Physico-chemical composition of bulk milk and experimental and control cheese samples through the lactation period is reported in **Table 4**. Administration of experimental feed influenced the milk yield that was significantly higher in the E group (p ≤ 0.05). The significantly highest fat and protein amount (g/d) (p ≤ 0.05) were observed in E group. A significant (p ≤ 0.05) increase was observed for clotting time (RCT) in the E group, while no significant differences were observed for curd firming time (k20) parameter. Milk urea content was lower for E group, compared to C group (p ≤ 0.05), resulting in a better nitrogen utilization in presence of citrus by-products supplementation. The readily fermentable molasses and orange pulp could have helped ewes to use the N diet more efficiently and increased the response. This gives the rumen microbial population a possibility to match the inflow of protein with carbohydrates. Overall, significant differences (p ≤ 0.05) between control and experimental samples were detected, indicating that the experimental fed significantly affected all the parameters studied, except aw. In detail, the experimental cheese samples presented lower pH values and moisture content during the whole lactation period (**Table 4**). Significant differences (p ≤ 0.05) in salt content were observed, with values ranging from 1.84 g/100g to 2.45 g/100 g. The diet significantly affected cheese fat content, observing a higher content in experimental cheese samples than in control samples. An increase of protein level was also registered in experimental samples, with values ranged from 31.26 g/100 g, in March to 34.52 g/100 g, in February (**Table 4**).

## Cholesterol and Fatty Acids Content in Cheese Samples

The total cholesterol content, expressed as mg 100/g of fat, was significantly lower in experimental cheese, mostly in February, March and April (−16.58%, −21.74% and −6.05%, respectively, **Table 4**). The fatty acids (FA) composition and nutritional indices of experimental cheeses are shown in **Table 5**. In general, fatty acid profiles of cheeses were qualitatively similar, especially regarding to unsaturated fatty acids (MUFA). The saturated fatty acid (SFA) dominated the fatty acid profiles of control samples produced in January and in March, while the replacement of the cane molasses and beet pulp by molasses and blond orange pulp produced a significant decrease of these compounds (p ≤ 0.05) in experimental cheese in the same period. The lowest content of SFA was observed in experimental cheese produced in March (58.14%), whereas the highest ratio of these fatty acids was revealed in control samples produced in January (77.04%).

Regarding the MUFAs, nothing different was observed between the two cheese groups, except for control cheese manufactured in January, which showed the lowest registered value. Similar trend of polyunsaturated fatty acid (PUFA) content was observed in the whole experimental period except for experimental sample of March, in which the content was the highest (p ≤ 0.05) registered (**Table 5**). The content of ω-3 fatty acid was significant lower (p ≤ 0.05) in experimental cheese produced in January and February, while the content of ω-6 acid in this group was the highest during the whole experimentation period (**Table 5**). Because of different fatty acid compositions, cheeses were characterized by significant (p ≤ 0.05) different health lipid indices, such as index of atherogenicity (AI), index of thrombogenicity (IT) (**Table 5**). Due to the lowest content of USFAs (22.31%), control cheese produced in January was characterized by the most unfavorable AI and TI values. On the contrary, the health lipid indices reported above were observed in experimental cheese produced in March (AI 2.34; TI 1.70). The conjugated linoleic acids (CLAs) content revealed that the experimental cheeses was high in all months of cheese making and significantly higher than in control cheeses (p ≤ 0.05) in February, March and April (**Table 5**).

## Thiobarbituric Acid-Reactive Substances

The influence of dietary supplementation with citrus by-products on lipid oxidation in cheese is presented in **Table 6**. The TBARs value of the control samples was higher than that found in experimental samples (**Table 6**), after 40 days of retention and after the 60 days of ripening (p ≤ 0.05).

## Microbiological Data

Results of microbiological analyses carried out on both control and experimental cheese samples at different sampling periods, expressed as an average of three replicates with standard deviation are shown in **Table 7**. Overall, no difference among the main groups was detected. Lactic acid bacteria (LAB) achieved an average value of about 7.30 log cfu/g in control samples and of 7.40 log cfu/g in experimental cheese, with a slight dominance of thermophilic lactobacilli in both cheese samples. Statistical data revealed that the experimental fed significantly (p ≤ 0.05) influenced the level of streptococci, which showed higher values, and the level of staphylococci that was significantly reduced by the addition of citrus by-products (**Table 7**). Regarding streptococci group, the average value ranged from 6.15 log cfu/g to 7.44 log cfu/g in control cheese. The highest value was revealed in experimental cheese. Staphylococci group suffered a decrease of about 2 log units for coagulase positive staphylococci and of about 1 log unit for coagulase negative in experimental cheese samples, reaching a final value of about 1 log CFU/g. Enterobacteriaceae, yeast and mold and total mesophilic bacteria showed similar value in both samples, reaching

TABLE 4 | Physico-chemical and coagulation parameters of both control and experimental ewe's milk and cheese during at each sampling time.

a,bMean values with different letter in superscript within rows indicates significant differences (p ≤ 0.05) due to fed. SEM, standard error of least square means.

r, clotting time, min; a30, curd firmness, mm; K20, curd firming time, min.

average values of 4.05, 3.11, and 5.50 log cfu/g, respectively (**Table 7**).

which was not detected in the samples produced in March (line 7).

## PCR-DGGE

PCR-DGGE results of the 16S rDNA of bacterial population of control and experimental cheeses at different sampling periods are reported in **Figure 1**. In order to identify the dominant species, the DGGE profiles generated from control and experimental cheeses were compared to those of strains, previously isolated from Pecorino cheese and identified (data not shown). Overall, the microbial community of both cheese samples remained quite stable with several bands in common. In detail, the bands A and B, dominated all cheese samples and showed an identical profile of Enterococcus faecalis and Streptococcus thermophilus, respectively (**Figure 1**). In addition, the weak bands C and D were detected in all cheese samples analyzed, and were even more pronounced in the control cheese. They showed identical profile to Enterococcus durans and Enterococcus faecium, respectively. It is interesting to note the appearance of several bands, E, F, and H only in the experimental cheeses, which were identified as Lactobacillus rhamnosus/casei group, with the exception of the band G, correspondent to Lactobacillus fermentum species,

## DISCUSSION

The present study investigated the effect of citrus by products as sheep dietary supplementation on physico-chemical, microbiological and fatty acid composition of Comisana sheep's milk and cheese. Despite several studies have investigated the effect of including in various forms olive by-products (18–20) or tomato wastes (21–23) in the diet of ruminants, to our knowledge there is no information available on feeding dairy Comisana sheep with fodder made from a mixture of pelleted feed integrated with molasses and blond orange pulp. Overall, results showed that experimental fed significantly affected the physico-chemical and microbiological parameters, in contrast with findings reported by Fegeros et al. (24) who, evaluating the use of dry citrus pulp (11%) in the ration of dairy ewes, did not observe any significant effect on the composition of milk. Results of the present study firstly demonstrated that the experimental feeding system significantly decreased pH and moisture of the experimental cheeses. The low pH value could be related to the high occurrence of LAB at 60 days of ripening. It is noteworthy

a,bMean values with different letter in superscript within rows indicates significant differences (p ≤ 0.05) due to fed. SEM. standard error of least square means.

SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; n−3, omega 3 fatty acid series; n−6, omega 6 fatty acid series; CLAs, Conjugated linoleic acids.

AI, atherogenic index; TI, thrombogenic index.

TABLE 6 | Lipid oxidative markers (TBARs) of control and experimental feed at each sampling time (values are expressed in mg MDA/kg) at 60 days of cheese ripening.

a,bMean values with different letter in superscript within rows indicates significant differences (p ≤ 0.05) due to fed. SEM. standard error of least square means.

Time, days of storage at 4◦C.

Means of six cheeses for each group with five replicates of measurements at the stated time period.

TBARs, thiobarbituric acid-reactive substances; MDA, malonaldehyde.

that LAB produce large amount of lactic acid, as a consequence of their metabolism (25), which could explain the pH lowering in the experimental cheese. In addition, the pH decrease could be related to the loss of colloidal calcium phosphate from casein submicelles with a progressive dissociation of submicelles into smaller casein aggregates, leading to a greater proteolytic effect (26). In cheese, proteolysis can have a significant effect on several attributes, including development of texture and flavor (27, 28), which highly affect the overall acceptability of the final products. In the present study, it clearly showed that the experimental feed improved the microbial diversity in derived cheese samples, particularly in terms of high occurrence of lactobacilli. In fact, the application of culture-independent approach, such as DGGE, lead to the detection of L. rhamnosus/casei group and L. fermentum species in the experimental 60 days ripened cheese, confirming their importance in the cheese proteolysis and lipolysis. It is already well established that the peptidase activity of lactobacilli could increase the levels of free amino acids (FFA), affecting the development of flavor properties of cheese (28).

