Lactic Fermentation of Broccoli (Brassica oleracea var. italica) to Enhance the Antioxidant and Antiproliferative Activities

Round 1

Reviewer 1 Report

The authors present an interesting study that aims to identify selected health benefits of fermented broccoli. Even though the topic is of interest to the scientific community, the manuscript needs a lot of work in the areas of data presentation and discussion before it should be considered for publication. 

Specific comments

Line 20   The abstract needs more actual results. Right now, it could also be an abstract for a review paper.

Line 56   Please elaborate on the fermentation conditions for vegetable fermentations and broccoli specifically. The authors compare broccoli fermentation to cabbage fermentation in multiple sections of the manuscript but those are typically very different regarding their conditions such as salt concentration or the use of starter cultures.

Line 61   The objective is not specific enough. It should be precise and ideally separated into its own paragraph.

Line 65   I would like to know more about the broccoli used for this study. i do not believe that it is good scientific practice to buy produce on a farmers market without knowing anything about the way it was grown.

Line 66   Why was the broccoli sanitized? This is very unusual, especially since you were using uninocculated fermentation as a variable.

Line 67   This is true throughout the manuscript, please add the complete manufacturer information for all the chemicals, supplies and materials you used.

Line 76   This is one of the biggest problems of this study. The authors compare a mixed culture fermentation to a natural uninocculated fermentation but do not have a real control or single-strain version to support their conclusions. At least single strain fermentations with L. brevis and L. lactis would be necessary.

Line 79   6% brine seems very high. What is the range in the industry?

Line 91   Please add a reference for your procedure.

Line 96   The titration method is outdated. There are many specific methods that actually analyze lactic acid instead of titratable acidity.

Line 107  Should be 1:50 dilution.

Line 114  Please add references to this section.

Line 123  The column was washed and reconditioned after each sample? Why? Also, please add references to this section.

Line 134  This is not the standard FolinC method. Please provide a source and explain your modifications.

Line 138  Please add a reference and elaborate on the method. This is way too short.

Line 139  What are you trying to analyze here? The section headers are inconsistent.

Line 188  This is true for the whole manuscript, please explain abbreviations when you first use them. Most assay descriptions use abbreviations without explanantion.

Line 196  Please include that single sentence/paragraph in the description above.

Line 200  This whole section is missing references.

Line 210  What does 1.0_104 cells per well mean?

Line 219  "Media was changed every day" does not make sense.

Line 230  What software and which tests did you use?

Line 235  Please use Figure 1 instead if Fig. 1

Line 245  Are the differences between s-FB and i-FB significant?

Line 247  What do the error bars represent?

Line 251  You did not quantify lactic acid, you used a titration for total acidity.

Line 254  This is true for the whole manuscript, you do not need to add p<0.05 every time you state that something is significant. You explained that part in methods and materials.

Line 261  It should at least be mentioned that the target is to be below pH 4.6 for food safety reasons.

Line 273  I do not see the value of this whole section. Why would you expect moisture, ash and protein content to be different? This does not add anything to the objective of this study.

Line 289  Please put the value and the standard deviation in one cell, so it is easier to read. Also, please add statistics to this table.

Line 307  Is there an explanation for this?

Line 316  There is no real control for any of this data or, if there is, it is not discussed here. The only discussion talks about the comparison between 2 treatments (inocculated vs. natural). This needs to be addressed.

Line 354  At what concentrations do polyphenols have these effects? Do your concentrations meet the threshold? There needs to be a lot more discussion here. This is simply not enough.

Line 377  Have you actually checked for the release of functional compounds?

Line 380  The Figure description is too short and not sufficient.

Line 382  The first few sentences belong in the introduction.

Line 400  Did you have a positive control? I do not see that discussed anywhere else.

Line 411  This is not your own data and needs to be referenced.

Line 446  The prevention of chronic diseases was not part of your study. please focus on your own results and only make claims that are supported within your study.

Author Response

Reviewer 1

The authors present an interesting study that aims to identify selected health benefits of fermented broccoli. Even though the topic is of interest to the scientific community, the manuscript needs a lot of work in the areas of data presentation and discussion before it should be considered for publication. 

Specific comments

Line 20   The abstract needs more actual results. Right now, it could also be an abstract for a review paper.

R= The abstract was restructured

“Lactic acid bacteria (LAB) have been used for centuries to produce fermented foods. Cruciferous vegetables contain large amounts of health-promoting compounds such as glucosinolates (GLS) and phenolics. GLS and phenolics have been linked to antioxidant, anticancer, and immunosuppressive effects. However, it has been reported that some BAL strains are able to metabolize and enhance the activity and amounts of biomolecules, through decarboxylation and/or reduction activities, with positive impacts on human diet and colorectal cancer (CRC) prevention. In the present work, the bioprocessing of broccoli by lactic fermentation was evaluated to produce a functional food, using both spontaneous and induced fermentation (Levilactobacillus brevis and Lactococcus lactis as starter co-culture). Changes in proximal, GLS and phenolic content, as well as the antioxidant, antiproliferative and immunosuppressive effect of the fermented product were evaluated in in vitro cellular models to validate their potential in CRC chemoprevention. The results demonstrated that fermented broccoli extracts increase antioxidant activity in Caco2 cells and inhibit proliferation of HT29 and HT116 cell lines in a concentration-dependent manner, with the best results for day 6 at a concentration of 600 µg/mL. Our finding also provides evidence that fermented broccoli could have an anti-inflammatory effect.”

