vehicle for probiotics as an alternative to dairy products. Earlier, Martins et al., 2013 reported that vegetables have been presented as an alternative for the insertion of these bacteria in the diet, since fruits and vegetables have nutrients that help the growth of probiotics. In addition, they are essential in the human diet, since their consumption promotes health benefits due to the presence of micronutrients and fibers (Ramos, Miller, Brandão, Teixeira, & Silva, 2013). Fruits and vegetables also have prebiotic growth promoting ingredients and act as protectors for probiotic microorganisms, protecting their viability during the life of the product when passing through the gastrointestinal tract (Cerdó, García-Santos, Bermúdez, & Campoy, 2019; Galgano, Condelli, Caruso, Colangelo, & Favati, 2015, chap. 10). In addition, raw and fermented vegetables represent an excellent vehicle for probiotic bacteria (Zhao et al., 2019) because of their natural structure that allows for easy availability of nutrients for microbial growth (Espírito-Santo, Perego, Converti, & Oliveira, 2011; Peres, Peres, Mendoza, & Malcata, 2012). The plant tissues are composed of

Corresponding author. Tel: +55 32 3571-5742. E-mail address: [email protected] (E.M.F. Martins).

https://doi.org/10.1016/j.lwt.2019.108512 Received 29 January 2019; Received in revised form 25 July 2019; Accepted 14 August 2019 Available online 15 August 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

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Amaya (2001, p. 64) at times 0, 30, 60 e 90 days of storage at 8 °C. Colour analysis was evaluated using a Konica Minolta CR10 (Tecnal, BR) colorimeter. The colour was determined by direct reading of the reflectance of the coordinates L* (lightness and varies from 0 (black) to 100 (white)) and b* (saturation and varies from +b (yellow) to -b (blue)) using the CIELAB L* scale adopted as standard by the International Commission on Illumination, at times 0, 30, 60 e 90 days of refrigerated storage.

intracellular spaces, pores and capillaries, and microorganisms can penetrate through injuries in these tissues (Brackett & Splittstoesser, 2001). Plant products fermented with probiotic lactic acid bacteria are consumed in the world, which are in the form of pastes, antipasti, canned vegetables, sauerkraut, kimchi, drink and others (Alves et al., 2015; Campos et al., 2019; Ji et al., 2013; Yu et al., 2012). However, the tolerance of the gastrointestinal tract conditions is one of the necessary characteristics of any bacteria to exert probiotic function in the intestine, and the in vitro assay is used to predict its survival in the in vivo condition (Rubio, Martín, Aymerich, & Garriga, 2014). The in vitro assay is widely used because they are fast, safe, and do not exhibit the same ethical constraints as many methods in vivo (You, Zhao, Regenstein, & Ren, 2010). The main objective of this study was to evaluated the resistance of Lactobacillus plantarum LP299v or Lactobacillus rhamnosus GG in vegetable appetizer to the gastrointestinal tract (GI) simulated in vitro. In addition the physico-chemical, microbiological and sensorial characteristics were evaluated.

2.4. Determination of the viability of L. plantarum or L. rhamnosus in the appetizer of vegetables

2. Material and methods

The viability of L. plantarum LP299v or L. rhamnosus GG was determined after the fermentation period (time 0 day), and after 15, 30, 60 and 90 days of storage at 8 °C. These probiotic bacterium were quantified according to Richter and Vedamuthu (2001) in the de Man Rogosa and Sharpe (MRS, Merck, Darmstadt, Germany) culture medium. The diluted samples mixtures were plated in MRS agar by the pour plate technique, and the Petri dishes incubated in anaerobic jars at 37 °C for 72 h.

2.1. Processing of vegetables and preparation of appetizer

2.5. Evaluation of the microbiological quality of the appetizer

Fresh apples, eggplants, cucumbers, cabbages, peppers and onions were selected, washed in running water, sanitized in chlorine solution containing 200 mg/L of active chlorine for 15 min and rinsed in potable water containing 10 mg/L of active chlorine. They were then cut manually into cubes with the aid of stainless steel knives. Grape raisins, olive, linseed and chia were purchased ready to use. After cutting, the eggplant was bleached in water at 85 °C for 5 min. Later, this vegetable was drained for the elaboration of the appetizer which consisted of 18% of olive oil, 4% of onion, 3% of yellow pepper, 3% of red pepper, 1.5% of flaxseed, 1.5% of chia, 7% of grape raisins, 20% of eggplant, 8% of olive, 15% of apple, 5% of cucumber and 14% of cabbage. The ingredients were homogenized and transferred to a stainless steel vat that was left in the oven at 180 °C for 30 min.