One of the most important results of the present study concerns the enhancement of the daily milk production and the fat content in experimental samples, in accordance with a recent study (29). It is well known that milk fat quality is affected by the composition of feed diets (30), and that feed ingredients influence the composition of milk fatty acids (FA) (31). In this context, nutritional quality is becoming a major issue in food choices because of rising consumer awareness of the link between diet and health, increasing market demand for functional foods. An overall improvement of the nutritional composition of experimental cheese, compared to controls was achieved. Our data showed a significant increase of CLAs, which are considered bioactive compounds, with beneficial effect on human health, as demonstrated in several studies (32, 33). Yang et al. (34) reviewed the array of benefits associated with CLA, including positive effects on immune function and protective effects against cancer, obesity, diabetes, and atherosclerosis in animal and human cell line studies. However, no significant difference was revealed for MUFA, PUFA, n-3 and n-6 for the whole trial period, even if an increasing trend was observed in several sampling time. The nutritional composition of the experimental cheese was also improved by the low thrombogenicity antiatherogenic index scores, which enhanced the health benefit of the product. In addition, our data clearly demonstrated a positive effect of the experimental feed on cholesterol content, which could be related to the antioxidant properties of citrus by-product (35). The reduction of cholesterol in foods is of nutritional interest because its high level in human plasma

Frontiers in Nutrition | www.frontiersin.org

TABLE

7

Microbial

counts

expressed

as

log10

CFU/mL

or

CFU/g

and

standard

deviation

(SD)

of

the

main

microbial

groups

detected

on

sheep's

milk

and

60

days

old

cheese

samples

at

different

manufactures

times.

is associated with an increasing risk of cardiovascular disease (36, 37). The lipid oxidation in cheese, expressed by the TBARs value, was higher in control cheese, and this could lead to the formation of flavor defects and nutritional quality losses (38, 39).

Citrus fruit waste is rich in biologically active compounds, including natural antioxidants, such as phenolic acids and flavonoids (40), that show several therapeutic properties and act as antioxidant, anticancer, antitumor, and anti-inflammatory agents. Hence, the inclusion of citrus by-products in the diets for dairy ewes could increase the intake of total polyphenols, which modifying rumen fermentation (30, 41). In the present study, it has been found that LAB population clearly dominated, while staphylococci were detected at lower level in the experimental cheese. This is probably due both to the high acidification rate of LAB population and to the antimicrobial activity of polyphenols.

## CONCLUSION

Citrus by-products are cheap raw material, widely available in Mediterranean countries and their potential as a feed ingredient is promising especially for sheep and cattle which are the dominant farm animal species in the area. In the present study the dietary mixture of pelleted feed integrated with molasses and blond orange pulp is a promising strategy in the diet of lactating sheep, which could improve milk and cheese nutritional quality and, therefore, enhancing the health properties of final products.

## AUTHOR CONTRIBUTIONS

LL, NR, AZ, and ARDR performed the experiments. LL and CR analyzed data. LL, CR and CC wrote the manuscript. LL, CR, CC and VC designed the study and contributed to data interpretation. All authors revised the manuscript.

## FUNDING

This study was funded by a grant from the BIO4BIO Project PON02\_00451\_3362376, CUP B61C12000910005, Biomolecular valorization of residual biomass of the agro-industrial sector

## REFERENCES

received from Ministero dell' Istruzione, dell' Università e della Ricerca (MIUR). The 2014-2020 National Operational Program Research and Innovation (PONRI) is the tool with which Italy contributes to the implementation of the European Union's cohesion policy in favor of its most disadvantaged areas.

tract of healthy volunteers. Int J Food Microbiol. (2017) 252:42–52. doi: 10.1016/j.ijfoodmicro.2017.04.012

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Liotta, Randazzo, Russo, Zumbo, Di Rosa, Caggia and Chiofalo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Effect of Sequential Inoculum of Beta-Glucosidase Positive and Probiotic Strains on Brine Fermentation to Obtain Low Salt Sicilian Table Olives

Alessandra Pino1 , Amanda Vaccalluzzo1 , Lisa Solieri 2 , Flora V. Romeo3 , Aldo Todaro4 , Cinzia Caggia1 , Francisco Noé Arroyo-López 5 , Joaquin Bautista-Gallego5 and Cinzia L. Randazzo1 \*

#### *Edited by:*

Rosalba Lanciotti, University of Bologna, Italy

#### *Reviewed by:*

Aldo Corsetti, Università Degli Studi di Teramo, Italy Gianluca Bleve, Consiglio Nazionale Delle Ricerche, Italy

> *\*Correspondence:* Cinzia L. Randazzo<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

*Received:* 30 October 2018 *Accepted:* 22 January 2019 *Published:* 08 February 2019

#### *Citation:*

Pino A, Vaccalluzzo A, Solieri L, Romeo FV, Todaro A, Caggia C, Arroyo-López FN, Bautista-Gallego J and Randazzo CL (2019) Effect of Sequential Inoculum of Beta-Glucosidase Positive and Probiotic Strains on Brine Fermentation to Obtain Low Salt Sicilian Table Olives. Front. Microbiol. 10:174. doi: 10.3389/fmicb.2019.00174 1 Department of Agricultural, Food and Environment, University of Catania, Catania, Italy, 2 Department of Life Sciences, University of Modena and Reggio Emilia, Reggio Emilia, Italy, 3 Council for Agricultural Research and Economics, Research Centre for Olive, Citrus and Tree Fruit, Acireale, Italy, 4 Department of Agricultural, Food and Forest Science, University of Palermo, Palermo, Italy, 5 Food Biotechnology Department, Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Universidad Pablo de Olavide, Seville, Spain

In the present study, the β-glucosidase positive strain Lactobacillus plantarum F3. 3 was used as starter during the fermentation of Sicilian table olives (Nocellara Etnea cultivar) at two different salt concentrations (5 and 8%), in order to accelerate the debittering process. The latter was monitored through the increase of hydroxytyrosol compound. In addition, the potential probiotic Lactobacillus paracasei N24 strain was added after 60 days of fermentation. Un-inoculated brine samples at 5 and 8% of salt were used as control. The fermentation was monitored till 120 days through physico-chemical and microbiological analyses. In addition, volatile organic compounds and sensorial analyses were performed during the process and at the end of the fermentation, respectively. Lactic acid bacteria and yeasts were, in depth, studied by molecular methods and the occurrence of the potential probiotic N24 strain in the final products was determined. Results highlighted that inoculated brines exhibited a higher acidification and debittering rate than control ones. In addition, inoculated brines at 5% of salt exhibited higher polyphenols (hydoxytyrosol, tyrosol, and verbascoside) content compared to samples at 8% of NaCl, suggesting a stronger oleuropeinolytic activity of the starter at low salt concentration. Lactobacilli and yeasts dominated during the fermentation process, with the highest occurrence of L. plantarum and Wickerhamomyces anomalus, respectively. Moreover, the potential probiotic L. paracasei N24 strain was able to survive in the final product. Hence, the sequential inoculum of beta-glucosidase positive and potential probiotic strains could be proposed as a suitable technology to produce low salt Sicilian table olives.

Keywords: NaCl reduction, microbial debittering, starter cultures, healthy olives, molecular approach

## INTRODUCTION

Among fermented vegetables, table olives are widespread in the Mediterranean area with increasing consumption in both European and non-European countries (International Olive Council, 2016). Olives are intrinsically health thanks to the high content of fiber, vitamins, and polyphenols which play a very important role, exhibiting pharmacological properties and antioxidants effects. In particular, hydroxytyrosol scavenges free radicals, inhibits human low-density lipoprotein (LDL) oxidation, inhibits platelet aggregation, and discloses anticancer activity by means of pro-apoptotic mechanisms (Raederstorff, 2009; Allouche et al., 2011; Buckland and Gonzalez, 2015).

In Sicily, table olives fermentation is mainly performed under traditional methods exploiting the fermentative action of the autochthonous microbiota. Olives are directly brined without previous debittering treatment; therefore, the indigenous microorganisms and the effect of the physico-chemical conditions of brine (pH, salt, presence/absence O2, etc.) are mainly responsible of the hydrolysis of the oleuropein and of other β-glucosides. The oleuropein, a β-glucoside compound lending the strong "bitterness" aroma to the olive fruit, is hydrolyzed by β-glucosidases enzymes with the release of glucose and aglycones which are degraded, by an esterase, in the no-bitter phenols hydroxytyrosol, and elenolic acid (Bianchi, 2003). The spontaneous debittering is time consuming and not predictable, and it is strongly influenced by physico-chemical parameters, by the presence of fermentable substrates, and by the autochthonous microbiota. Starter cultures with oleuropein degrading activity were extensively applied in order to reduce the debittering time and to control the fermentation process (Panagou et al., 2003, 2008; Marsilio et al., 2005; Servili et al., 2006; Bevilacqua et al., 2013; Bonatsou et al., 2015), and, among them, the use of β-glucosidase positive strains could be promising (Ghabbour et al., 2011; Tataridou and Kotzekidou, 2015).