Line 56   Please elaborate on the fermentation conditions for vegetable fermentations and broccoli specifically. The authors compare broccoli fermentation to cabbage fermentation in multiple sections of the manuscript but those are typically very different regarding their conditions such as salt concentration or the use of starter cultures.

R= The introduction, results and discussion were approached considering works on fermented cruciferous vegetables, but the information on foods based on fermented broccoli was enriched. New references were added for work on broccoli fermentation.

Line 61   The objective is not specific enough. It should be precise and ideally separated into its own paragraph.

R= The introduction was modified as well as the wording of the objective.

 

“Each year 8.2 million persons death from cancer. Colorectal cancer (CRC) is the third most prevalent form of cancer worldwide and it usually manifests itself in elderly people [1, 2]. Most cases of colorectal cancer have not family history [3]. However, many studies have found that certain extrinsic factors, like diet, obesity, smoking, alcohol intake, consumption of processed and red meat represent modifiable risks factors linked to colorectal carcinoma. Approaches to reduce CRC incidence and mortality have included primary prevention strategies, such as dietary changes or physical activity increment [4]. Chemoprevention is the use of pharmacological or natural agents to treat the pre-cancerous condition and to inhibit the initiation of tumorigenesis. Non-steroidal anti-inflammatory drugs, vitamins, and antioxidants have been used as chemopreventive agents [5]. Phytochemicals of plants such as glucosinolates (GLS), isothiocyanates (ITC), indoles, flavonoids, and phenolic compounds are a source of natural bioactive metabolites that act as anticancer molecules. These phytochemicals compounds are present in the cruciferous vegetable group, including broccoli, cauliflower, cabbage, radish, and brussels sprouts [6, 7]. Nevertheless, many of these metabolites must undergo an enzymatic or microbial conversion to turn to their bioactive form. For example, GLS need to be hydrolyzed by myrosinase in their biologically active products, such as ITC and indoles [8, 9]. Although these vegetables can be eaten fresh, they are usually cooked for consumption [10]. However, it has been demonstrated that cooking methods, like boiling and steam boiling lead to a negative effect in the myrosinase biological activity [11], resulting in a physicochemical properties changes, as well as partially or totally inhibit the formation of health promoting compounds, such as GLS, polyphenols or vitamin C [12]. As a result of these, GLS must undergo hydrolysis to their breakdown products only by gut microbiota which is characterized by a quite low efficiency [13]. On the other hand, it has been previously reported that strategies such as fermentation, have shown a potentially beneficial production of breakdown products [14], since it favors the phenolics and GLS hydrolysis to several beneficial breakdown products [15]. Lactic acid bacteria (LAB) have an important role in food fermentation since they contribute to sensory characteristics, preservative effects and probiotic properties [16]; they constitute a small diversity of the autochthonous microbiota of raw vegetables (2.0 -2.4 log CFU/g) [17]. When positive conditions of anaerobiosis, water activity, salt concentration, and temperature occur, raw vegetables may be subjected to spontaneous lactic-acid fermentation. Although, the selection of a starter culture offers an advantage contributing to maintain or increase the antioxidant activities, the complete degradation of GLS, and increased content of health-promoting compounds [18, 19]. It has been reported that some strains, like Levilactobacillus brevis, Limosilactobacillus fermentum and Lactiplantibacillus plantarum [20] are able to metabolize and improve the activity and amounts of biomolecules, like phenolic compounds and GLS, through decarboxylation and/or reduction activities, with positive impacts in human diet and CRC prevention [7, 16]. Broccoli is one of the main dietary sources of phenolics compounds that have shown to have several effects in health, such as scavenging free radicals and the inhibition of human low-density lipoprotein oxidation [21]. In addition, is a good source of GLS, fibers, vitamins, and minerals [20]. It is normally consumed after going through a thermal treatment process, which can cause the degradation or leaching of GLS and phenolics [12] and, unlike other cruciferous vegetables such as cabbage, its fermentation has not been widely studied. Therefore, its bioprocessing through lactic fermentation represents an interesting strategy to produce a functional food. In the present work, both spontaneous and induced fermentation with an inoculum composed of lactic acid strains previously isolated in the working group, from Agave sap, and their effect on the antioxidant and antiproliferative effect in vitro cellular models as a strategy to CRC prevention were evaluated.“

Line 65   I would like to know more about the broccoli used for this study. i do not believe that it is good scientific practice to buy produce on a farmers market without knowing anything about the way it was grown.

R= In order to justify the use of broccoli in the present work, the introduction was modified. It should be noted that, unlike other cruciferous vegetables, broccoli fermentation has been little explored.

Line 66   Why was the broccoli sanitized? This is very unusual, especially since you were using uninocculated fermentation as a variable.

R= The broccoli used in the present work does not correspond to pre-washed or disinfected (minimally processed) broccoli florets, but to complete broccoli "heads" acquired in a local market and therefore, should be disinfected like any other fruit or vegetable. This vegetable has been associated with Aeromonas sp. contamination, so in Mexico it is common to wash and disinfect this and other fruits and vegetables first to remove soil, dirt and plant debris, and then to avoid the presence of pathogenic microorganisms.

Line 67   This is true throughout the manuscript, please add the complete manufacturer information for all the chemicals, supplies and materials you used.

R= This information was reviewed to ensure that it was included in all sections of the document.

