Algae Chlorella vulgaris as a factor conditioning the survival of Lactobacillus spp. in adverse environmental conditions

LWT - Food Science and Technology 133 (2020) 109936

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Algae Chlorella vulgaris as a factor conditioning the survival of Lactobacillus spp. in adverse environmental conditions ´ Scieszka Sylwia *, Klewicka Elz˙ bieta Institute of Fermentation Technology and Microbiology, Lodz University of Technology, W´ olcza´ nska 171/173, 90-924, Ł´ od´z, Poland

A R T I C L E I N F O

A B S T R A C T

Keywords: Algae Chlorella vulgaris Lactic acid bacteria Bile salts Low pH Phenol

This paper presents the impact of selected adverse environmental conditions of the human gastrointestinal tract on the survival of Lactobacillus spp. strains in the presence of the algae Chlorella vulgaris. The tested strains of Lactobacillus brevis (ŁOCK 0944, 0980, 0992, and MG451814) demonstrated high survival rates in bile salts (0.4% and 2.0%) and phenol (0.4%). The largest decrease in the number of lactic acid bacteria was observed in a low-pH solution. Moreover, the addition of Chlorella vulgaris to the growth environment of the tested bacteria increased their survival rates in the presence of bile salts and for 24 h when 0.4% (v/v) phenol was in the environment. The protective effect of algae at low pH is a strain-dependent feature. Furthermore, to increase the survival of the Lactobacillus brevis in this study, it was beneficial to add algae to the bacterial culture after 24 h. The survival rate of Lactobacillus spp. grown in the presence of Chlorella vulgaris in adverse environmental conditions supports the use of algae as a component of fermented food products.

1. Introduction Global demand for macroalgal and microalgal foods is growing (Wells et al., 2017). It is estimated that about 5000 tons of Chlorella is ´n, 2017). Microalgae can cultivated per year (García, de Vicente, & Gala be used in many commercial applications, including human nutrition. Some algae species, such as Chlorella and Spirulina, are already commercially used in food to provide bioactive compounds in dietary supplements (Zhang, He, Luo, & Chen, 2020). Chlorella is one of the algae classified as a food source which falls into the category of Generally Recognized As Safe (GRAS) (Bleakley & Hayes, 2017; Love­ day, 2019). Moreover, microalgal protein powder and a lipid ingredient derived from Chlorella have been classified by the U. S. Food and Drug Administration as GRAS (García et al., 2017). Chlorella vulgaris is the most commonly used industrial species due to its high protein content (51%–58% dry weight) and well-balanced amino acid profile (Caporgno & Mathys, 2018). Furthermore, Chlorella vulgaris contains many other beneficial nutrients, including carotenoids – such as β-carotene, lutein, ´, Marounek, Skˇrivan, & Duˇskova ´, 2020) – and zeaxanthin (Englmaierova vitamins and minerals (Bleakley & Hayes, 2017), pigments, flavonoids, and fatty acids (ω-3 and ω-6 fatty acids) (Koyande et al., 2019). Algae are a great source of biologically active compounds, and may be used for development of functional food. Chlorella have been introduced into

functional spreadable processed cheese (Tohamy, Ali, Shaaban, Moha­ mad, & Hasanain, 2018), processed cheese (Jeon, 2006), and Appenz­ eller cheese (Heo et al., 2006). Moreover, the count of lactic acid bacteria (LAB) was higher in the algae-enriched cheese compared to the control samples (Heo et al., 2006). Furthermore, high-salt content seaweed sauce by lactic acid fermentation was developed (Uchida et al., 2017). Combination of Chlorella vulgaris with fermented products of­ fering a high content of (LAB) allows to create a brand new segment of fermented food. Lactobacillus spp. have a significant effect on the human digestive tract. Among them are many probiotic strains which confer beneficial effects to the host’s health (Harper, Naghibi, & Garcha, 2018; Khare & ´ zewska, 2017). Numerous studies have Gaur, 2020; Markowiak & Sli˙ assessed the use of probiotics in the prevention and treatment of many diseases, especially those associated with intestinal disorders such as lactose intolerance, diarrhea, allergies (Ellis et al., 2019), and atopic dermatitis (Huang et al., 2017). In addition, probiotics may reduce the risk of multiple chronic diseases, including cancer, those associated with high serum cholesterol, and HIV (Nazir, Hussain, Abdul Hamid, & Song, 2018). In order to produce these beneficial effects in the host, the po­ tential probiotic strain should possess a number of desirable character­ istics, such as tolerance to low pH, bile salts, and phenol (Divisekera et al., 2019). Traditionally, LAB strains were isolated from raw milk and

* Corresponding author. ´ Sylwia). E-mail address: [email protected] (S. https://doi.org/10.1016/j.lwt.2020.109936 Received 8 May 2020; Received in revised form 20 July 2020; Accepted 22 July 2020 Available online 26 July 2020 0023-6438/© 2020 Elsevier Ltd. All rights reserved.