Analysis of filamentous fungi and yeasts were performed using surface plating in Dichloran 18 Glycerol Agar (Acumedia, Michigan, USA - DG 18), according to Beuchat and Cousin (2001), chap. 20. Total and thermotolerant coliforms were determined by the Most Probable Number (MPN) technique according to Kornacki and Johnson (2001). The presence or absence of Salmonella sp. was determined in 25 g of the samples, homogenized in 225 mL of lactose broth (MicroMed/Isofar, Duque de Caxias, Rio de Janeiro, Brazil) using the methodology of Andrews, Flower, Silliker, and Bailey (2001). Vegetables appetizer were evaluated immediately after fermentation (time 0 day) and after 90 days of refrigerated storage at 8 °C.

2.2. Inoculation of probiotic cultures to the appetizer

Appetizers from control treatment and containing probiotic cultures were evaluated by 65 volunteers. The hedonic scale of nine points was used, varying from “extremely liked” (score 9) to “disliked extremely” (score 1) for the following attributes: appearance, acidity, taste, colour, texture and overall impression, according to Stone, Bleibaum, and Thomas (2012), at zero time (T0) and after 90 days of storage at 8 °C. Moreover, the consumer attitude with respect to purchasing intention of the products was assessed using a five-point scale ranging from “definitely would buy” (score 5) to “certainly would not buy” (score 1), according to Meilgaard, Civille, and Carr (2006, p. 281), after the fermentation period (time 0 day), and after 90 days of refrigerated storage.

2.6. Sensorial quality of vegetables appetizers

A ready-to-use freeze-dried culture of Lactobacillus plantarum LP299v (Digestive Care™) or L. rhamnosus GG (Culturelle®) was aseptically added to the vegetables to produce the appetizers. A capsule containing 1.0 × 1010 cells of each culture was added individually, under aseptic conditions, in a portion of approximately 30 mL of olive oil, representing 1.03% of the 18% of olive oil added, being homogenized in 500 g of the appetizer at 40 °C. The appetizers were packed in sterile glass vials and incubated for 24 h at 37 °C in order to achieve a count of approximately 8.0 Log CFU/g of the probiotic bacteria. After this fermentation period, the canned product was stored in a stove B·O.D (Novatecnica NT 704, Piracicaba, São Paulo, Brazil) at 8 °C for up to 90 days for the analysis. The control (without addition of probiotic culture) was also elaborated, being maintained under the same conditions.

2.7. Resistance in vitro of L. plantarum LP299v or L. rhamnosus GG to simulated gastrointestinal conditions The resistance of L. plantarum LP299v or L. rhamnosus GG to the in vitro gastrointestinal system was evaluated by oral, gastric and enteric I and enteric II phases, at zero times, 30, 60 and 90 days. As the first step of the in vitro study, the oral phase was performed, according to the methodology described by Mills et al. (2008) and Gullon et al. (2015), with some modifications. The simulation of gastric and enteric (I and II) juices with enzymes of the gastrointestinal tract was performed according to the methodology of Bedani, Rossi, and Saad (2013).