Line 76   This is one of the biggest problems of this study. The authors compare a mixed culture fermentation to a natural uninocculated fermentation but do not have a real control or single-strain version to support their conclusions. At least single strain fermentations with L. brevis and L. lactis would be necessary.

R= It was decided to work with both strains as fermentation inducers due to the benefits that each one of them presents individually. Information in this regard was added in the first paragraph of the results. The purpose of the present work was to compare the effect of using an inoculum in contrast to spontaneous fermentation.

“Broccoli spontaneous fermentation (s-FB) was produced by utilizing the endogenous microbiota of the broccoli. On the other hand, a starter culture was used to perform the induced fermentation of broccoli (i-FB), Levilactobacillus brevis (3M1) and Lactococcus lactis (3M8) which were previously isolated from Agave sap and which have previously shown resistance to simulated gastro-intestinal tract conditions and antiproliferative activity vs colon cancer cell lines (HT29 and HTC 116).”

Line 79   6% brine seems very high. What is the range in the industry?

R= Different authors report the use of saline concentrations from 2 to 6 %. For example, Kiczorowski et al (2022) carried out the fermentation of vegetables such as broccoli, carrots, cucumber, peppers and beets using a concentration of 2.5 %, although authors such as Salas-Millán et al (2022) fermented broccoli by-products using concentrations of 6 %. The 6 % concentration was chosen to prevent the growth of contaminating microorganisms and because this concentration of NaCl is tolerated by the strains used in the inoculated fermentation. Similar results were reported by Xiong et al 2016, whose research has shown that 2% (w/v) salt accelerated the maturation of sauerkraut, but could not effectively inhibit the growth of harmful microorganisms. In contrast, 5% salt favored the inhibition of fungi and E. coli, while concentrations of 8% salt not only delayed sauerkraut ripening, but also minimized sucrose utilization and slowed LAB metabolism.

Salas-Millán JA, Aznar A, Conesa E, Conesa-Bueno A, Aguayo E. Functional food obtained from fermentation of broccoli by-products (stalk): Metagenomics profile and glucosinolate and phenolic compounds characterization by LC-ESI-QqQ-MS/MS. LWT,169, 113915 (2022). https://doi.org/10.1016/j.lwt.2022.113915.

Kiczorowski, P., Kiczorowska, B., Samolińska, W. et al. Effect of fermentation of chosen vegetables on the nutrient, mineral, and biocomponent profile in human and animal nutrition. Sci Rep 12, 13422 (2022). https://doi.org/10.1038/s41598-022-17782-z

Xiong T, Fan Liang, Junbo Li, et al. Effects of salt concentration on Chinese sauerkraut fermentation Lebensmittel-wissenschaft + [i.e. Und] Technologie. Food Science + technology. Science + Technologie Alimentaire. 2016 Jun;69:169-174. DOI: 10.1016/j.lwt.2015.12.057.

Line 91   Please add a reference for your procedure.

R= This is a general microbiological procedure, so no specific reference to the procedure was added.

Line 96   The titration method is outdated. There are many specific methods that actually analyze lactic acid instead of titratable acidity.

R= It is true that procedures such as gas chromatography exist for the determination and quantification of organic acids; however, the infrastructure to make such a determination is not available. Nevertheless, the determination of pH and total acidity, generally expressed as g equivalents of lactic acid, continues to be a response variable in the processing of these fermented vegetable products due to its relationship with sensory aspects but also with microbial dynamics. It was clarified in the methodology that what was determined was titratable acidity expressed as grams of lactic acid/L.

“2.5.1. Determination of pH and total titratable acidity

The pH value of the samples was recorded using a pH meter (Metler-Toledo, Country). pH was measured directly in the sample immediately after opening the jar. For Total Titratable Acidity (TA), NaOH (0.1 N) was used and 10 ml of brine sample according to the standard procedures of AOAC (1984). As an indicator for titration, 2 drops of phenolphthalein (C₂₀H₁₄O₄) 1% were used. TA was expressed as gram equivalent of lactic acid per litre, using the acid factor for lactic acid is 0.009. “

Line 107  Should be 1:50 dilution.

R= The wording was amended as suggested

Line 114 Please add references to this section.

R= The pertinent references have been added to this section:

 

“Briefly, extraction modified procedure from Salas-Millán [22] was used. Four hundred (400) mg of the freeze-dried broccoli powder was extracted using 2 mL of a 70% (v/v) aqueous methanol solution. The solutions were sonicated for 5 minutes and centrifuged 4000 rpm for 6 minutes at 4°C. The supernatant was taken and 1:50 dilution was made with 70% aqueous methanol solution and stored at -20 °C. All extracts were prepared in duplicate and were used for total phenolic content, GLS analysis, antioxidant capacity and for the IL-8 Immunomodulation assays. For the cellular antioxidant activity (CAA) assay [23] and proliferation by Sulforhodamine (SRB) assay [24] on cancer cell lines, five milligrams (5 mg) were taken with 200 μL of dimethyl sulfoxide (DMSO, Sigma). On the day of the experiment, samples were diluted from 0 to 600 μg/mL in serum-free culture media; the final samples solutions contain <2% DMSO.”

Line 123  The column was washed and reconditioned after each sample? Why? Also, please add references to this section.

R= This sentence was removed because the column was only washed and reconditioned at the end of sample injection.

Line 134  This is not the standard FolinC method. Please provide a source and explain your modifications.

R= The reference corresponding to the methodology for the determination of hydrolyzable polyphenols was added.