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LWT 133 (2020) 109936

naturally fermented dairy products (Wang et al., 2016). In recent years, the interest of LAB with potential probiotic properties from unconven­ tional sources, such as non-dairy fermented food products, and non-intestinal sources, like fermented drinks, fruits, vegetables, and soil increased significantly (Sornplang & Piyadeatsoontorn, 2016). There­ fore, it is reasonable to search for new strains with probiotic properties from plant sources, for instance, Lactobacillus brevis ŁOCK 0944, which ´ zewska, & was isolated from beetroot silage (Klewicka, Libudzisz, Sli˙ Otlewska, 2013). The authors isolated new strains of Lactobacillus brevis ŁOCK 0980, ŁOCK 0992, and MG451814 and characterised their pro­ biotic features (unpublished studies). The aim of this study was to evaluate the effect of Chlorella vulgaris algae on the survival of Lactobacillus brevis in adverse environmental conditions: in the presence of bile salts, phenol, and at low pH.

sample preparation depending on the presence of bile salts and Chlorella vulgaris. The control samples were 24-h Lactobacillus brevis cultures with and without algae (samples 1 and 2; according to Table 1) and LAB with bile salts (samples 3 and 6). The survival rate of the Lactobacillus brevis strains was tested by the plate method at specified intervals (0, 1, 2, 3, and 4 h). For the microbiological analysis, the bacteria were plated on MRS agar (Merck, Germany) and the samples were incubated for 48 h at 30 ◦ C. The results are presented as log colony-forming units (CFU) per millilitre. 2.4.2. Low pH The effect of Chlorella vulgaris on the survival of Lactobacillus spp. at different stomach pH levels (1.5, 2.0, and 2.5) was tested by adding algae at a concentration of 1.5% (w/v) to a 24-h bacterial culture (with a density of 109 CFU/ml) and reducing the pH using 1M hydrochloric acid (HCl). The control samples were LAB culture in MRS broth (pH 5.7) without and with the addition of Chlorella vulgaris. The survival of the Lactobacillus brevis strains in low pH was determined by the plate method at specified intervals (0, 1, 2, and 3 h) during incubation at 30 ◦ C. After incubation, the cultures were serially diluted, spread on MRS agar plates, and incubated for 48 h at 30 ◦ C. The cell viability (log CFU/ ml) was calculated by the plate count method.

2. Materials and methods 2.1. Bacterial strains and growth conditions Four strains belonging to the species Lactobacillus brevis, which were isolated from vegetable silages, were used for the study. Three strains of Lactobacillus brevis which are deposited with the pure culture collection of the Institute of Fermentation Technology and Microbiology ŁOCK 105, at Lodz University of Technology (Poland) were used: Lactobacillus brevis ŁOCK 0944, Lactobacillus brevis ŁOCK 0980, and Lactobacillus brevis ŁOCK 0992. Lactobacillus brevis ŁOCK 0944 have a patent deposit number (B/00035) in the Polish Collection of Microorganisms. The Lactobacillus brevis MG451814 was a newly isolated strain derived from sauerkraut, whose nucleotide sequence was deposited in the GenBank National Centre for Biotechnology Information database under acces­ sion number MG451814. Stock cultures of Lactobacillus brevis strains were cultured in MRS broth (Merck, Germany) with 20% (v/v) glycerol for long-term preser­ vation in a freezer (− 20 ◦ C). The strains were activated by transfer to the MRS broth medium and cultivated at 30 ◦ C for 24 h.