2.3. Determination of the physicochemical characteristics The pH value, titratable acidity (lactic acid/100 g of product) and total soluble solids (TSS) of the vegetables appetizer were determined according to AOAC (2016). The physico-chemical analyzes of pH and total soluble solids (in ° Brix) of the appetizer were performed after fermentation (time 0) after seven days, 15, 30, 45, 60, 75 and 90 days. The titratable total acidity (% lactic acid) was performed at time 0, 7, 15, 30, 60, 75 and 90 days of storage at 8 °C. Total carotenoid content was analyzed for the control and for appetizer of vegetables inoculated with L. plantarum or L. rhamnosus. Total carotenoids were extracted from samples according to Rodriguez-

2.8. Statistical analysis The results of physical chemical analyzes, microbiological, viability and in vitro assay were evaluated using analysis of variance and the completely randomized design, with 3 replicates. For pH, acidity and 2

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acidic compounds increasing the acidity of the medium (Chim, Zambiazi, & Rodrigues, 2013). This behavior was observed for the acidity of the appetizer containing probiotic cultures, which presented significant alteration (p < 0.05) between the beginning and the end of the storage period. Carotenoids play an important role in the diet because they are precursors of vitamin A and have antioxidant capacity (Mattietto & Lopes, 2011, chap. 27; Saini, Nile, & Park, 2015) related to lycopene and β-carotene. For the total carotenoid content, there was no significant difference (p > 0.05) between treatments, however significant degradation (p < 0.05) of these pigments was observed throughout the storage period (Table 1). In the literature it is reported that foods that are composed of more than 0.02 mg of carotenoids/g of product are rich sources of this important pigment for health (Rodriguez - Amaya, Kimura, & Amayra-Farfan, 2008; Rodriguez-Amaya, Kimura, & AmayaFarfan, 2001). Therefore, vegetable appetizer can be considered a good source of carotenoids if consumed in larger portions, because the intake of 100 g may offer the consumer at least 0.29 mg of carotenoids at the end of storage time. There was no difference between the treatments and time interference (p > 0.05) over the total soluble solids (TSS) values during the 90 days of refrigerated storage (Fig. 1). Chim et al. (2013) have reported that the storage temperature exerts influence on the action of microorganisms and, consequently, on the maintenance of SST.

total soluble solids a factorial scheme 3 × 8 (treatments x times) was adopted and for colour and carotenoids a 3 × 4 factorial scheme was used. A 2 × 5 factorial scheme was used for the viability experiment. The in vitro simulation of the GIT was conducted in a 2 × 4 x 4 factorial scheme (treatments x phases x times). The acceptance and purchase intention data were analyzed by a completely randomized block design, in a 3 × 2 factorial scheme. The results were evaluated through analysis of variance and the means compared by the Tukey test. All statistical procedures were carried out considering a 5% level of probability and using the statistical software STATISTICA 7.0 (StatiSoft, Inc., Tulsa, Okla., USA). 3. Results and discussion 3.1. pH values, acidity, total carotenoids and TSS contents of the vegetable appetizer The pH values differed significantly (p < 0.05) from the control treatment for that added of probiotic cultures, and the time factor was also significant (p < 0.05). However, when analyzing pH values, only for the appetizer added of probiotic cultures, it was verified that there was no difference (p > 0.05) between the treatments (Table 1). The fermentation process of the appetizer added of probiotic bacteria promoted reduction of pH values over time (p < 0.05), a fact already expected due to the production of acids by bacteria. In relation to the acidity in % of lactic acid, there was a difference (p < 0.05) in the control treatment for that with probiotic cultures, with a significant time factor (p < 0.05) (Table 1). Acidity is also considered an important quality feature. The reactions involved in the decomposition, such as fermentation, generate

3.2. Colour There was no significant difference (p > 0.05) for luminosity (L *) between the vegetable appetizer of the control treatments and inoculated with probiotic bacteria. There was also no influence (p > 0.05) of the storage time on L *, evidencing that the addition of L. plantarum LP299v or L. rhamnosus GG did not alter the luminosity of the appetizer. The b * coordinate, which measures the degree of variation between yellow and blue, also did not differ (p > 0.05) between the appetizer and there was no influence of storage time (p > 0.05) (Table 2), being possible to affirm that the appetizer tended to the yellow colour.