“Hydrolyzed phenolic content in the extracts was determined by the commercial reagent Folin-Ciocalteu method described by Wong et al. [25] using gallic acid as the standard. Briefly, a dilution 1:200 from the extract was prepared with methanol 70%. The reaction occurred with 20 µl of each sample, 20 µl of Folin-Ciocalteu reagent and 20 µl of a solution of sodium carbonate. Finally, the solution was diluted with 125 µl of distilled water and the absorbance was measured in an EPOCH plate reader at 760 nm. The results were expressed as mg of gallic acid equivalent per g of dry weight.”

 

Line 138  Please add a reference and elaborate on the method. This is way too short.

R= The reference was added and extended description of the methodology used.

“The methodology of HCL-Butanol according to Shay et al. [26]; the standard curve was performed with catechin. Briefly, 500 μL of sample extracts were transferred into screw cap test tubes. Then, 3 mL of HCl-Butanol 1:10 solution were added to the mixture and 100 μL of a 1:9 ferric solution. The tubes were mixed and placed in a water bath at 100 ◦C for 1 h and then, cooled down at room temperature. The absorbance was measured at 460 nm (EPOCH plate reader) and the results were expressed as mg of catechin equivalent per g of dry weight.”

Line 139  What are you trying to analyze here? The section headers are inconsistent.

This was performed to identify polyphenols in fermented samples. Header was modified as follows:

“2.8.3. HPLC-ESI-MS for qualitative analysis of polyphenols

For the qualitative analysis of polyphenols, a Reverse Phase-High Performance Liquid Chromatography was performed on a Varian HPLC system including an autosampler (Varian ProStar 410, USA), a ternary pump (Varian ProStar 230I, USA) and a PDA detector (Varian ProStar 330, USA).”

Line 188  This is true for the whole manuscript, please explain abbreviations when you first use them. Most assay descriptions use abbreviations without explanantion.

R= Description of abbreviations was added.

Line 196  Please include that single sentence/paragraph in the description above.

R= This sentence was integrated in the previous paragraph

Line 200  This whole section is missing references.

R= The references corresponding to the methodology were integrated

Line 210  What does 1.0_104 cells per well mean?

R= It was a drafting error; it has been corrected.

“SRB assay. At 24 h before stimulation, the cells were seeded on 96-well microplates at 2x10⁴ cells per well.”

Line 219  "Media was changed every day" does not make sense.

R= The medium was changed daily until the cells reached confluence to run the experiment.

Line 230  What software and which tests did you use?

R= This information was added

“All fermentations were carried out in triplicate. Student’s t-test was performed on the data obtained every 48 h. Statistical analysis were performed using GraphPad Software, San Diego, CA, USA. and ANOVA was performed; p < 0.05 was considered to be a statistically significant level. “

Line 235  Please use Figure 1 instead if Fig. 1

R= Corrected

Line 245  Are the differences between s-FB and i-FB significant?

R= Only at the beginning and end of fermentation; this information was incorporated into the document.

“Broccoli spontaneous fermentation (s-FB) was produced by utilizing the endogenous microbiota of the broccoli. On the other hand, a starter culture was used to perform the induced fermentation of broccoli (i-FB), Levilactobacillus brevis (3M1) and Lactococcus lactis (3M8) which were previously isolated from Agave sap and which have previously shown resistance to simulated gastro-intestinal tract conditions and antiproliferative activity vs colon cancer cell lines (HT29 and HTC 116). As shown in Figure 1, the native population of microorganisms, mainly lactic acid bacteria, was <1 log CFU/mL in s-FB, while in i-FB it was ~ 7.7 log CFU/mL, having a significant statistical difference. Di Cagno et al. [17] have reported LAB counts between 2.0-4.0 log CFU/g as part of the normal microbiota of raw vegetables. However, from day 2 and up to day 8, no significant statistical differences were presented between both fermentations, reaching the maximum value on day 2 of fermentation for both treatments (8.91 and 8.84 for s-FB and i-FB, respectively). After this maximum growth, a significant decrease was observed from day 4 (~8 log CFU/mL); between both fermentations, there was a significant statistical difference on day 10, where greater growth was observed in s-FB (7.77 log CFU/mL) with respect to i-FB (7.31 log CFU/mL).”

Line 247  What do the error bars represent?

R= They represent the standard deviations from the average. This description was added in figures

Line 251  You did not quantify lactic acid, you used a titration for total acidity.

R= The pertinent changes were made in the methodology, results and discussion to clarify that what was determined was titratable acidity.