2.4.3. Phenol The impact of algae on the survival of LAB in adverse environmental conditions was assessed by incubating the Lactobacillus brevis strains at a density of 108 CFU/ml in MRS broth (Merck, Germany) in the presence of Chlorella vulgaris (1.5% [w/v]) and 0.4% (v/v) phenol. The resistance of the Lactobacillus spp. to phenol was tested by the plate method. Serial dilutions were spread-plated onto MRS agar at time-points 0, 24, and 48 h of incubation at 30 ◦ C. The results are presented as the mean values of three independent trials in log (CFU/ml). 3. Results 3.1. The impact of Chlorella vulgaris on the survival of Lactobacillus spp. in bile salts

2.2. The research material

The results showed that all tested Lactobacillus spp. bacteria demonstrated high survivability in bile salts (more than 108 CFU/ml after 4 h of incubation; Table 2). However, the presence of 0.4% (w/v)

The research material was powdered Chlorella vulgaris, a dietary supplement from Bellis Food (BellisPharma Sp. z o. o., Jarosław, Poland). The algae concentration used in the study was 1.5% (w/v), which was taken from the daily recommendation for human consump­ tion given by the manufacturer (3.0 g per day in a divided dose of 1.5 g).

Table 1 Samples preparation for the determination of the survival of Lactobacillus brevis in bile salts.

2.3. Statistical analysis All analyses were performed in three independent trials. The results are shown as the arithmetic means of the three repetitions with standard deviations. To determine the statistical significance of differences be­ tween the results, one-way ANOVA was performed at p ≤ 0.05 using Tukey’s post hoc procedure. The software Origin Pro 2017 was used for analysis. 2.4. Survival of Lactobacillus brevis cultured in the presence of Chlorella vulgaris under adverse environmental conditions 2.4.1. Bile salts The ability of LAB to survive in adverse conditions was assessed by incubating the isolates (Lactobacillus brevis strains with a density of 109 CFU/ml) in MRS broth (Merck, Germany) in the presence of bile salts ´d´z, Poland) at concentrations of 0.4% (w/v) and 2.0% (w/v). (BTL, Ło The aqueous bile salt solutions were added to the 24-h bacterial culture. To confirm the protective effect of algae on LAB, Chlorella vulgaris was added to the tested samples (1.5% [w/v]) in two variants: with the bacterial inoculum and to 24-h bacterial culture together with bile salts in the right concentration. Table 1 presents the different variants of

Samples

Chlorella vulgaris added with LAB inoculum

Chlorella vulgaris added to 24-h LAB culture

Concentration of bile salt solution [%]

1 (control) LAB 2 (control) LAB with algae 3 (tested) LAB + bile 0.4% 4 (tested) LAB with algae + bile 0.4% 5 (tested) LAB + algae with bile 0.4% 6 (tested) LAB + bile 2.0% 7 (tested) LAB with algae + bile 2.0% 8 (tested) LAB + algae with bile 2.0%

– +

– –

– –

–

–

0.4

+

–

0.4

–

+

0.4

–

–

2.0

+

–

2.0

–

+

2.0

LAB- lactic acid bacteria. 2

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LWT 133 (2020) 109936

and 2.0% (w/v) bile salts in the growth environment of all tested Lactobacillus brevis strains statistically significantly reduced their numbers (comparing control samples with samples 3 or 6). The largest difference in bacterial survival in 2.0% (w/v) bile salts was observed for strains Lactobacillus brevis ŁOCK 0944 and MG451814, where after 4 h of incubation the number of bacteria had been reduced by 1.37 log CFU/ml (Lactobacillus brevis ŁOCK 0944) and 1.44 log CFU/ml (Lactobacillus brevis MG451814). Chlorella vulgaris was added to the tested samples (1.5% [w/v]) in two variants, first with the bacterial inoculum (samples 4 and 7). The number of Lactobacillus brevis ŁOCK 0992 and MG451814 cultivated with algae and 0.4% (w/v) bile salts (samples 4) after 4 h of incubation was statistically significantly different than in the control samples with bile salts (samples 3). Moreover, the count of Lactobacillus brevis ŁOCK 0944 and MG451814 cultivated with algae and bile salts at a concen­ tration of 2.0% (w/v) (samples 7) after 4 h of incubation statistically increased comparing to the control samples with 2.0% (w/v) bile salts (samples 6). For other strains of LAB cultivated with algae (samples 4 and 7) no statistically significant differences were found. The second variant of introducing Chlorella vulgaris was adding them to 24-h