Recently, salt intake consumption hypertension and cardiovasular diseases [U.S. Dept. of Agriculture and U.S. Dept. of Health and Human Services, 2010; World Health Organization (WHO), 2012].

Recently, high attention was paid to salt intake since its overmuch intake is considered a risk factor for the onset of hypertension and cardiovascular diseases [U.S. Dept. of Agriculture and U.S. Dept. of Health and Human Services, 2010; World Health Organization (WHO), 2012]. Indeed, the setup of table olives with low NaCl content is an issue of great interest for the sector (Bautista-Gallego et al., 2013a). However, a complete removal of salt may lead to an increased risk in the survival/ growth of spoilage or food pathogen microorganisms and may also alter food flavor, causing important economic losses. Hence, several studies evaluated the use of KCl, CaCl2, and ZnCl2 as NaCl replacers. It is well-demonstrated that table olives dealt with NaCl reduction and partial substitution with other salts have a more equilibrated mineral composition, enhancing the consumers' acceptance (Bautista-Gallego et al., 2010, 2011a, 2013b). Nevertheless, the effect of NaCl replaces on sensorial aspects is still controversial and strongly influenced by the concentration of the salt mixture used (Zinno et al., 2017). Recently, Pino et al. (2018a) demonstrated that the reduction of NaCl content to 5%, without any NaCl replacers, did not negatively affect the Nocellara Etnea table olives fermentation, obtaining a microbiologically safe product with appreciate sensorial traits.

Another challenge for vegetable product industry is to satisfy the increasing consumer demand for healthier products. Numerous studies demonstrated that table olives are promising carrier for probiotic strains being able to support their survival, probably thanks to the release of prebiotic substances from fruits. Additionally, their microstructure, in terms of roughness of olive surface, promotes the formation of biofilm that seem to protect probiotic bacteria from stressful conditions (such as acidic environment), favoring their survival through the human gastrointestinal tract (De Bellis et al., 2010; Arroyo-López et al., 2012a; Blana et al., 2014; Randazzo et al., 2017; Rodríguez-Gómez et al., 2017). According to that, the present study was aimed to evaluate the effect of a sequential inoculum of a β-glucosidase positive strain and probiotic bacteria on brine fermentation in order to set up a low salt Sicilian table olives.

## MATERIALS AND METHODS

## Pilot Scale Olives Processing

Traditionally Sicilian–style table olives from Nocellara Etnea cultivar, provided from a local company, located in Paternò (Sicily), were processed without any lye treatment. Olives were pre-treated as previously reported (Pino et al., 2018a) and directly immersed in sterilized brine, containing 5 or 8% (w/v) of NaCl. The β-glucosidase positive strain Lactobacillus plantarum F3.3, previously isolated from fermented table olives was used as starter. The strain was previously characterized for the presence of bglH gene according to Marasco et al. (1998) and its β-glucosidase activity was evaluated by enzymatic assay, according to Sestelo et al. (2004). To set-up probiotic table olives, the potential probiotic Lactobacillus paracasei N24, belonging to the Di3A microbial collection was added. This strain was selected according to its technological and probiotic features (Pitino et al., 2010; Randazzo et al., 2010) and for its good ability to survive in table olives (Randazzo et al., 2017; Pino et al., 2018a). Both microorganisms were applied as lyophilized strains.

The experimental fermentation design comprised 8 treatments: 4 fermentations at 5% of NaCl with (F5A; F5B) and without (F5C; F5D) the addition of L. plantarum F3.3 strain, and 4 fermentations at 8% of NaCl with (F8A; F8B) and without (F8C; F8D) the addition of L. plantarum F3.3. Brine samples F5A; F5B; F8A; F8B were inoculated with the L. plantarum starter culture, to a final cell density of 7 log cfu/ml, directly after brining. The potential probiotic L. paracasei N24 strain was inoculated in F5B; F5D; F8B; F8D samples after 60 days of brining (at final cell density of 9 log cfu/ml). All fermentations were done at room temperature (18 ± 2 ◦C), and followed up to 120 days. Marine salt was periodically added to maintain the initial concentration and fresh brine was supplied to keep olives totally dipped. Each fermentation was carried out in triplicate.

## Physico-Chemical Analysis

The pH of the brines was detected by using a MettlerDL25 pHmeter (MettlerDL25, Mettler-Toledo International Inc.). Titratable acidity was determined by titring brine samples with 0.1 N NaOH and was expressed as lactic acid (g/100 ml).

The olive brines were filtered through PTFE filters (Millipore, 0.45µm) and injected in the chromatographic system to analyze the phenol fraction. The HPLC instrument consisted of a chromatography Waters Alliance 2695 HPLC equipped with a Waters 996 photodiode array detector (PDA) set at 280 nm. The column used was a Luna C18 (250 mm × 4.6 mm i.d., 5µm, 100 Å, Phenomenex, Torrence, CA) which was maintained at 30◦C in an oven. The flow rate was 1 mL/min. Separation was obtained by elution gradient using an initial composition of 95% of A solution (2% acetic acid in water) and 5% of B solution (methanol). The concentration of B solution was increased to 30% in 15 min and to 70% in 25 min and then, after 2 min in isocratic condition, the mobile phase was set at the same initial concentration in 8 min. Phenolic compounds were identified by injecting the pure standards of oleuropein, verbascoside, tyrosol, and hydroxytyrosol and by comparing their retention time and UV-Vis spectra. All the analyses were performed in triplicate.

## Microbiological Analyses

Brine samples at 1, 30, 60, 90, and 120 days of fermentation were subjected to microbiological analysis as previously described (Pino et al., 2018a). Mannitol Salt Agar (MSA), incubated at 32◦C for 48 h, was used to isolate both coagulase positive and negative staphylococci. All media were provided from Oxoid (Italy) with the exception of YM provided from Difco (Italy). Microbiological analyses were performed in triplicate and results were expressed as log cfu/ml ± standard deviation.

## LAB Isolation and Identification

For each brine sample (F5A, F5B, F5C, F5D, F8A, F8B, F8C, and F8D) and each sampling time (1, 30, 60, 90, and 120 days), 20% of the total number of colonies, recovered on MRS agar plate, were randomly selected, purified, checked for catalase activity and Gram reaction, and microscopically examined before storing in liquid culture using 20% (v/v) glycerol at −80◦C. Six-hundred (600) LAB isolates were purified and subjected to total genomic DNA (gDNA) extraction following the method described by Pino et al. (2018b). gDNA concentration and quality were evaluated using the Fluorometer Qubit (Invitrogen, Carlsbad, 278 CA, USA). Multiplex RecA and Tuf gene species-specific PCR were performed as previously described (Torriani et al., 2001; Ventura et al., 2003), respectively. Strains not identified at species level with species-specific PCR were subjected to 16S rRNA gene PCR-RFLP analysis according to Pino et al. (2018b). For each PCR-RFLP cluster, the 16S rRNA gene PCR amplicon of one representative strain was purified using the Qiaquick PCR purification kit (Qiagen Hilden, Germany) and was subjected to sequencing and Blast analysis.

## Isolation and Genotypic Identification of Yeasts

From each brine samples at each sampling time, as previously reported, 200 colonies were randomly isolated from YM medium, purified, and microscopically examined prior to storing in liquid culture using 20% (v/v) glycerol at −80◦C. For the yeasts characterization, DNA was extracted according to

TABLE 1 | Results of pH and titratable acidity values in olive brines expressed as means and standard deviations at different time of fermentations.

Data in the same column with different letters are significantly different at P< 0.01.

Ruiz-Barba et al. (2005) and subjected to repetitive element palindromic (rep)-PCR analysis by using GTG5 primer. The PCR reaction was carried out in a final volume of 25 µl, containing: 5 µl of DNA, 5 µl 5X PCR Buffer, 1 µl of primer GTG5 (5-GTGGTGGTGGTGGTG-3), 13.9 µl of filtered water on 0.1 µl of Taq polymerase (Invitrogen, Italy). The amplification program was as follows; an initial denaturation (95◦C, 5 min) followed by 30 cycles of denaturation (95◦C, 30 s), annealing (40◦C, 1 min), and extension (65◦C, 8 min) with a single final extension (65◦C, 16 min). PCR products were electrophoresed in a 2 % agarose gel in 1X TAE buffer, stained with ethidium bromide (30 min) and visualized under ultraviolet light. The resulting fingerprints were digitally captured and analyzed with the Bionumerics 6.6 software package (Applied Maths, Kortrijk, Belgium). Dendrogram for clustering comparison was built with UPGMA (Unweighted Pair Group Method) method and Pearson correlation.