“3.2. Changes in pH and Titratable Acidity during fermentation

The pH is a critical indicator during fermentation, a decreasing of pH values occurs mainly due to the lactic acid formed by the degradation product of the LAB [19]. Fermentation was monitored by pH measures and quantification of TA (g/L). As shown in Figure 2a, the initial pH values were ~5.8 for both treatments (s-FB and i-FB), after 2 days a pH was decreasing rapidly to 4.53 and 3.99, respectively, indicating the onset of fermentation. At day 4 of fermentation, pH values of i-FB were significantly lower than s-FB, and was maintained until the end of fermentation (day 10). During the fermentation process, the pH values were significantly different between both treatments (s-FB and i-FB), and also, different between the different days within the same treatment; however, from day 2 onwards, the target pH of less than 4.6 was achieved in order to promote the safety of the fermented food. Shokri et al. [33] reported a decrease in pH of broccoli floret purees fermented from 6.8 (initial) to 3.8 (fermentation end point). In addition, they studied the effect of pretreatment of the florets with heat or thermosonication (TS), finding that this value was reached after 24.1 h for the untreated control samples and at shorter times of 8.25 and 9.9 h for the thermal and TS pretreatments, respectively. Similar results were found by Salas-Millán et al, [22], who reported an initial pH of 7.85 and TA <0.01 g/100 mL, decreasing on the third day of fermentation (4.73-4.96), followed by a second reduction on the sixth day (4.20-4.30), with no significant changes until the end of the experiment. Rapid acidification during fermentation is expected due to the increase in the production of organic acids, such as lactic and acetic; this is an important parameter since it minimizes the influence of bacteria of decomposition and contribute to good hygienic conditions of fermented foods [36]. Behavior of the lactic acid production in broccoli fermentation spontaneously (s-FB) or induced (i-FB) (Figure 2b) was correlated with pH as we expected, TA increased significantly from day 0 to day 2 in both treatments, and reaching their highest production at day 8 of fermentation for both treatments. However, the Total Acidity was significantly higher in i-FB (13.26 ± 0.5 g/L) than in s-FB (11.85 ± 0.5 g/L), due to the metabolic activities of lactic acid bacteria used. These results are also similar to those reported by Shokri et al. [33] in broccoli purees, where values of 10.9, 11.7 and 13.1 g/L were achieved for non-per-treated, thermal pre-treated and TS pre-treated broccoli, respectively.”

 

Line 254  This is true for the whole manuscript, you do not need to add p<0.05 every time you state that something is significant. You explained that part in methods and materials.

R= This information was removed

Line 261  It should at least be mentioned that the target is to be below pH 4.6 for food safety reasons.

R= This sentence was added.

“During the fermentation process, the pH values were significantly different between both treatments (s-FB and i-FB), and also, different between the different days within the same treatment; however, from day 2 onwards, the target pH of less than 4.6 was achieved in order to promote the safety of the fermented food.”

Line 273  I do not see the value of this whole section. Why would you expect moisture, ash and protein content to be different? This does not add anything to the objective of this study.

R= The determination of these parameters is relevant because the aim is not only to evaluate the bioactivity of the developed product, but also the effects of the different inoculums and fermentation on its nutritional content.

Line 289  Please put the value and the standard deviation in one cell, so it is easier to read. Also, please add statistics to this table.

R= Mean values and standard deviations were placed in one cell.

Line 316  There is no real control for any of this data or, if there is, it is not discussed here. The only discussion talks about the comparison between 2 treatments (inocculated vs. natural). This needs to be addressed.

R= Only a fermentation without inoculum (spontaneous) was carried out and compared with the induced fermentation, in which two lactic acid strains previously isolated and studied from Agave sap were inoculated.

Line 354  At what concentrations do polyphenols have these effects? Do your concentrations meet the threshold? There needs to be a lot more discussion here. This is simply not enough.

R= In the present work, only sinigrin and indole-3-carbinol were quantified. In the case of polyphenols, the diversity of compounds present in the different treatments was studied but not quantified. The concentration that has shown a beneficial effect on health with respect to the consumption of glucosinolates was added.

“Several clinical trials related broccoli intake to its bioactivity in relation to cancer, oxidative stress, and inflammation [40]; López-Chillón et al. [41] demonstrated a decrease in chronic inflammation in overweight subjects with a dietary ration of 120 mg daily of glucosinolates.“

 

Line 377  Have you actually checked for the release of functional compounds?

R= No, therefore this sentence was modified.

“3.8. Proliferation assay

In the present work, spontaneous and induced fermentation was used as a tool for the elaboration of a functional food as a strategy to assist in the chemoprevention of CRC.”

Line 380  The Figure description is too short and not sufficient.

R= Description was modified

“Figure 5. Cellular antioxidant activity (CAA) assay of fresh broccoli (upper-left corner), a commercial capsule of Indole.3-carbinol with cruciferous vegetables (100 mg, Solaray ®) (upper right), and spontaneous (lower-left corner) or induced-fermented broccoli (lower-right corner) extracts in Caco2 cells.”

Line 382  The first few sentences belong in the introduction.

R= This information was removed from the results section and some sentences were taken for the introduction.

Line 400  Did you have a positive control? I do not see that discussed anywhere else.

R= Yes, 5-fluoroacyl was used as a positive control and the commercial indole-3-carbinol capsule with cruciferous vegetables was used as a control test. This information was included in the methodology section and indicated in the figure captions.

Line 411  This is not your own data and needs to be referenced.

R= The corresponding reference was added

“… the upregulation of proapoptotic genes such as caspase8-p21 p53 and Bax and the downregulation of antiapoptotic genes: Bcl-2 and Hsp90 [48].”

Line 446  The prevention of chronic diseases was not part of your study. please focus on your own results and only make claims that are supported within your study.

R= The conclusion was restructured

“Fermented plant foods. can be employed as chemopreventives due to the presence of various nutrients such as fibers, vitamins, minerals and also phytochemicals. Particularly, lactic fermentation can enhance the health benefits of these foods. In the present study, glucosinolates and phenolic compounds from broccoli were evaluated during a 10-day fermentation. The highest sinigrin content was detected on day 6 of fermentation; however, both spontaneous and induced fermentation preserved the content of phenolic compounds and maintained their antioxidant activity. Evaluation of biological activity by cellular antioxidant assay in the Caco2 cell line confirmed the protective effect against induced oxidative stress. The highest antioxidant activity was detected with day 6 fermentation extracts. In addition, broccoli extracts reduced the proliferation of colon cancer cell lines and had an anti-inflammatory effect, demonstrating that lactic fermentation of broccoli is useful for the development of functional foods from this cruciferous vegetable.”