bacterial culture together with bile salts (samples 5 and 8). Comparing those two variants (samples 4 with 5 and samples 7 with 8), the authors observed that for the survival of the tested Lactobacillus brevis strains, it was preferable to add Chlorella vulgaris to the 24-h culture. Moreover, adding 0.4% (w/v) or 2.0% (w/v) bile salts together with the algae (samples 5 and 8) to all tested Lactobacillus brevis resulted in higher survival rates comparing to the control samples with bile salts (samples 3 and 6) after 4 h of incubation (statistically significant differences). The highest increased in the number of bacteria was found for the strain Lactobacillus brevis MG451814 (0.46 log CFU/ml in 0.4% (w/v) bile salt, and 1.23 log CFU/ml in 2.0% (w/v) bile salt. In addition, adding 0.4% (w/v) bile salts together with the algae (samples 5) to all tested Lacto­ bacillus spp. resulted in survival rates similar to those of the control culture (samples 2) after 4 h of incubation (no statistically significant differences). Furthermore, no statistically significant differences were observed between the survival of Lactobacillus brevis ŁOCK 0944, 0980, and MG451814 with algae and bile salts at a concentration of 2.0% (w/ v) (sample 8) and the control samples (samples 2). Therefore, the introduction of Chlorella vulgaris into the medium protected the LAB from dying in bile salts.

Table 2 The impact of Chlorella vulgaris on the survival of Lactobacillus brevis ŁOCK 0944, ŁOCK 0980, ŁOCK 0992, and MG451814 in bile salts at concentration 0.4% (w/v) and 2.0% (w/v). Strain

Time [h]