To validate the clustering analysis and for identification of strains, the 26S rDNA gene of all isolates was further sequenced. The gDNA amplification was performed according to Porrua et al. (2018). PCR products were resolved by electrophoresis on agarose gel (1% w/v) stained with ethidium

TABLE 2 | Results of the analyzed phenols in olive brines expressed as means (mg/l) and standard deviations at different time of fermentations.

a–f: for each phenols, data in the same column with different superscript letters are significantly different P < 0.01.

TABLE 3 | Microbial counts expressed as log10 CFU/ml of 3 replicates ± standard deviation of the main microbial groups detected in experimental brine samples during the fermentation process.

a–h: for each medium, data in the same column with different superscript letters are significantly different (P < 0.05).

bromide. DNA ladder plus (Invitrogen, USA) was used to evaluate the molecular weight of amplified DNA. PCR products were purified using Isolate DNA kit (Bioline, USA) according to the manufacturer's instructions and quantified by agarose gel electrophoresis (1% w/v) in 0.5X TBE buffer (89 mM Tris-borate, 2 mM EDTA pH 8). An amount of 10 µl of purified product with forward primer NL1 was used for sequencing by Stab Vida (Lisbon, Portugal). Nucleotide sequences were aligned with the software Molecular Evolution Genetic Analysis (MEGA).

## Rep-PCR for Detecting the Presence of Probiotic *L. paracasei* N24 Strain

Rep-PCR genomic fingerprinting was performed on 79 L. paracasei strains, isolated from F5B, F5D, F8B, and F8D brine samples at 120 days of fermentation, using the (GTG)5-primer, as described by Versalovic et al. (1994). PCR was carried out in a 20 µlreaction mixture containing 1x Thermo Green buffer (Thermo Scientific, Waltman, MA, USA), 3.0 mM MgCl2, 200µM of each dNTP (Fermentas), 1 U of Taq polymerase (Thermo Scientific, Waltman, MA, USA), 2µM (GTG)5 primer and 50 ng gDNA. Amplifications were performed in a MyCycler thermal cycler (BioRad, Hercules, CA). The PCR cycling parameters and gel running conditions were set according to Solieri et al. (2012). The only modification was the change of annealing temperature from 40 to 45◦C. The GeneRuler 100 bp Plus DNA Ladder (Thermo Scientific, Waltman, MA, USA) was used as a molecular size marker. BioDoc Gel Analyzer device (Biometra GmbH, Germany) was used to capture DNA fingerprint images which were then processed through the BioNumerics software v3.0 (Applied Maths, Sint-Martens-Latem, Belgium). Repeatability of rep-PCR was assessed using the inoculated strain N24 as internal control. Pearson's correlation similarity coefficient was chosen to calculate bands patterns similarity matrix with optimization and curve smoothening values at 1%. Unweighted pair group method with arithmetic mean (UPGMA) analysis was exploited to build the (GTG)5-based dendrogram.

## Analysis of Volatile Organic Compounds (VOCs)

VOCs analysis was performed on brine samples at 1, 60, and 120 days of fermentation following method and conditions previously described (Randazzo et al., 2017; Pino et al., 2018a) using a gas chromatography-mass-spectrometry (GC-MS). All analyses were performed in triplicate and the results were expressed as means in µg/l of brine.

## Sensory Evaluation of Table Olives

Table olives at 120 days of fermentation were subjected to sensory evaluation by trained panelists (6 females and 4 males, aged from 22 to 40 years). Sensory panel was conducted according to the International Olive Council method (International Olive Council, 2011). Descriptors related to negative sensations, gustatory, and kinaesthetic perceptions were evaluated as previously described (International Olive Council, 2011, 2016; Pino et al., 2018a). In addition, the overall acceptability descriptor, such an indication of the overall quality, was also scored. Sensory data were acquired by a direct computerized registration system (FIZZ Biosystemes. Couternon, France).

## Statistical Analysis

Microbiological and chemical (i.e., single phenol compounds, pH and acidity) data were analyzed by ANOVA (One-way Analysis of Variance) using Tukey's post-hoc test, in order to assess the overall differences among treatments. The reference level of significance was 0.01 for chemical assay and 0.05 for

VOCs and microbiolgical assays. All statistical analyses were performed using MATLAB software (MathWorks, version 8.5.0), while sensory data were analyzed using the software package Statgraphics R Centurion XVI (Statpoint Technologies, INC.) setting samples as treatments. Data correlations between brine samples differently treated and VOCs were computed using XLStat software (version 2016.1).

## RESULTS

## Physico-Chemical Data

In **Table 1**, the results of pH and titratable acidity detected in brine samples analyzed thought the fermentation are shown. At the beginning of fermentation, pH values ranged from 5.8 to 6.3, and then they decreased after 30 days. Differences among samples become more appreciable after 60 days of fermentation with the lowest values showed by F5B and F8C samples. At the end of fermentation (120 days) the pH fitted the hygienic limit of 4.3 in all samples (**Table 1**).

The titratable acidity values exhibited an increasing trend in all samples with the exception of F8A and F8B brines, which slightly decreased in acidity at 120 days. The highest acidity values were detected in brines at 5% of NaCl (F5A, F5B, F5C, and F5D). In addition, a significant increase in acidity was recorded after the addition of N24 strain (90 days) mainly in F5B and F5D samples (**Table 1**).

Results of hydroxytyrosol, tyrosol, oleuropein, and verbascoside quantification are shown in **Table 2**. Overall, all polyphenols analyzed showed an increasing trend during the fermentation mainly in 5% NaCl brine samples inoculated with starter culture. The highest values of hydroxytyrosol, tyrosol, and verbascoside were mainly recorded in brines inoculated with L. plantarum F3.3 starter culture already at 30 days. Similar behavior was observed for oleuropein.

## Microbial Count

**Table 3** shown microbial counts of brine samples at both 5 and 8% of NaCl, which is expressed as log cfu/ml. Viable mesophilic bacteria showed different trend among samples. In detail, brines at 5% of NaCl (F5A–F5D), from an initial average value of 7.11 log unit, exhibited a steady trend during the fermentation with slight decrease of cell density after 60 days. Similar behavior was observed for brine samples at 8% of NaCl (F8A–F8D) which showed a mean initial value of 6.73 log unit and a final mean value of 5.56 log unit (**Table 3**). Regarding LAB population, all brine samples inoculated with starter culture (F5A, F5B, F8A, F8B) presented, at the beginning of fermentation, higher cell density than spontaneous ones (F5C, F5D, F8C, F8D). From 60 to 120 days, LAB reached the highest values in samples inoculated with the potential probiotic N24 strain (F5B, F5D, F8B, F8D). Yeasts were present at an initial level of about 3 log cfu/ml in all experimental brines with the exception of F5A and F5B samples which exhibited initial value of 4.04 and 4.37 log cfu/ml, respectively (**Table 3**). The yeasts cell densities significantly increased thought the fermentation process, achieving, at 120 days, an average value of 7.37 log unit and 6.64 log unit in brine samples at 5 and 8% of NaCl, respectively. Regarding the staphylococci count, only coagulase negative staphylococci, forming red colonies in the medium, were enumerated and their level, at the beginning of the fermentation, was quite similar among all samples. After a slight increase till 60 days, a decrease to final average values of 3.0 and 3.8 log cfu/ml was achieved in 5 and 8% brine samples, respectively. Similar behavior was observed for enterobacteria counts, which significantly decreased through the fermentation process. At the end of fermentation (120 days) this microbial group was detected, at value below 2 log, in brine samples at 8% of NaCl and below the detection limit in samples at 5% of NaCl, with the exception of the F5D sample. E. coli was never detected in any brine samples analyzed (data not shown).