Reviewer 2 Report

This study  investigate the changes in proximal, GLS and phenolic content, and antioxidant activities of spontaneous and induced (Lactobacillus brevis and Lactococcus lactis as a starter co-culture) fermented broccoli(s-FB and i-FB ).  the effect of broccoli extracts on colon cancer cell lines, including, antioxidant, antiproliferative and immunosuppressive activities was evaluated.  The highest antioxidant activity was detected with extracts from day 6 of fermentation, In a concentration dependent manner, fermented broccoli extracts increase the antioxidant activity in Caco2 cells and inhibit proliferation of HT29 and HT116 cell lines. This study have some creative, give us some valuable date,  the research is relatively comprehensive.

I think this manuscript can be published after the authors make some revise. My revision opinions are as follows:

1. Fig 1. microbial  population  reaching the maximum value at day 2 of fermentation for both s-FB and i-FB，and a significant decrease after 3.5 days, can author give some explanation?

2. Table 1.Protein 49.78-70.81(g/ 100 g of sample)，this date is incredible, high than meat, please check this date.

3.  Compare the effect of  s-FB with i-FB, it seems  s-FB is better than  i-FB, what is basis or principle for using Lactobacillus brevis and Lactococcus lactis as a starter?

4. Sulforaphane(SFN) is one of the important function compounds of  broccoli , it will  be better  if authors can check the SPN changes  after fermentation

5. It will be better the author can condensed some important date in abstract .

Author Response

Reviewer 2

This study investigate the changes in proximal, GLS and phenolic content, and antioxidant activities of spontaneous and induced (Lactobacillus brevis and Lactococcus lactis as a starter co-culture) fermented broccoli(s-FB and i-FB ).  the effect of broccoli extracts on colon cancer cell lines, including, antioxidant, antiproliferative and immunosuppressive activities was evaluated.  The highest antioxidant activity was detected with extracts from day 6 of fermentation, In a concentration dependent manner, fermented broccoli extracts increase the antioxidant activity in Caco2 cells and inhibit proliferation of HT29 and HT116 cell lines. This study have some creative, give us some valuable date,  the research is relatively comprehensive.

I think this manuscript can be published after the authors make some revise. My revision opinions are as follows: 

1. Fig 1. microbial population reaching the maximum value at day 2 of fermentation for both s-FB and i-FB，and a significant decrease after 3.5 days, can author give some explanation?

R= Section 3.1, corresponding to growth, was modified as follows:

“3.1. Growth of bacteria during fermentation

Broccoli spontaneous fermentation (s-FB) was produced by utilizing the endogenous microbiota of the broccoli. On the other hand, a starter culture was used to perform the induced fermentation of broccoli (i-FB), Levilactobacillus brevis (3M1) and Lactococcus lactis (3M8) which were previously isolated from Agave sap and which have previously shown resistance to simulated gastro-intestinal tract conditions and antiproliferative activity vs colon cancer cell lines (HT29 and HTC 116). As shown in Figure 1, the native population of microorganisms, mainly lactic acid bacteria, was <1 log CFU/mL in s-FB, while in i-FB it was ~ 7.7 log CFU/mL, having a significant statistical difference. Di Cagno et al. [17] have reported LAB counts between 2.0-4.0 log CFU/g as part of the normal microbiota of raw vegetables. However, from day 2 and up to day 8, no significant statistical differences were presented between both fermentations, reaching the maximum value on day 2 of fermentation for both treatments (8.91 and 8.84 for s-FB and i-FB, respectively). After this maximum growth, a significant decrease was observed from day 4 (~8 log CFU/mL); between both fermentations, there was a significant statistical difference on day 10, where greater growth was observed in s-FB (7.77 log CFU/mL) with respect to i-FB (7.31 log CFU/mL). In a study by Shokri et al. [33] with broccoli puree fermented with a mixed culture (Leuconostoc mesenteroides (BF1, BF2; 1:1) and Lactiplantibacillus plantarum (B1)), maximum counts of 7.99 log CFU/g were also achieved. Salas-Millán et al. [22] also reported a significant increase in LAB count after three days of spontaneous fermentation of broccoli stalks, without dressing or dressed with garlic or mustard, reaching 8.04-8.50 log CFU/g. Regarding spontaneous fermentation of broccoli, Chen et al. [34] reported the presence of Weisella, Lactococcus and Lactobacillus as the most common genera in both fresh broccoli and Yan-tsai-chin (fermented broccoli stems), with potential to produce bacteriocin-like inhibitory substance (BLIS). In addition to contributing to the flavor, aroma and texture of fermented products, LAB present in fermented products also lower the pH, effectively promoting their quality and safety [35].”

2. Table 1.Protein 49.78-70.81(g/ 100 g of sample)，this date is incredible, high than meat, please check this date. 

R= The authors are grateful that this was pointed out and we verified that there was an error. The original data were reviewed, as well as the formulas used, and an error was detected, so the protein contents were modified.

3. Compare the effect of s-FB with i-FB, it seems  s-FB is better than  i-FB, what is basis or principle for using Lactobacillus brevis and Lactococcus lactis as a starter?

R= The information corresponding to the use of the strains was added at the beginning of the results and the effect of the type of fermentation was compared according to the statistical results.