1 LAB

2 LAB with algae

3 LAB + bile 0.4%

ŁOCK 0944

0

9.70 ± 0.18 9.64 ± 0.09a 9.71 ± 0.10a 9.59 ± 0.12a 9.59 ± 0.10a

9.44 ± 0.19

9.57 ± 0.18

9.51 ± 0.08ab 9.48 ± 0.14ab 9.55 ± 0.06a 9.49 ± 0.16ab

9.42 ± 0.12

9.40 ± 0.26

9.59 ± 0.14

8.90 ± 0.12

9.00 ± 0.16

9.36 ± 0.10b

9.38 ± 0.16bc

9.64 ± 0.12ac

8.60 ± 0.09d

8.90 ± 0.06e

9.44 ± 0.14bc

9.26 ± 0.09b

9.33 ± 0.08b

9.60 ± 0.10a

8.40 ± 0.13c

8.8 ± 0.14d

9.36 ± 0.18ab

9.14 ± 0.13c

9.29 ± 0.09bc

9.58 ± 0.13a

8.18 ± 0.16d

8.66 ± 0.22e

9.28 ± 0.20ac

9.25 ± 0.06 9.21 ± 0.04a 9.16 ± 0.12 9.24 ± 0.08a 9.25 ± 0.06a

9.16 ± 0.10

9.24 ± 0.08

9.17 ± 0.11

9.15 ± 0.10

9.24 ± 0.07

9.14 ± 0.11

9.16 ± 0.10

1 2 3 4

ŁOCK 0980

0 1 2 3 4

ŁOCK 0992

0 1 2 3 4

MG 451814

0 1 2 3 4

b

4 LAB with algae + bile 0.4%

5 LAB + algae + bile 0.4%

6 LAB + bile 2.0%

9.40 ± 0.29

9.56 ± 0.19

9.55 ± 0.18

abc

ab

ab

ab

d

b

7 LAB with algae + bile 2.0%

8 LAB + algae + bile 2.0%

9.49 ± 0.18

9.54 ± 0.20

b

9.42 ± 0.09b

9.12 ± 0.05ab

9.18 ± 0.12ab 9.08 ± 0.14

9.08 ± 0.16ab 9.01 ± 0.09

9.13 ± 0.09

9.16 ± 0.08

9.01 ± 0.11

9.05 ± 0.11

9.12 ± 0.07

8.99 ± 0.14

9.01 ± 0.11

9.20 ± 0.11

9.13 ± 0.08a 9.11 ± 0.11abc

8.96 ± 0.06b

8.98 ± 0.08bc

9.08 ± 0.09ab

8.71 ± 0.13d

8.96 ± 0.07b

9.12 ± 0.07ac

8.94 ± 0.04d

8.96 ± 0.06cd

9.09 ± 0.05b

8.64 ± 0.15e

8.87 ± 0.09de

9.07 ± 0.07bc

9.19 ± 0.11 9.12 ± 0.12 9.05 ± 0.11ab 9.10 ± 0.09a 9.06 ± 0.10a

9.08 ± 0.14

9.20 ± 0.10

9.10 ± 0.15

9.09 ± 0.14

9.18 ± 0.12

9.08 ± 0.15

9.09 ± 0.14

9.04 ± 0.11

9.09 ± 0.14

9.11 ± 0.09

9.01 ± 0.14

9.01 ± 0.11

9.03 ± 0.13

9.01 ± 0.08

9.01 ± 0.06ab 8.94 ± 0.12ab 8.88 ± 0.09ab

9.01 ± 0.11abc 8.66 ± 0.05cd 8.53 ± 0.07c

9.12 ± 0.10a

9.14 ± 0.09a

8.80 ± 0.11c

8.88 ± 0.11bc

8.88 ± 0.16ae

8.96 ± 0.10a

8.91 ± 0.06bc 8.56 ± 0.07c

8.64 ± 0.13ce

8.74 ± 0.10bde

8.71 ± 0.08bd

8.89 ± 0.06a

8.40 ± 0.03e

8.42 ± 0.04ce

8.67 ± 0.06d

9.66 ± 0.07 9.62 ± 0.06a 9.72 ± 0.07a 9.59 ± 0.10a 9.54 ± 0.09a

9.60 ± 0.05

9.60 ± 0.03

9.54 ± 0.11

9.56 ± 0.08

9,61 ± 0.05

9.54 ± 0.07

9.60 ± 0.06

9.59 ± 0.04a 9.62 ± 0.11ab 9.51 ± 0.08ab 9.47 ± 0.09ab

9.41 ± 0.13bc 9.32 ± 0.11cde 9.30 ± 0.14bc 8.97 ± 0.02c

abc

ab

c

9.06 ± 0.09

cd

9.44 ± 0.12

ac

9.56 ± 0.07ab

9.46 ± 0.13

9.58 ± 0.07

9.26 ± 0.08

9.42 ± 0.09bc

9.49 ± 0.12bc

9.20 ± 0.09d

9.31 ± 0.08cd

9.51 ± 0.09be

9.40 ± 0.08bc

9.51 ± 0.10ab

8.60 ± 0.16d

9.24 ± 0.09c

9.46 ± 0.10ab

9.30 ± 0.07b

9.43 ± 0.04a

8.17 ± 0.11d

9.06 ± 0.06e

9.40 ± 0.08ab

Data are represented as mean number of bacteria (log CFU/ml) ± SD; LAB –lactic acid bacteria; a, b, c, d, e – the statistically significant differences between samples of the same strains within one time interval; p ≤ 0.05; the number of samples (n = 3). 3

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LWT 133 (2020) 109936

medium of tested Lactobacillus strains, it protected the bacteria during the 24 h of incubation (no statistically significant differences between the control samples and those with phenol). After 48 h of incubation, all tested LAB cultivated with Chlorella vulgaris indicated a statistically lower survivability in phenol than bacteria without the addition of algae. However, the LAB still had high survival rates (more than 107 CFU/ml after 48 h of incubation).

3.2. The effect of algae on the survival of Lactobacillus spp. in low pH The results revealed that low pH negatively affects the survival of the tested bacteria (Table 3). The largest decreased in the number of bac­ teria after the first hour of incubation at a pH of 2.5 was observed for Lactobacillus brevis ŁOCK 0980. However, adding algae statistically significantly increased the survival of Lactobacillus brevis ŁOCK 0980 after 1 h of incubation by 0.80 log CFU/ml. For the other tested bacteria, the level of multiplication remained more than 8 log CFU/ml. In each subsequent hour of incubation, the number of LAB decreased. However, depending on the bacterial strain being tested, the addition of algae caused an increase or decrease in the number of Lactobacillus spp. For instance, it was observed that the addition of Chlorella vulgaris to the growth environment of Lactobacillus brevis ŁOCK 0980 and MG451814 caused a statistically significant increase in their survival at a pH of 2.5 after 1 and 2 h of incubation. Introducing Chlorella vulgaris to Lactoba­ cillus brevis ŁOCK 0944 at a pH of 2.5 caused no statistically significant differences in their survival rates. However, algae added to the culture of Lactobacillus brevis ŁOCK 0992 caused a statistically significant reduc­ tion in their survivability. Therefore, the protective effect of algae at low pH was dependent on the strain of bacteria. With lower pH (2.0 and 1.5), the survival rates of LAB decreased (Table 3). Lactobacillus brevis ŁOCK 0944 was the only strain that sur­ vived in a pH of 2.0. After 1 h of incubation, the number of Lactobacillus brevis ŁOCK 0944 was 6.23 log CFU/ml and decreased with each sub­ sequent hour. Moreover, adding Chlorella vulgaris did not protect Lactobacillus brevis ŁOCK 0944 from dying (no statistically significant differences). However, the introduction of Chlorella vulgaris into the medium inhibited the dying of Lactobacillus brevis ŁOCK 0992 in pH 2.0, since after 2 h the effect was not visible. Complete bacterial death was observed at a pH of 1.5, which was too low for all of the tested Lacto­ bacillus brevis strains.