Table 1 pH, acidity (lactic acid/100 g of product) and carotenoids averages in the appetizer of the different treatments during the storage period. Treatments

Time (days)

Physicochemical Characteristics pH

Acidity (lactic acid/100 g)

Carotenoids (μg/g)

0 7 15 30 45 60 75 90

4.48DEde 4.46DEde 4.52Ee 4.56Ee 4.42DEde 4.32CDEcde 4.30CDEcde 4.30CDEcde

0.13Aa 0.23ABab 0.21ABab 0.21ABab – 0.24ABab 0.25ABCabc 0.22ABab

4.86Aa – – 4.63Aa – 3.31Aa – 2.85Aa

AVP

0 7 15 30 45 60 75 90

3.93ABCDEabcde 3.79ABCDabcd 3.69ABCabd 3.66ABCabc 3.61ABCabc 3.57ABab 3.47ABab 3.48ABab

0.32ABCDabcd 0.47ABCDEabcde 0.55ABCDEabcde 0.64BCDEbcde – 0.50ABCDEabcde 0.67BCDEbcde 0.73CDEcde

5.31Aa – – 3.92Aa – 3.41Aa – 3.27Aa

AVR

0 7 15 30 45 60 75 90

4.09BCDEbcde 3.79ABCDabcd 3.66ABCabc 3.51ABab 3.48ABab 3.54ABab 3.36Aa 3.35Aa

0.23ABab 0.42ABCDEabcde 0.53ABCDEabcde 0.64BCDEbcde – 0.79Dede 0.85Ee 0.90Ee

5.50Aa – – 4.20Aa – 2.99Aa – 2.70Aa

AVC

3.3. Viability of L. plantarum LP299v or L. Rhamnossus GG in the vegetable appetizer The canned appetizer enriched with L. plantarum LP299v or L.

Capital letters reflect comparisons between different treatments for the same time. Small letters reflect comparisons between different times for the same treatment. Means followed by the same letter in the same column do not differ from each other according to the Tukey test at 5% of probability. AVC: appetizer of vegetable control; AVP: vegetable appetizer containing L. plantarum LP299v; AVR: vegetable appetizer containing L. rhamnosus GG.

Fig. 1. Mean values of total soluble solids (°Brix) of vegetable appetizer of the control treatments and inoculated with L. plantarum LP299v (LP299v) or L. rhamnosus GG (LRGG) stored at 8 °C for 90 days. The confidence interval (p < 0.05) is represented by the vertical lines. 3

The vegetable appetizer with different probiotic bacteria and the control treatment showed absence of Salmonella sp. in 25 g of the 4

7,43 7,55 7,45 7,48A 7,37 7,48 7,66 7,50A 7,38a 7,55a 7,35a 7,91 7,95 7,57 7,81B 6,86 7,15 7,12 7,05A 7,45a 7,49a 7,63a 7,32 7,57 7,55 7,48A 7,57 7,41 7,71 7,56A 7,30a 7,25a 7,66b 7,32 7,34 7,49 7,38A 7,28 7,17 7,83 7,42A 7,23b 6,41a 7,35b 7,61 6,34 7,46 7,14B 6,85 6,45 7,25 6,86A

90 days

Means followed by the same lowercase letter in the column and upper case in the row, respectively, did not differ by Tukey's test (p < 0.05). AVC: appetizer of vegetable control; AVP: vegetable appetizer containing L. plantarum LP299v; AVR: vegetable appetizer containing L. rhamnosus GG.

3.4. Evaluation of the microbiological quality of the appetizer

7,31a 7,58 ab 7,65b

rhamnosus GG presented counts higher than 7.42 Log CFU/g and 8.84 Log CFU/g (p < 0.05), respectively, over the 90 days of storage (Fig. 2), with no significant influence (p > 0.05) of the time. There was a reduction of approximately 1.5 log cycles in the L. plantarum LP299v count when compared to L. rhamnosus GG (p > 0.05) at 60 and 90 days of storage of the appetizer and, despite a reduction of the count, the appetizer still is a good carrier of this strain, since it showed counts above 107 CFU/g. Some authors consider that the level of probiotic cultures in food should be high, being in the range of 106–108 CFU/mL (Hussain, Patil, Yadav, Singh, & Singh, 2016), while others suggest between 109 and 1010 CFU/g (Martins, Ramos, Martins, & Rodrigues, 2015; Naidu, Adam, & Govender, 2012). In this way, the ingestion of 100 g of the vegetable appetizer containing L. plantarum LP299v may provide the consumer with a minimum of 9.42 log CFU per serving while the appetizer containing L. rhamnosus GG may offer 10.84 Log UFC, indicating that the developed vegetable appetizer are an excellent carrier of these bacteria.