“3.1. Growth of bacteria during fermentation

Broccoli spontaneous fermentation (s-FB) was produced by utilizing the endogenous microbiota of the broccoli. On the other hand, a starter culture was used to perform the induced fermentation of broccoli (i-FB), Levilactobacillus brevis (3M1) and Lactococcus lactis (3M8) which were previously isolated from Agave sap and which have previously shown resistance to simulated gastro-intestinal tract conditions and antiproliferative activity vs colon cancer cell lines (HT29 and HTC 116).”

4. Sulforaphane(SFN) is one of the important function compounds of  broccoli , it will  be better  if authors can check the SPN changes  after fermentation 

R= We appreciate your comment, and we are aware that there are other compounds of interest that could have biological activity in broccoli, such as glucoraphanin or sulforaphane; however, these determinations were not carried out due to the lack of standards at the time of experimentation, although we consider it relevant to study their behavior in subsequent studies. 

5. It will be better the author can condensed some important date in abstract 

R= The abstract was restructured

“Lactic acid bacteria (LAB) have been used for centuries to produce fermented foods. Cruciferous vegetables contain large amounts of health-promoting compounds such as glucosinolates (GLS) and phenolics. GLS and phenolics have been linked to antioxidant, anticancer, and immunosuppressive effects. However, it has been reported that some LAB strains are able to metabolize and enhance the activity and amounts of biomolecules, through decarboxylation and/or reduction activities, with positive impacts on human diet and colorectal cancer (CRC) prevention. In the present work, the bioprocessing of broccoli by lactic fermentation was evaluated to produce a functional food, using both spontaneous and induced fermentation (Levilactobacillus brevis and Lactococcus lactis as starter co-culture). Changes in proximal, GLS and phenolic content, as well as the antioxidant, antiproliferative and immunosuppressive effect of the fermented product were evaluated in in vitro cellular models to validate their potential in CRC chemoprevention. The results demonstrated that fermented broccoli extracts increase antioxidant activity in Caco2 cells and inhibit proliferation of HT29 and HT116 cell lines in a concentration-dependent manner, with the best results for day 6 at a concentration of 600 µg/mL. Our finding also provides evidence that fermented broccoli could have an anti-inflammatory effect.”

Reviewer 3 Report

Introduction: Please add more information about the importance of using broccoli in this study.

Line 58 “to have several effech in health, such as…”. Please add this information.

Please add a bit information about the strains (L. brevis and L. lactis) in the introduction

Please change the name of Lactobacillus according to the latest taxonomy name, change Lactobacillus brevis to Levilactobacillus brevis.

Line 74, why 4430 RCF?

Line 84, why did you perform the fermentation at room temperature for 10 days?

Line 112 how much is the concentration of DMSO did you use In the assay?

In the method section, please mention the source of the protocols.  (except for antioxidant activity).

Figure 1. the data in Y-axis is quite confusing, please convert it to log cfu.

Sect 3 it should be “Results and discussion”.

Line 390 Glucosinolate content: is there any explanation, why sinigrin content in 6 days showed no significant differences with day 2-10? While the sinigrin content in 6 days is the highest. Please add more references in this section.

Section 3.5, please mention the unit.

Figure 5, please add more explanation about the graph. What do you mean by “capsule”?

 

 

Author Response

Reviewer 3

Introduction: Please add more information about the importance of using broccoli in this study.

R= Information to justify the use of broccoli in the introduction was added.

“…Lactic acid bacteria (LAB) have an important role in food fermentation since they contribute to sensory characteristics, preservative effects and probiotic properties [16]; they constitute a small diversity of the autochthonous microbiota of raw vegetables (2.0 -2.4 log CFU/g) [17]. When positive conditions of anaerobiosis, water activity, salt concentration, and temperature occur, raw vegetables may be subjected to spontaneous lactic-acid fermentation. Although, the selection of a starter culture offers an advantage contributing to maintain or increase the antioxidant activities, the complete degradation of GLS, and increased content of health-promoting compounds [18, 19]. It has been reported that some strains, like Levilactobacillus brevis, Limosilactobacillus fermentum and Lactiplantibacillus plantarum [20] are able to metabolize and improve the activity and amounts of biomolecules, like phenolic compounds and GLS, through decarboxylation and/or reduction activities, with positive impacts in human diet and CRC prevention [7, 16]. Broccoli is one of the main dietary sources of phenolics compounds that have shown to have several effects in health, such as scavenging free radicals and the inhibition of human low-density lipoprotein oxidation [21]. In addition, is a good source of GLS, fibers, vitamins, and minerals [20]. It is normally consumed after going through a thermal treatment process, which can cause the degradation or leaching of GLS and phenolics [12] and, unlike other cruciferous vegetables such as cabbage, its fermentation has not been widely studied. Therefore, its bioprocessing through lactic fermentation represents an interesting strategy to produce a functional food. In the present work, both spontaneous and induced fermentation with an inoculum composed of lactic acid strains previously isolated in the working group, from Agave sap, and their effect on the antioxidant and antiproliferative effect in vitro cellular models as a strategy to CRC prevention were evaluated.”

Line 58 “to have several effech in health, such as…”. Please add this information.

R= This information was added

“…Broccoli is one of the main dietary sources of phenolics compounds that have shown to have several effects in health, such as scavenging free radicals and the inhibition of human low-density lipoprotein oxidation [21].”