4. Discussion The average concentration of bile salts in the small intestine is about 0.2%–0.3%, and it can increase up to 2.0% (w/v) depending on the host’s physiology and the type and amount of food ingested (Menconi, Morgan, Pumford, Hargis, & Tellez, 2013). Healthy humans commonly have about 0.3% bile, so commercial probiotic bacteria must tolerate at least 0.3% bile (Divisekera et al., 2019). The authors examined the survival of LAB in 0.4% (w/v) and 2.0% (w/v) bile salts and the results showed that all tested strains of Lactobacillus spp. demonstrated high survival rates in bile salts (more than 108 CFU/ml). Similar results were obtained by Reuben, Roy, Sarkar, Alam, and Jahid (2019), who confirmed that the LAB strains (isolated from chickens) they tested were able to tolerate 0.3% bile salt concentrations after 6 h of incubation. Despite the high survival rates of the tested Lactobacillus brevis strains (ŁOCK 0944, 0980, 0992, and MG451814) in bile salts, adding the compound did statistically significantly reduce the number of bacteria. The authors developed a solution to this problem by introducing Chlorella vulgaris to 24-h culture of the LAB. Therefore, the adverse effect of bile salts was minimised because the algae protected the bacteria from dying. The ability of the potential probiotic strains to tolerate bile salts is a major requirement in probiotic selection (Reuben et al., 2019), and the tested Lactobacillus strains cultured in the presence of Chlorella vulgaris all met this requirement. Apart from the ability of potential probiotics to tolerate bile salts, it is also expected that the probiotics will tolerate an acidic environment (Reuben et al., 2019). All tested strains of Lactobacillus brevis survived at a pH of 2.5, even after 3 h of incubation. As the pH was lowered, the survival of LAB decreased. The only strains that survived in pH 2.0 were Lactobacillus brevis ŁOCK 0944; for the remaining strains the pH was too low, and it caused the complete death of the bacterial colony. Similar results were obtained by Reuben et al. (2019), who demonstrated that the survival of potential probiotic LAB strains (isolated from chicken) in simulated gastric juice (pH 2.0) depends on the strain. However, Klingberg, Axelsson, Naterstad, Elsser, and Budde (2005) showed that

3.3. Survival of Lactobacillus spp. in presence of Chlorella vulgaris and phenol The LAB isolates’ tolerance to a 0.4% (v/v) phenol concentration is presented in Fig. 1. All of the tested Lactobacillus strains demonstrated high survival in the presence of 0.4% (v/v) phenol (more than 108 CFU/ ml after 24 h of incubation). However, the presence of 0.4% (v/v) phenol in the growth environment of LAB statistically significantly reduced the number of all tested Lactobacillus brevis strains over 24 h of incubation. Moreover, when Chlorella vulgaris was added to the growth

Table 3 The impact of Chlorella vulgaris on the survival of Lactobacillus brevis ŁOCK 0944, ŁOCK 0980, ŁOCK 0992, and MG451814 at a pH of 2.5, pH of 2.0, and pH of 1.5. Strain

Time [h]