7,40 7,74 7,72 7,62A

Fig. 2. Viability of probiotic microorganisms L. plantarum LP299v (LP299v) or L. rhamnosus GG (LRGG) over 90 days of storage of vegetable appetizer. The confidence interval (p < 0.05) is represented by vertical lines.

7,21 7,43 7,58 7,41A

Capital letters reflect comparisons between different treatments for the same time. Small letters reflect comparisons between different times for the same treatment. Means followed by the same letter in the same column do not differ from each other according to the Tukey test at 5% of probability. AVC: appetizer of vegetable control; AVP: vegetable appetizer containing L. plantarum LP299v; AVR: vegetable appetizer containing L. rhamnosus GG.

AVC AVP AVR Média

0,47Aa 0,52Aa 1,09Aa 1,17Aa

0 day

± ± ± ±

Average

1,55 2,38 2,88 3,44

90 days

0,56Aa 1,10Aa 2,08Aa 3,30Aa

0 day

± ± ± ±

4,29Aa 1,82Aa 1,48Aa 5,57Aa

Average

14,10 12,40 11,67 10,50

± ± ± ±

90 days

32,27 32,23 28,90 27,12

0 day

0 30 60 90

Average

AVR

90 days

1,19Aa 0,45Aa 0,84Aa 1,82Aa

0 day

± ± ± ±

Average

1,20 1,69 1,77 2,47

90 days

0,17Aa 1,42Aa 2,72Aa 0,98Aa

0 day

± ± ± ±

Average

13,00 12,40 14,40 12,00

90 days

27,6 ± 4,37Aa 26,73 ± 4,37Aa 32,27 ± 3,68Aa 29,83 ± 1,51Aa

0 day

0 30 60 90

Time

AVP

Time

2,59 ± 3,50Aa −0,21 ± 6,51Aa −0,59 ± 3,90Aa −0,36 ± 3,63Aa

Time

0,96Aa 1,21Aa 3,67Aa 1,60Aa

Time

± ± ± ±

Time

12,33 11,10 14,27 10,90

Time

26,0 ± 1,44Aa 23,93 ± 4,37Aa 31,33 ± 4,39Aa 28,03 ± 5,84Aa

Texture

0 30 60 90

Colour

AVC

Taste

b*

Acidity

L*

Global Impression

Colour index (CI)

Appearance

Colour coordinates

Appetizer

Time (days)

Table 3 Averages of the scores obtained for the sensorial attributes (appearance, acidity, taste, colour, texture and global impression) of the appetizer of the control and added treatment of probiotic bacteria.

Treatments

Average

Table 2 Average colour determination results for the coordinates L *, b * and colour index (CI) of the vegetable appetizer.

7,40a 7,51a 7,55a

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analyzed samples and < 3.0 × 101 NMP/g of thermotolerant coliforms, being in accordance with the Brazilian legislation (Brazil, 2001). It also showed counts < 1.0 × 101 CFU/g of filamentous fungi and yeasts, indicating that the preparations were microbiologically safe for human consumption, during the 90 days of storage. 3.5. Sensory quality of appetizer It was verified that the mean values of the sensorial attributes of the appetizer, after fermentation (time 0) and after 90 days of refrigerated storage, presented scores between 7 and 8, which correspond to the terms “moderately liked” to “liked very much” except for the attribute acidity that ranged from 6 to 8, which equates to “I liked it slightly” and “I liked it a lot” in the hedonic scale of 9 points, demonstrating the market potential of the elaborated product (Table 3). It is suggested that the addition of probiotic cultures did not affect the sensory quality of the product, which is evidenced by the good acceptability of the appetizer, but the one added of L. rhamnosus GG presented a slightly higher mean scores, mainly for the flavor and acidity, demonstrating greater preference on the part of the judges. The acidity attribute differed (p < 0.05) between treatments and storage times, presenting a lower average compared to the other attributes evaluated. It was observed that acidity was an important factor in reducing the acceptability of the appetizer added of L. plantarum LP299v, since there was a reduction of the pH value and an increase of the acidity in % of lactic acid, resulting from the fermentation process, promoting thus reducing the purchase intention notes by the tasters. As regards to purchase intention, there was a difference (p < 0.05) between treatments, being the time significant (p < 0.05). The appetizer added of L. plantarum LP299v or L. rhamnosus GG presented notes at time 0 and after 90 days, ranging from 3.0 to 5.0 on the 5-point scale, being the appetizer with L. rhamnosus GG (p < 0.05) the preferred, indicating that the tasters would “probably buy” and “certainly buy” this product (Table 4).