## Molecular Identification of LAB

Six hundred isolates from MRS agar plates were considered LAB based on their positive Gram reaction, non-motility, absence of catalase activity and spore formation, as well as rod or coccal shape. RecA and Tuf gene species-specific PCRs revealed the presence of strains belonging to L. plantarum, Lactobacillus pentosus, L. paracasei, and Lactobacillus casei species. The distribution of lactobacilli at different sampling times is reported in **Figure 1A** and their occurrence in the different brine samples is showed in **Figure 1B**. Isolates not identified at species level were indicated as "others." Evaluating the distribution of LAB strains through the fermentation, results indicated that L. plantarum and L. pentosus represented the dominant species at the beginning of the process (till 30th days) and a high occurrence of L. paracasei species was detected up to 60 days. Whereas, the highest occurrence of L. casei strains was achieved at 120 days (**Figure 1A**). Zooming on the fermentation at different salt content (5 and 8%) differently inoculated, results

TABLE

showed the dominance of L. plantarum strains in samples inoculated with starter, the highest occurrence of L. casei and L. pentosus in spontaneous samples (F5C, F8C, F5D, F8D) and of L. paracasei in samples inoculated with the probiotic strain N24 (**Figure 1B**).

Isolates not identified initially with recA and Tuf gene primer pairs were subjected to PCR-RFLP analysis of the 16SrDNA and clustered into four different groups (data not shown). One representative isolate for each cluster was identified by 16S rRNA gene sequencing and were deposited in the GenBank database. The species attribution and the accession numbers of the sequenced strains were as follows (isolates code in parentheses): Leuconostoc mesenteroides MK085109 (F5C.1), Lactococcus lactis MK085110 (F5B.38), Lactobacillus brevis MK085111 (F5D.44), and Enterococcus faecium MK085112 (F5A.21).

### Molecular Identification of Yeasts

Two-hundred yeast isolates were randomly obtained during the fermentation process. The dendogram generated by rep-PCR with primer GTG5 showed that the isolates formed 17 groups clearly differentiated. The most numerous groups belonged to the Wickerhamomyces anomalus and Candida boidinii species, although representatives of Candida diddensiae, Pichia kluyveri and Meyerozyma guillermondii were also identified (**Figure 2A**).

The evolution of the different yeast species throughout the fermentative process is presented in **Figure 2B**. At this regard, W. anomalus and C. boidinii formed a stable dual species consortium through the fermentation, since they were both detected more frequently than others species, with a mean frequency of 49 and 37%, respectively. Indeed, these species were dominant in all brine samples differently treated (**Figure 2C**). The rest of the species were isolated at very low mean frequencies; in particular, M. guillermondii (1.97%) was detected only in samples at 8% of NaCl till 30th days of fermentation, whereas, P. kluyveri (2.98%) in samples F5A, F5D, and F8D till 60 days and C. diddensiae (5.87%) was detected starting from the 30th day of fermentation only in brines at 8% of NaCl (**Figure 2C**).

## Detection of *L. paracasei* N24 Strain at 120 Days of Fermentation

The presence of inoculated strain N24 was assessed at 120 days of fermentation by rep-PCR with primer GTG5 on a pool of 79 L. paracasei isolates from samples F5B, F5D, F8B, and F8D. Preliminarily, the rep-PCR repeatability was evaluated using gDNA from strain N24 as internal control in four different gels, obtaining a similarity of 74.3% (data not shown). Accordingly, this value was retained as similarity threshold to establish the identity of isolates compared to the rep-PCR profile of promising probiotic strain N24. The dendrogram generated using the GTG5-based patterns of L. paracasei isolates revealed the presence of four major clusters (from A to D) and four singleton L. paracasei isolates below 74.3% similarity (**Figure 3**; **Supplementary Figure 1**). The cluster analysis showed that the major cluster A grouped both the inoculated strain N24 and 48 out of 79 isolates, indicating that these isolates were assimilated

TABLE

4

Continued

a–h: for each compound different superscript letters in the same raw are significantly different

 <

 Bold value indicates

 class and the total of them.

to the N24 strain profile. The majority of them were isolated from samples F5B and F8B, over the indigenous L. paracasei isolates. In detail, out of 48, 18 strains were isolated from F5B, 12 from F8B, 10 from F5D, and 8 from F8D. The remaining isolates belonged to clusters B, C, and D.

## Volatile Organic Compounds (VOCs)

Results of VOCs of different brine samples at 60 and 120 days of fermentation are reported in **Table 4**. Overall, 46 compounds as acids, alcohols, esters, aldehydes, and phenols were identified, exhibiting a growing trend through the fermentation, with the exception of samples F5A, F5D, F8A, and F8D. The highest value was registered in samples F8C, with a value of 2,739.17 µg/l. Alcohols were the most abundant compounds, followed by esters and acids, whereas aldehydes and phenols were detected at lower concentrations. Focusing on each compound, among alcohols, ethanol dominated the fermentation process showing an increase only in spontaneous samples (F5C and F8C), followed by isoamylalcohol and phenylethylalcohol, which registered a variable trend through the fermentation (**Table 4**). Ethyl-acetate and methyl 2-methylbutanoate were the main detected esters. Among acids, the acetic acid was the most abundant compound, with the highest value in F5C and F8C samples. The most abundant aldehydes and phenols were nonanal, benzaldehyde and creasol, respectively (**Table 4**).

**Figure 4** shows correlation between VOCs and brine samples differently treated. Overall, it is possible to point out that the salt concentration did not influence the VOCs formation through fermentation in brine samples, which were mainly grouped based on the treatment (starter and/or probiotic addition and spontaneous). In detail, samples inoculated with starter (F5A and F8A) were clustered together, showing a negative correlation with alcohol and ester compounds; spontaneous brine samples inoculated with the probiotic strain N24 (F5D and F8D) were negatively correlated to phenols, aldehydes and alcohols. Different correlations were detected for samples inoculated with both starter and probiotic strains (F5B and F8B). In particular, sample F8B at 60 and 120 days of fermentation were grouped together, exhibiting a positive correlation with alcohols and acids and a negative correlation with phenols and aldehydes (**Figure 4**). Evaluating sample F5B, it is possible to assert that VOCs formation was strongly influenced by the fermentation time. In fact, the sample F5B at 120 days revealed a distinct VOCs profile, displaying a positive correlation with esters, acids, and phenols. Similarly, spontaneous fermentation samples (F5C and F8C) were grouped based on fermentation time, showing a divergent VOCs profile through the fermentation.

## Sensory Data

**Table 5** shown results of sensory analysis. Overall, none negative sensation was perceived, as deduced by the low scores attributed by panelist to these descriptors. No statistically significant differences were achieved among samples for hardness, fibrousness, and crunchiness. Among gustatory descriptors, higher scores for acidity were attributed to uninoculated brine samples at both 5 and 8% of NaCl (F5C and F8C), while higher bitterness score was observed in samples without the addition of the β-glucosidase L. plantarum strain

eachdescriptor,inthesamecolumn,withdifferent superscriptlettersaresignificantlydifferent(P<0.05).

(F5C, F5D, F8C, and F8D). Finally, F5B and F8B samples received higher scores for the overall acceptability descriptor.

## DISCUSSION

A current challenge in the processing technology of table olives is the selection of starter cultures able to fasten and safely drive the fermentation process. In contrast to industrial starter cultures, autochthonous strains, that naturally dominate spontaneous fermentation, tend to have high metabolic capacities, which can beneficially affect the quality of the final product. In addition, it was already established that the microbial dynamics through fermentation is influenced by the technology applied (e.g., salt reduction). One of the most widely employed strategies to reduce sodium content in table olives is the use of NaCl substitutes, which can be added alone or in combination with other salts (Bautista-Gallego et al., 2013a). Few studies have evaluated the possibility to setup low NaCl table olives without any salt replacement. Based on our previously reported data (Randazzo et al., 2017; Pino et al., 2018a), in the present study a wild βglucosidase positive strain was used both as debittering and as driven agent during olive fermentations at lowered salt content (5%). It is well-known that β-glucosidase enzyme is important for oleuropein hydrolysis and, among lactobacilli, L. plantarum species has been successfully used as starter for its strong ability to break the glycosidic bond of oleuropein (Ciafardini et al., 1994; Tataridou and Kotzekidou, 2015) and for its high versatility. Overall, our data revealed that all brine samples reached a pH value ≤4.3 and exhibited a good acidification rate, indicating the success of the fermentation and ensuring the microbiological safety of the final product, in accordance to other researches (Corsetti et al., 2012; Martorana et al., 2017). In particular, samples inoculated with the β-glucosidase positive strain exhibited a more pronounced reduction of the fermentation time, with a higher content of hydroxytyrosol, tyrosol, and verbascoside compounds from 30 days of fermentation than un-inoculated ones, according to other studies (Romero et al., 2004; Ben Othman et al., 2009; Pistarino et al., 2013; Kaltsa et al., 2015). In addition, the autochthonous strain exhibited a better adaptation/growth rate in brine samples at 5% of salt. It is well-established that autochthonous strains are generally more adapted to harsh conditions of raw material than allochthonous ones, and, therefore, to dominate the microbiota, driving the fermentation and counteracting spoilage microorganisms (Di Cagno et al., 2008; Bevilacqua et al., 2015). Microbiological data indicated a significant reduction of Enterobacteriaceae starting from 30 days of fermentation, with an improvement of the safety of the final product, as previously reported (Pino et al., 2018a). It is interesting to point out that results revealed a high occurrence of yeasts as part of table olive natural microbiota, coexisting with LAB during the whole fermentation process (Arroyo-López et al., 2008, 2012a,c), which could be related to the geographic area, and cultivars (Bleve et al., 2014). Our data registered an unusual yeast count at the end of the process, higher than Spanishstyle and Sicilian-style table olives, which generally reached value of 4-5- log CFU/ml. This could be linked to the processing

TABLE 5 | Sensory data obtained for the evaluation of fruits in the different treatments

 assayed.