LAB

LAB + algae

LAB pH 2.5

LAB + algae pH 2.5

LAB pH 2.0

LAB + algae pH 2.0

LAB pH 1.5

LAB + algae pH 1.5

ŁOCK 0944

0 1 2 3

9.72 ± 0.06 9.70 ± 0.02a 9.74 ± 0.04a 9.68 ± 0.06a

9.72 ± 0.04 9.70 ± 0.01a 9.66 ± 0.05a 9.62 ± 0.07a

9.72 ± 0.05 9.41 ± 0.03b 7.14 ± 0.16b 6.56 ± 0.09b

9.70 9.29 7.23 6.46

± 0.06 ± 0.13b ± 0.33b ± 0.06b

9.67 ± 0.07 6.23 ± 0.09c 4.38 ± 0.07c 1.85 ± 0.16c

9.58 ± 6.01 ± 4.39 ± 2.11 ±

9.66 ± 0.04 N N N

9.62 ± 0.07 N N N

ŁOCK 0980

0 1 2 3

9.52 ± 0.07 9.56 ± 0.08a 9.48 ± 0.04a 9.43 ± 0.02a

9.43 ± 0.06 9.42 ± 0.06a 9.44 ± 0.04a 9.49 ± 0.05a

9.53 ± 0.06 5.27 ± 0.22b 3.96 ± 0.09b 3.63 ± 0.11b

9,51 6.07 4.64 3.69

± 0.05 ± 0.09c ± 0.27c ± 0.22b

9.51 ± 0.03 N N N

9.55 ± 0.07 N N N

9.48 ± 0.02 N N N

9.42 ± 0.09 N N N

ŁOCK 0992

0 1 2 3

9.29 ± 0.10 9.35 ± 0.01a 9.45 ± 0.05a 9.37 ± 0.03a

9.39 ± 0.02 9.31 ± 0.04a 9.37 ± 0.04a 9.39 ± 0.02a

9.36 ± 0.03 8.92 ± 0.29b 8.87 ± 0.14b 7.63 ± 0.10b

9.41 8.27 5.86 5.24

± 0.05 ± 0.04c ± 0.12c ± 0.07c

9.39 ± 0.06 N N N

9.44 ± 0.06 1.83 ± 0.63d N N

9.37 ± 0.02 N N N

9.44 ± 0.06 N N N

MG 451814

0 1 2 3

9.69 ± 0.07 9.73 ± 0.05a 9.56 ± 0.06a 9.66 ± 0.02a

9.77 ± 0.02 9.80 ± 0.03a 9.66 ± 0.05a 9.67 ± 0.03a

9.75 ± 0.05 8.45 ± 0.19b 6.40 ± 0.34b 5.21 ± 0.20b

9.71 8.83 7.75 5.40

± 0.06 ± 0.06c ± 0.14c ± 0.21b

9.75 ± 0.05 N N N

9.81 ± 0.06 N N N

9.73 ± 0.04 N N N

9.83 ± 0.08 N N N

0.13 0.09c 0.07c 0.16c

Data are represented as mean number of bacteria (log CFU/ml) ± SD; LAB –lactic acid bacteria; N- no growth; a, b, c, d –the statistically significant differences between samples of the same strains within one time interval; p ≤ 0.05; the number of samples (n = 3). 4

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LWT 133 (2020) 109936

Fig. 1. The survival of Lactobacillus brevis ŁOCK a) 0944 b) 0980 c) 0992 d) MG451814 in 0.4% (v/v) phenol. LAB –lactic acid bacteria; a, b, c, d –the statistically significant differences between samples with the same strains within one time interval; p ≤ 0,05; the number of samples (n = 3); White- LAB; Black- LAB + algae; Dots- LAB + phenol; Stripes- LAB + algae + phenol.

exposure to a pH of 2.5 was a very discriminating factor, with 11 out of 27 strains of Lactobacillus spp. still surviving after 1 h, and only 4 of them surviving after 4 h of exposure. The study of the effect of Chlorella vul­ garis on LAB survival showed that the protective effect of algae at low pH is dependent on the strain. For Lactobacillus brevis ŁOCK 0980 and MG451814, higher survival rates were observed at a pH of 2.5 when algae were present in the environment, while for Lactobacillus brevis ŁOCK 0992 at the same level of pH algae caused statistically significant decrease in their survivability. Some aromatic amino acids derived from dietary or endogenously produced proteins can be deaminated in the gut by bacteria, leading to the formation of phenols (Kumar, Ghosh, & Ganguli, 2012). These compounds may exert a bacteriostatic effect against some strains of Lactobacillus spp. (Pinto, Franz, Schillinger, & Holzapfel, 2006). There­ fore, the ability of Lactobacillus spp. strains to grow in the presence of phenol was examined. All tested Lactobacillus strains demonstrated high survival rates in the presence of 0.4% (v/v) phenol, both after 24 h of incubation (more than 108 CFU/ml) and after 48 h (more than 107 CFU/ml). A high tolerance to 0.4% phenol was also observed by Somashekaraiah, Shruthi, Deepthi, and Sreenivasa (2019) for LAB iso­ lated from the sap extract of coconut palm inflorescence – Neera (a naturally fermented drink) – during 24 h of incubation. However, Pinto et al. (2006) showed that L. johnsonii strains were highly sensitive to 0.4% phenol, whereas L. plantarum strains – despite being unable to grow in the presence of phenol over a 24-h incubation – were generally moderately tolerant to this compound. Phenol compounds have bacte­ riostatic properties and can inhibit the growth of probiotic LAB. Therefore, phenol tolerance is essential for their survival in the gastro­ intestinal tract (Divisekera et al., 2019). Although the tested Lactoba­ cillus brevis strains demonstrated high survival rates in the presence of 0.4% (v/v) phenol, a decrease in their number was observed after 24 h of