Fig. 3. Resistance of L. plantarum LP299v (LP299v) or L. rhamnosus GG (LRGG) to the GIT assay on vegetable appetizer during 90 days of refrigerated storage. Barras demonstrate the viability of the strains in the appetizer not exposed to gastric stress, followed by the oral, gastric, enteric I and enteric II phases. The confidence interval (p < 0.05) is represented by the vertical lines.

first exposed to salivary enzymes at neutral pH, and then submitted to the action of gastric pepsin at an acidic pH, finally reaching the intestine, where it again comes into contact with a pH value close to neutrality and with the bile salts. When evaluating the behavior of the microorganisms in the different phases, it was verified for all times (0, 30, 60 and 90 days) that the viability of L. rhamnosus GG, before the in vitro assay, did not differ (p > 0.05) the viability of the oral phase, different from that observed for L. plantarum LP299v (p < 0.05). The viability of L. plantarum LP299v or L. rhamnosus GG at all times of storage before exposure to gastric stress differed (p < 0.05) from viability at end of gastric phase, that is after 2 h incubation at pH 2.0–2.5. In enteric phase II, where the large intestine is simulated, the product inoculated with L. rhamnosus GG started from a count of 4.15 Log CFU/g (time 0) to 4.34 Log CFU/g and 4.92 Log CFU/g in the first and second month of storage, respectively. However, it is suggested that there was an adaptation of the strain to the low pH, with expression of the alternative sigma factor in response to the sublethal acid stress between the 30 and 60 days, since at the end of the shelf life this strain presented 7.53 Log UFC/g (Fig. 3). A similar behavior to the present study was observed by Campos et al. (2019) when evaluating the survival of L. rhamnosus GG in a mixed pineapple and jussara juice to the simulated gastrointestinal conditions in vitro. The authors observed an adaptation of the strain in the enteric stages I and II, at times 7 and 14 days, presenting higher counts at the end of storage period (time 28 days). The viability of probiotic microorganisms can be affected by several factors such as temperature, pH, dissolved oxygen and resistance along the gastrointestinal tract. In addition, these microorganisms may be in a physiological state called “Viable But Non-culturable” (VBNC), in which the cells undergo physiological and morphological changes, being able to provide a resistance phenotype and may lose its capacity to multiply in the culture media (Davis, 2014). Thus, it is suggested that L. rhamnosus GG presented at 0, 30 and 60 days of storage, in a VBNC state. On the other hand, another possibility would be to make a maturation of the appetizer added to L. rhamnosus GG prior to consumption, so that it was consumed with approximately 90 days of storage, promoting the ingestion the minimum quantity of recommended in any storage time. The bacteria need to be able to survive the acidic conditions in the stomach and to resist bile acids in the small intestine in order to act as a probiotic in the gastrointestinal tract (Monteagudo-Mera et al., 2012). L. plantarum LP299v, in enteric stage II, presented counts of 6.67 log CFU/g at time 0, maintaining this same value at the end of the 90 days

3.6. Evaluation of resistance of L. plantarum LP299v or L. rhamnosus GG to simulated gastrointestinal conditions in vitro The incorporation of probiotic microorganisms into a food matrix requires studies on their survival during industrial processing and storage conditions, and during the stress conditions imposed during gastrointestinal transit to the site of action (Bernat, Cháfer, Chiralt, & González-Martínez, 2015), hence the importance of performing in vitro studies simulating digestion, in order to guarantee the effective action of the probiotics. The in vitro resistance test of L. plantarum LP299v or L. rhamnosus GG in the appetizer submitted to gastrointestinal conditions presented a significant interaction (p < 0.05) between probiotic microorganisms, phases and storage times of the appetizer (Fig. 3). According to Minekus et al. (2014), during digestion, the food is Table 4 Values of intention to buy the appetizer of the different treatments. Appetizer