Descriptors

technology applied in the present study. Yeasts favor bacteria growth, enhancing lactic acid production to inhibit spoilage microorganisms and affect flavor and texture of the final products (Arroyo-López et al., 2008, 2012b; Bevilacqua et al., 2013). As reported in a recent review, Candida boidinii, Debaryomyces hansenii, and Pichia membranifaciens were revealed as the most geographically diffused species (Campus et al., 2018). Evaluating yeast behavior, although it is noteworthy that yeast development is related to high salt level and phenolic compounds or low pH. In the present study, W. anomalus, and C. boidinii were the species mainly detected in brines processed at 5% of salt, whereas C. diddensae and M. guilliermondii were mainly revealed in brines at 8% NaCl. Several studies reported strong β-glucosidase activity for W. anomalus species (Bautista-Gallego et al., 2011b; Arroyo-López et al., 2012c; Romero-Gil et al., 2013; Bonatsou et al., 2015), and strong lipase and esterase activities for C. boidinii species, which positive impacts to fruity and olive flavor (Hernández et al., 2007; Bautista-Gallego et al., 2011b; Arroyo-López et al., 2012c; Pereira et al., 2015). In contrast to previously published data (Hurtado et al., 2008; Bautista-Gallego et al., 2011b), C. diddensae, which is generally associated to the early stage of fermentation, was detected at the highest frequency at 120 days. Focusing on LAB population, L. plantarum, L. pentosus, and L. paracasei were the main species found in all brine samples, confirming both their key role in table olive fermentation and biofilm formation with yeasts. In addition, a high survivability of the potential probiotic L. paracasei N24 strain was depicted in the final products, mainly in samples at 5% of NaCl. This evidence confirms its suitability to growth in harsh environment, such as brines, and that table olives are able to support probiotic survival (Lavermicocca et al., 2005; Rodríguez-Gómez et al., 2017; Pino et al., 2018a). In fact, nutrients and prebiotics released into the brines favor the biofilm formation, protecting bacteria from acidic environment and enhancing their passage through human gastrointestinal (GI) tract (De Bellis et al., 2010; Ranadheera et al., 2010; Arroyo-López et al., 2012a; Blana et al., 2014; Rodríguez-Gómez et al., 2014a,b, 2017; Grounta et al., 2015). It is interesting to point out that the addition of the potential probiotic strain at 60 days of fermentation in brine samples processed at low salt content and with starter, significantly modified the VOCs pattern. In particular, compounds responsible for floral and fruity notes, such as phenylethyl alcohol and methyl 2-methylbutanoate, highly increased, while ethanol and isoamyl-alcohol significantly deceased compared to un-inoculated samples. The high content of alcohols in un-inoculated brine samples could be related to yeast metabolic activities (Bleve et al., 2014; Randazzo et al., 2017). This evidence was in accordance to sensory data since

## REFERENCES

Allouche, Y., Warleta, F., Campos, M., Sanchez-Quesada, C., Uceda, M., Beltran, G., et al. (2011). Antioxidant, antiproliferative, and pro-apoptotic capacities of pentacyclic triterpenes found in the skin of olives on MCF-7 human breast cancer cells and their effects on DNA damage. J. Agric. Food Chem. 59, 121–130. doi: 10.1021/jf102319y

panelists attributed the higher score to the bitterness and acid descriptors in un-inoculated samples. Finally, data obtained from correlation between VOCs and brine samples differently treated revealed that the VOCs formation was mainly influenced by the starter and/or probiotic addition instead of salt content.

## CONCLUSION

The effects of a sequential inoculum of β-glucosidase positive and potential probiotic strains on the fermentation of Sicilian table olives were investigated. Remarkably, results demonstrate that the technology applied, based on the sequential inoculum and the brines fermentation at low salt content, without any salt replacement, did not increase the risk of microbial spoilage, nor the overgrowth of foodborne pathogens. Indeed, the composition and the dynamics of brine microbiota, mainly constituted by LAB and yeasts consortium, significantly affected the composition of the VOCs and the sensorial traits of the final products, which were confirmed by a panel of trained assessors. Hence, the results of the present study are promising, suggesting the possibility to formulate table olives with reduced salt content.

## AUTHOR CONTRIBUTIONS

AP, FR, AT, AV, and LS performed the experiments and analyzed the data. AP, JB-G, and CR wrote the manuscript. CR, JB-G, and CC designed the study. CR, JB-G, and FA-L contributed to data interpretation. All authors revised the manuscript.

## ACKNOWLEDGMENTS

We thanks Dr. Vincenzo Alfeo for his advice on VOCs determination and statistical analysis. LS was supported by the Italian Ministry of Education, University, and Research (MIUR), within the framework of the Italian National Grant for Fundamental Research (FFABR 2017). JB-G thanks MINECO and CSIC for his JDC-Incorp. fellowship. The research was supported by the Project Valutazione della sostenibilità dei sistemi agroalimentari locali e selezione di markers molecolari e biologici nella gestione della qualità di prodotti agroalimentari funded by the University of Catania (Italy).

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.00174/full#supplementary-material

Improvement of the storage process for cracked table olives. J. Food Eng. 89, 479–487. doi: 10.1016/j.jfoodeng.2008.06.001

paracasei IMPC2.1 in an industrial plant. Int. J. Food Microbiol. 140, 6–13. doi: 10.1016/j.ijfoodmicro.2010.02.024

process time of black table olives (Cv. Itrana and Leccino): a pilot-scale application. J. Agric. Food Chem. 54, 3869–3875. doi: 10.1021/jf053206y

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Pino, Vaccalluzzo, Solieri, Romeo, Todaro, Caggia, Arroyo-López, Bautista-Gallego and Randazzo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Wholegrain Durum Wheat Bread Fortified With Citrus Fibers: Evaluation of Quality Parameters During Long Storage

Alfio Spina1 , Selina Brighina2 , Serena Muccilli 3 , Agata Mazzaglia2 , Simona Fabroni 3 , Biagio Fallico2 , Paolo Rapisarda3 and Elena Arena2 \*

1 Centro di Ricerca Cerealicoltura e Colture Industriali, Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria (CREA), Acireale, Italy, 2 Dipartimento di Agricoltura, Alimentazione, Ambiente (Di3A), University of Catania, Catania, Italy, 3 Centro di Ricerca Olivicoltura, Frutticoltura e Agrumicoltura, Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria (CREA), Acireale, Italy

#### Edited by:

Esra Capanoglu, Istanbul Technical University, Turkey

#### Reviewed by:

Juan E. Andrade, University of Illinois at Urbana-Champaign, United States Antonella Pasqualone, Università degli Studi di Bari, Italy

> \*Correspondence: Elena Arena<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Nutrition and Food Science Technology, a section of the journal Frontiers in Nutrition

Received: 03 September 2018 Accepted: 24 January 2019 Published: 13 February 2019

#### Citation:

Spina A, Brighina S, Muccilli S, Mazzaglia A, Fabroni S, Fallico B, Rapisarda P and Arena E (2019) Wholegrain Durum Wheat Bread Fortified With Citrus Fibers: Evaluation of Quality Parameters During Long Storage. Front. Nutr. 6:13. doi: 10.3389/fnut.2019.00013 The aim of this work was to evaluate the effect of the addition of citrus fibers, from blood orange and lemon peels to produce a functional durum wheat bread. Breads fortified in fiber were packaged under a modified atmosphere (MAP) and stored at 25◦C up to 120 days. No significant differences were observed with respect to the specific volume and weight, internal structure, pH and titratable acidity among the bread samples obtained using different types and percentages of fibers. Storage time, at 30 up to 90 days, affected significantly the bread firmness and caused significant differences in 5-hydroxymethylfurfural (HMF) levels in all bread samples. In fortified breads with citrus fibers the yeast and mold counts showed values of approximately 1 log10 cfu/g for the first 30 days and 3.5 log10 cfu/g at the end of storage. The results of the sensory evaluation highlight that loaves enriched with blood orange and lemon fibers showed a citrus flavor but had a similar overall evaluation respect to control bread produced without addition of citrus fiber. The results of this study showed that the addition up to 2% of blood orange and lemon fibers in wheat whole durum flour is a possible strategy to produce "high fibre" bread.