incubation. The adverse effect of phenol at 24 h of incubation was ameliorated when algae were introduced into the medium. With Chlorella vulgaris added to the culture of the tested Lactobacillus brevis strains, there was a protective effect; no statistically significant differ­ ences were found between the tested samples with the addition of algae and phenol and the control samples during the 24 h of incubation. However, after 48 h of incubation tested Lactobacillus spp. cultivated with Chlorella vulgaris showed statistically lower survivability in phenol than bacteria without the addition of algae. According to Guang-Hua, Chao, and Xiao-Ling (2008) the toxicity of phenols to algae was related mainly to their electronic properties and hydrophobicity. Phenols are polar narcotic chemicals, which exhibit a higher toxic potency than that estimated by their hydrophobicity due to the existence of polar sub­ stituents in the molecules. Scragg (2006) examined the growth of Chlorella vulgaris and Chlorella VT-1 in the presence of phenol (0–400 mg l− 1, 4.24 mM) and found that both algae could tolerate phenol but Chlorella VT-1 was more tolerant being able to grow albeit slowly in the presence of 400 mg l− 1 phenol whereas this concentration inhibited the growth of Chlorella vulgaris. The toxicity of phenols has been attributed to the disruption of membrane structures due to hydrophobic in­ teractions. The partitioning of lipophilic compounds into the membrane may cause significant changes in the structure and function of the membranes. The results indicated that the presence of bile salts, phenol, and especially low pH levels negatively affects the survival of the bacteria tested. The largest decrease in the numbers of LAB was observed at low pH levels. In the other adverse environmental conditions, the tested Lactobacillus spp. strains displayed high survivability. Recently, the FAO and the WHO have defined probiotics as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’ (a joint FAO/WHO report, Canada, 2002). 5

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Traditionally, the minimum amount of probiotics should be at least 106 CFU/g or ml of food product to confer health benefits (Guimar˜ aes et al., 2019; Sornplang & Piyadeatsoontorn, 2016). The tested LAB strains showed high survival rates (more than 107 CFU/ml) in an environment with bile salts and phenol. Moreover, the addition of Chlorella vulgaris increased their survival in bile salts, during 24 h of incubation in phenol, and depending on the bacteria strain at a pH of 2.5. These results point to the combination of LAB and algae in the formulation of an innovative functional product. However, considering the different bacterial sur­ vival cultivated with Chlorella vulgaris especially at low pH levels the selection of appropriate LAB strains is necessary.

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5. Conclusion Chlorella vulgaris increased the survival rates of the tested Lactoba­ cillus brevis strains in the presence of 0.4% (w/v) and 2.0% (w/v) bile salts, protecting them against this factor. Moreover, for the survival of the tested Lactobacillus spp. it was preferable to add algae to the culture after 24 h. The protective effect of algae at low pH is a strain-dependent feature. Furthermore, algae added to the culture of all tested Lactoba­ cillus brevis strains protected the bacteria for 24 h when 0.4% (v/v) phenol was in the environment. Nevertheless, longer incubation (48 h) of bacteria with algae in the presence of phenol caused a decrease in LAB survival, which may be related to their electronic properties, hydro­ phobicity, and disruption of membrane structures. However, these re­ sults proved that the LAB strains tested in this study have a chance to survive adverse gastrointestinal conditions. High-survival lactic acid bacteria cultured in presence of Chlorella vulgaris in adverse conditions (especially bile salts and phenol) allow the use of algae in fermented dietary products, and thus the creation of a new product. Credit author statement The authors declare no conflict of interests of any kind in the manuscript submitted for LWT - Food Science and Technology consid­ eration as a research article. The manuscript is entitled: Algae Chlorella vulgaris as a factor conditioning the survival of Lactobacillusspp. in adverse environmental conditions. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Ethical statement All authors of this paper have read and approved the final version submitted. The contents of this manuscript have not been copyrighted or pub­ lished previously. 1. The contents of this manuscript are not now under consideration for publication elsewhere. 2. The contents of this manuscript will not be copyrighted, submitted, or published elsewhere, while acceptance by the Journal is under consideration. 3. The authors have no conflicts of interest to disclose. 4. All procedures performed in this studies have not been conducted in human participants and/or animals Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 6

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