Please add a bit information about the strains (L. brevis and L. lactis) in the introduction

R= Information about the strains was included both in the introduction and in the results and discussion section.

“…In the present work, both spontaneous and induced fermentation with an inoculum composed of lactic acid strains previously isolated in the working group, from Agave sap, and their effect on the antioxidant and antiproliferative effect in vitro cellular models as a strategy to CRC prevention were evaluated.”

3.1. Growth of bacteria during fermentation

Broccoli spontaneous fermentation (s-FB) was produced by utilizing the endogenous microbiota of the broccoli. On the other hand, a starter culture was used to perform the induced fermentation of broccoli (i-FB), Levilactobacillus brevis (3M1) and Lactococcus lactis (3M8) which were previously isolated from Agave sap and which have previously shown resistance to simulated gastro-intestinal tract conditions and antiproliferative activity vs colon cancer cell lines (HT29 and HTC 116).”

Please change the name of Lactobacillus according to the latest taxonomy name, change Lactobacillus brevis to Levilactobacillus brevis.

R= The taxonomy was updated throughout the document.

Line 74, why 4430 RCF?

R= This information was verified and corrected

“Lactic acid bacteria (LAB) cells were harvested by collecting cell pellets after centrifugation at 6000 rpm for 6 min at 4 °C and then washed twice with a sterile 0.9% saline solution.”

Line 84, why did you perform the fermentation at room temperature for 10 days?

R= Different authors report different conditions for the production of fermented broccoli, varying in inoculum, broccoli pretreatment, fermentation time, brine concentration or use of spices or additives. However, most of the experiments coincide in the use of room temperature for the process and periods of between 6-10 days. Some references of related works are presented below;

(25 ºC, 6 days) Salas-Millán JA, Aznar A, Conesa E, Conesa-Bueno A, Aguayo E. Functional food obtained from fermentation of broccoli by-products (stalk): Metagenomics profile and glucosinolate and phenolic compounds characterization by LC-ESI-QqQ-MS/MS. LWT,169, 113915 (2022). https://doi.org/10.1016/j.lwt.2022.113915.

(24 °C, 3 weeks) Kiczorowski, P., Kiczorowska, B., Samolińska, W. et al. Effect of fermentation of chosen vegetables on the nutrient, mineral, and biocomponent profile in human and animal nutrition. Sci Rep 12, 13422 (2022). https://doi.org/10.1038/s41598-022-17782-z.

(20-25 ªC, 7 days) Xiong T, Fan Liang, Junbo Li, et al. Effects of salt concentration on Chinese sauerkraut fermentation Lebensmittel-wissenschaft + [i.e. Und] Technologie. Food Science + technology. Science + Technologie Alimentaire. 2016 Jun;69:169-174. DOI: 10.1016/j.lwt.2015.12.057.

 

Line 112 how much is the concentration of DMSO did you use In the assay?

R= The DMSO used is concentrated and was used to dissolve the fermented and freeze-dried broccoli samples (5 mg/200 uL); subsequently, they were diluted to obtain the concentrations evaluated (0-600 ug/mL) and thus having concentrations lower than 2 % DMSO.

In the method section, please mention the source of the protocols.  (except for antioxidant activity).

R= The corresponding references were added in each section

Figure 1. the data in Y-axis is quite confusing, please convert it to log cfu.

R= The axis was modified to log CFU/mL.

Sect 3 it should be “Results and discussion”.

R= The title of this section was modified

Line 390 Glucosinolate content: is there any explanation, why sinigrin content in 6 days showed no significant differences with day 2-10? While the sinigrin content in 6 days is the highest. Please add more references in this section.

R= Discussion and references were added to this section

“3.4. Glucosinolates content changes

Broccoli is a rich source of phytochemicals including glucosinolates, which constitute the rich secondary metabolites in sulfur derivatives of sugars or amino acids, and their hydrolysis products (isothiocyanates) are responsible for the beneficial health effects through antioxidant and antiproliferative activity [39]. The changes in the number of bioactive compounds of fermented extracts during treatments were compared. Figure 3 and Figure 4 present the content of the GLS sinigrin and the GLS breakdown product indole-3-carbinol in the two different fermentations. Similar values of sinigrin were obtained for s-FB and i-FB at the different fermentation times, only in day two of fermentation a significant difference were found between fermentations in which i-FB had a higher value than s-FB. We found the highest content of sinigrin in day 6 of fermentation for both treatments. However, it was not statistically different from day 0, 2, 4, 8, and 10 of fermentation. In the case of the glucosinolate breakdown product indole-3-carbinol, the highest content was found in the s-FB day 10 however, there is no significant difference among other days from the same treatment. Also, no significant difference was found among the different fermentation days of i-FB. Baenas et al. [9] reported that after 7 and 14 days of storage at 5 or 10 °C, individual and total glucosinolates decreased in broccoli sprouts. Several clinical trials related broccoli intake to its bioactivity in relation to cancer, oxidative stress, and inflammation [40]; López-Chillón et al. [41] demonstrated a decrease in chronic inflammation in overweight subjects with a dietary ration of 120 mg daily of glucosinolates. “

Section 3.5, please mention the unit.

R= Units were added to express antioxidant activity

Figure 5, please add more explanation about the graph. What do you mean by “capsule”?

R= The information regarding the capsule was added in the methodology section. It refers to a commercial Indole-3-carbinol capsule with cruciferous vegetables. This was also clarified in the figure caption and in the body text of the results.

Round 2

Reviewer 3 Report

The authors have improved their manuscript