Keywords: durum wheat bread, citrus fibers, storage, microbiological assay, HMF, sensory attributes

## INTRODUCTION

Dietary fiber have an important role in maintaining good health and prevention of disease. The increase of the dietary fiber intakes was associated with the reduction of cardiovascular disease and the incidence of type 2 diabetes (1–4) and with the prevention of excessive weight gain, thanks to the role played on the regulation of appetite (5, 6).

The majority of the world's population consumes bread daily and it represents the ideal food to act as a vehicle for healthy substances. The addition of dietary fiber, generally modified the physicochemical characteristics both of dough and bread (7). Several authors reported the successful use of fruit and vegetable fiber in bakery products (8–11). In southern Italy, particularly in Sicily, citrus industry produces approximately 500,000 t/year of "pastazzo," a by-product derived from the industrial squeezing of citrus fruit, which currently presents serious disposal problems.

**63**

After numerous washings and purifications, it is possible to obtain citrus flour rich in dietary fiber, which can be added to food products.

Several authors used citrus fiber to fortify baked goods. A decrease of bread volume without significant worsening of the crumb texture and various detrimental effects on dough handling and bread quality with the replacement of flour with dietary fiber was reported (12, 13). Nassar et al. (14), suggested that 15% orange peel and pulp could be incorporated in biscuits, as a source of dietary fiber containing bioactive compounds (flavonoids, carotenoids, etc.). Biscuits of good technological quality, good level of acceptance and a decreased energy value, were obtained by replacing up to 15 g/100 g of the wheat flour with extruded orange pulp (15). A reduction both of energy content and in vitro protein digestibility was reported for cookies added with apple or lemon or wheat bran fiber (16); the partial replacement of wheat flour with a dietary fiber-rich orange bagasse allowed to produce baked products containing high levels of total dietary fiber and indigestible fraction and a decrease of glycemic index of the products (17). During baking process, the heat transmission favors, mainly in the bread crust, the formation of Maillard reaction products, that improve the flavor, color and texture of food products (18). 5-Hydroxymethylfurfural (5- HMF) is a product both of the Maillard reaction and of the acidcatalyzed thermal dehydration of hexose. In several processed foods is used as index of thermal abuse (19–21) and in baking products HMF levels make possible to monitor the heating processes (22, 23). Estimated dietary intakes range between 4 and 350 mg per person and day, and among foods, cereal and cereal products, in particular bread, may serve as a major source of HMF exposure. In cereal products the average concentrations of HMF ranged from 14 to 53 mg/kg (24, 25) and the average intake can vary greatly with regard to low or high consumption. Even if, seems, that the toxic potential of HMF is rather low several strategies were used to reduce the HMF level in bakery products (26–28).

The aim of this work was to evaluate the effect of the addition of different level of citrus fiber on the physicochemical, microbiological, and sensory properties of durum wheat whole bread. Moreover, the evolution both of the quality parameters and the sensory attributes were studied during a long storage time under MAP conditions.

## MATERIALS AND METHODS

## Materials

Durum wheat whole semolina was provided by the "Valle del Dittaino" Agricultural Cooperative Society a.r.l. (Assoro, Enna, Italy), a local industrial bakery. The bread ingredients were food grade. The citrus fibers were derived from blood oranges (C. sinensis L. Osbeck) and lemons (C. limon L. Burm f.) and were kindly donated by Ortogel S.p.A., Caltagirone, Italy. The compressed yeast (Mauri Extra Classic) was obtained from "AB Mauri Italy S.p.A.," Pavia, Italy and the NaCl was from "Mulino S. Giuseppe," Catenanuova, Italy.

## Methods

### Physicochemical Analysis of Durum Wheat Whole Flour

Ash content was obtained following the ISO method 2171 (2007). The gluten index was determined using a Glutomatic 2200 apparatus (Perten Instruments AB, Huddinge, Sweden) according to the method UNI 10690 (1979). The α-amylase activity was determined using the Falling Number 1500 apparatus (Perten Instruments AB, Huddinge, Sweden), following the method UNI EN ISO 3093 (2010).

Total, soluble and insoluble dietary fiber content was determined using a K-TDFR kit from Megazyme (Megazyme International, Bray, Ireland) (29). The protein content and color indexes were determined according to the methods described for durum wheat whole flour (30).

Rheological dough properties were evaluated by mixograph (National Mfg. Co, Nebraska, USA) and farinograph curves (Brabender instrument, Duisburg, Germany) (31). All the analyses were conducted in triplicate.

## Chemical Characterization of Citrus Fiber

Total, soluble and insoluble dietary fiber content was determined as already reported above (29). The moisture content was determined according to the ISO method 712:2009. The ash content was obtained according to the ISO method 2171:2007. The alkalinity of the total ash was carried out following ISO method 5520:1981 and was expressed as the alkalinity index (the number of milliliters of 1 N acid solution required to neutralize 1 g of the ash obtained from the sample). The protein amount was determined by the Kjeldhal method. The sugars were determined by using HPLC with a refractive index detector (32). The pectin content was titrimetrically quantified as galacturonic acid according to the legal pectin specification (33). The water binding capacity (WBC), the water tendency to associate with hydrophilic substances, was determined (34). All the analyses were conducted in triplicate.

### Bread-Making Process and Storage Conditions

The breads were produced in an industrial bakery ("Valle del Dittaino"—Agricultural Cooperative Society a.r.l., Assoro, Italy). For each dough, 68 kg of durum wheat whole flour was mixed with tap water (78 ± 3%), compressed yeast (0.5%), NaCl (2.6%) and blood orange (OF) and/or lemon fibers (LF). In the control dough no citrus fiber were added; whereas in the other four doughs citrus fiber were added in variable amount: 1.5 and 2.0% of orange fiber (code samples 1.5OF and 2.0OF, respectively) or lemon fiber (code samples 1.5LF and 2.0LF, respectively) or a mix of orange and lemon fiber (1.0 + 1.0%) (code samples 1.0 + 1.0OLF). These levels were chosen to obtain breads with a total dietary fiber content higher than 6 g/100 g, which is the minimum required limit to indicate that a food is "high in fibre" (35). All the ingredients were mixed for 13 min using a mixer with beater arms (IBT 300, Pietroberto S.p.A, Marano Vicentino, Italy). The tap water temperature was 22◦C, and at the end of mixing, the dough temperature was 25.9 ± 0.4◦C. All the other ingredients were expressed as a % of the weight of whole wheat durum flour.

Afterward, the dough was broken into approximately 1,150 g pieces using volumetric dividing (Omega, Pietroberto S.p.A, Marano Vicentino, Italy), obtaining approximately 100 loaves for each dough. The loaves were rounded in a conical rounder (CO 3000, 1500 pieces/h, Turri F.lli S.r.l., Costa di Rovigo, Italy) and leavened in a tray proofing cell (Pavailler Engineering S.r.l., Galliate, Italy) for 2 h and 45 min at 32◦C with a relative humidity (RH) of 65%.

The loaves were baked for 1 h at 210◦C in a continuous oven (Pavailler Engineering S.r.l., Galliate, Italy). After 2 h of cooling in a conditioned room at 20◦C with a RH of 60%, the breads were packaged under a modified atmosphere in T6011B film (275µm thickness) (Sealed Air, Cryovac, Italy), and T9250B film (125µm thickness) (Sealed Air, Cryovac, Italy) was used to cover and seal the package. The containers were vacuumed, filled with mixed gas and sealed with a Multivac R-230 packaging machine (Germany) equipped with a modified atmosphere (MAP) mix 9,000 gas-mixer (PBI– Dansensor A/S, Ringsted, Denmark). The trays were packed using 70% N2:30 % CO2 gas combinations. All packaged bread samples were stored at 25 ± 2 ◦C and 60 ± 2% RH for 4 months and were periodically (0, 30, 60, 90, and 120 days of storage) sampled. Nine loaves for each formulation were withdrawn each month and immediately analyzed. The CO2 and O2 concentrations in the samples were measured using Check Point (Dansensor PBI, Ringsted, Denmark) gas analyzing equipment.

The following properties were tested for each bread sample during each sampling: volume, height, weight, diameter, crumb porosity, internal structure, loaf firmness, crust thickness, crust and crumb color, moisture, pH, acidity, HMF content, microbiological, and sensory analysis. All the analyses were conducted in triplicate.