Time

Average

0 day

90 days

AVC AVP AVR

3,97 3,95 4,15

3,66 3,66 4,07

3,81a 3,81a 4,11b

Average

4,02A

3,80B

3,91

Means followed by the same lowercase letter in the column and upper case in the row, respectively, did not differ by Tukey's test (p < 0.05). AVC: appetizer of vegetable control; AVP: vegetable appetizer with L plantarum LP299v; AVR: vegetable appetizer with L. rhamnosus GG. 5

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of shelf life (Fig. 3). L. plantarum LP299v was more tolerant to GIT stress factors, because this bacterium has a promising ability as a probiotic in food. Some strains have good resistance to passage through the gastrointestinal tract by adaptation to low pH (Turchi et al., 2013) in which its expression and production confer resistance to bile salts and the action of pancreatic enzymes that may exert an effect on the cell wall or membrane components that affect its viability (Ferrando, Quiberoni, Reinheimer, & Suárez, 2016). It is recommended that between 106 and 108 CFU/g or mL of viable probiotic cells should reach the intestinal colon for food to have therapeutic effect (Hussain et al., 2016). Therefore, a 100 g portion of the vegetable appetizer containing L. plantarum LP299v or L. rhamnosus GG offers above 107 CFU/g, sufficient quantity to promote consumer benefits, which makes the appetizer potentially probiotic. This study shows that fermentation in a plant matrix rich in nutrients and at the optimum incubation temperature provided the growth of the bacterial strains to the matrix, which also favored the counting of the probiotic bacteria evaluated.

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4. Conclusions The probiotic strains of L. plantarum LP299v or L. rhamnosus GG were successfully grown on vegetable appetizer, as the counts were higher than those required for probiotics in foods and according to international organizations as FDA. The formulations were safe for human consumption and contained considerable amounts of total carotenoids, presenting as a good source of this beneficial compound to the body. Canned appetizers were considered to be sensorially promising because they presented average scores above 6.0 in the nine point hedonic scale, and that one added with L. rhamnosus GG was preferred by consumers because they had higher purchase intentions. At least 8.67 Log CFU per 100 g portion of L. plantarum LP299v or L. rhamnosus GG, reached the large intestine at the end of shelf life, indicating the potentiality of the carrier matrix. A comparison of the in vitro assay results between the two species showed that L. plantarum LP299v showed better resistance over the entire storage period and L. rhamnosus GG needed to adapt to the medium to later resume growth, proving that the elaborated matrix is a good carrier of L. plantarum LP299v or L. rhamnosus GG. The appetizer is considered a new non-dairy product option, with technological potential for the canning industry, besides having functional appeal meeting the new demands of consumers. Conflicts of interest Declarations of interest: none. Acknowledgements The authors are thankful to the IF Sudeste MG, Rio Pomba Campus for financial support. References Alves, M., Peres, C. M., Mendonza, A. H., Bronze, M. R., Peres, C., & Malcata, F. C. (2015). Olive paste as vehicle for delivery of potential probiotic Lactobacillus plantarum 33. Food Research International, 75, 61–70. Andrews, W. H., Flower, R. S., Silliker, J., & Bailey, J. S. (2001). Salmonella. In F. P. Downes, & K. Ito (Eds.). Compendium of methods for the microbilological examination of foods (pp. 357–380). (4.ed.). Washington, DC: American Public Health Association – APHA. AOAC - Association of Official Analytical Chemists (2016). Official methods of analysis of the association of official analytical chemists (20.ed). Washington, D.C). Bedani, R., Rossi, E. A., & Saad, S. M. I. (2013). Impact of inulin and okara on Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 viability in a fermented soy product and probiotic survival under in vitro simulated gastrointestinal conditions. Food Microbiology, 34, 382–389. Bernat, N., Cháfer, M., Chiralt, A., & González-Martínez, C. (2015). Development of a non-

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