## Bread Quality Evaluation

### Determination of the Physicochemical Properties of Breads

The physicochemical properties of bread samples were evaluated according to Spina et al. (31).

The volume was determined according to the rapeseed displacement in a loaf volume meter, and the specific volume (cubic meters per gram) was calculated as a ratio of the loaf volume and the bread weight. The internal structure was visually estimated, and the crumb porosity was estimated using the Mohs scale. Loaf crust thickness was measured using a digital caliper (Digi-MaxTM, Sciencewarer, NJ, U.S.A.), on the loaf basis of three central slices, after removing the crumb. The loaf firmness was measured using a texture analyser (Zwick Z 0.5 Roëll, Germany) equipped with an aluminum 8-mm- diameter cylindrical probe. The resulting peak force was measured in kilograms per cubic centimeter.

The CIE L∗ a ∗b ∗ color parameters were measured for the crumb, in the transversely cut bread and on the crust surface, averaging ten distinct points in each case, using a Chroma Meter (CR-200, Konica Minolta, Osaka, Japan) with illuminant D65.

The moisture content was determined in triplicate by gravimetric analysis. The bread samples were ground in a home grinder (La Moulinette, Moulinex, 2002), and then portions of the ground bread sample were placed in an oven at 105◦C until the dry weight was constant.

The pH and the total titratable acidity (TTA) were measured in triplicate, using a pHmeter (Mettler Toledo, MP 220). The TTA results were expressed, for the dry matter, as milliliters of 0.1 M NaOH consumed (36).

Total, soluble and insoluble dietary fiber content was measured in 1 g of dried sample using a K-TDFR kit from Megazyme (Megazyme International, Bray, Ireland) (29).

## HMF Extraction and HPLC Analysis

The HMF was determined according to Spina et al. (31). The bread samples were ground in a home grinder (La Moulinette, Moulinex, 2002); subsequently, an aliquot of the ground sample (5 g) was transferred into a volumetric flask (50 ml), and 25 ml of water was added (JT. Baker, Deventer, Holland). The solution was stirred for 10 min, and then the sample was diluted up to 50 ml with water (JT. Baker, Deventer, Holland) and centrifuged for 45 min at 5,000 rpm. An aliquot of the supernatant was filtered through a 0.45-µm filter (Albet) and injected into an HPLC system (Shimadzu Class VP LC-10ADvp) equipped with a DAD (Shimadzu SPD-M10Avp). The column was a Gemini NX C18 (150 × 4.6 mm, 5µm) (Phenomenex), fitted with a guard cartridge packed with the same stationary phase. The HPLC conditions were the following: isocratic mobile phase, 90% water at 1% of acetic acid and 10% methanol; flow rate, 0.7 ml/min; injection volume, 20 µl (20). All of the solvents used were HPLC purity grade: water from J.T. Baker, and acetic acid and methanol from Merck. The wavelength range was 220–660 nm, and the chromatograms were monitored at 283 nm.

HMF was identified by splitting the peak of the HMF from the bread-solution sample with a standard of HMF (P > 98% Sigma-Aldrich, St. Louis, Mo., U.S.A.) and by comparison of the UV spectra of the HMF standard with that of the bread samples. All analyses were performed in duplicate, including the extraction procedure, and the reported HMF concentration is therefore the average of four values. The results were expressed as mg of HMF per kilogram of the bread dry matter.

### Microbiological Analysis

The microbiological analysis of the bread samples was performed in triplicate at 0, 30, 60, 90, and 120 days of storage, according to Spina et al. (31). Ten grams of each loaf were aseptically weighed and homogenized using a Stomacher (Brinkmann, Westbury, NY, USA) for 5 min and were serially diluted in a sterile physiological solution (0.9% NaCl). Serial dilutions of the suspension were pourplated in duplicate onto the following media: plate count agar (PCA) (Oxoid) for total viable count; violet red bile glucose agar (VRBGA) (Oxoid) for Enterobacteriaceae; and Saboraud dextrose agar for yeasts and molds. The VRBGA plates were incubated at 32◦C for 48 h under aerobic conditions. The PCA plates were incubated at 30◦C for 4 days, and the yeast-count plates were incubated at 25◦C for 4 days.

## Sensory Evaluation

The sensory profile was determined by applying the UNI 10957 (2003) method and was defined by a panel of 12 judges (six females and six males) recruited among student and University staff that have chosen to participate to the research and signed the informed consent as our institution do not have an ethics committee for the taste and food quality evaluation studies. The judges were submitted to training over 4 weeks to generate attributes using handmade and industrial breads (31, 37, 38) and to familiarize themselves with the scales and procedures (UNI EN ISO 8586, 2012). The judges, using a scale between 1 (absence of the sensation) and 9 (extremely intense), evaluated the intensity of the eighteen sensory attributes selected on the basis of frequency (%) (39) (**Table 1**). The evaluation sessions, which were performed at 0, 30, 60, 90, 120 days of storage, were conducted in an equipped laboratory (UNI EN ISO 8589, 2010) from 11:00 to 12:00 a.m. in individual booths illuminated with white light. All data were acquired by a direct computerized registration system (FIZZ Biosystemes, ver. 2.00 M, Couternon, France). The data reported were expressed as the mean ± standard deviation.

## Statistical Analysis

Statistical analysis of the results was performed using the STATSOFT 6.0 program (Vigonza, Italy). The significant differences were evaluated by Two-Way analysis of variance (ANOVA), and the mean separation was determined using the Tukey test with respect to time x thesis factor. The sensory data for each attribute were submitted to ANOVA using the software package Statgraphicsr Centurion XVI (Statpoint Technologies, INC.) using the samples as factors. Significant differences were determined at the alpha level of 0.01 and 0.05 (only for citrus fibers protein and sensory parameters).

#### TABLE 1 | Descriptive terms used for sensory profiling of bread.

## RESULTS AND DISCUSSION

## Physicochemical Characterization of the Durum Wheat Whole Flour

Chemical characteristics of whole flour were: moisture 13.15 ± 0.07 g/100 g, ash 1.75 ± 0.10 g/100 g, protein 12.5 ± 0.10 g/100 g. These quality parameters fulfilled the legal requirements (40). Total dietary fiber value was 6.45 ± 0.6 g/100 g, which was constituted by 3.93 ± 0.4 g/100 g of insoluble dietary fiber and 2.52 ± 0.2 g/100 g of soluble dietary fiber. Dry gluten content was 8.4 ± 0.2 g/100 g. Gluten index value was 95.9 ± 5.0 and the value of amylase activity at falling number was low (633 ± 2.0 s). With regard to dry gluten content and relative qualitative index, the whole flour sample exhibited regular gluten quantities but a very high gluten tenacity. As regards color parameters, the values were: brown index (100—L∗ ) 15.8 ± 0.2, red index (a∗ ) 0.12 ± 0.01, yellow index (b∗ ) 18.03 ± 0.04. As for dough rheological characteristics at Mixograph apparatus they were high: mixing time 360 ± 6.0 s and peak dough height 8.0 ± 2.0 M.U. (Mixograph Unit). Consequently, the classification scale (1-8) was good (7 ± 0.0). At Farinograph apparatus the quantity of water absorbed (%) at a 500 B.U. (Brabender Unit) dough consistency (64.6 ± 0.02 %) indicates that the whole flour had a high ability to absorb water due to high fiber content. The values of dough development time 270 ± 6.0 s and dough stability 1,080 ± 11 s were very long due to the good whole flour quality and were in agreement with those reported by other authors (41).

## Characterization of Citrus Fiber

**Table 2** reports the chemical characterization of the lemon and blood orange fibers used for the baking process. Both lemon and blood orange had highest levels of total dietary fiber (about 70%), with a balanced content of soluble (about 32%) and insoluble fibers (about 37.5%), differently from data reported

TABLE 2 | Chemical characterization of citrus (lemon and blood orange) fiber used in bread making.

Data expressed as the mean ± standard deviation. Different letters in the same row are significantly different lower case, p < 0.01; upper case, p< 0.05; n.s., not significant. WBC, water binding capacity.

for grapefruit, lemon and orange fibers by other authors (12), who reported lower levels of soluble fibers. Citrus fruit are characterized by distinctive concentrations of different minerals, mainly K, Ca, and P (42). Thus, the technological process applied for the preparation of the citrus fibers produced dried flours with 6.63 ± 0.15 g/100 g and 5.67 ± 0.15 g/100 g ash content with a relevant alkalinity index of 55.5 ± 0.08 and 52.7 ± 0.06 for the lemon and blood orange fibers, respectively. The moisture, total protein and sugar values were approximately in lines with values reported by other authors (12, 17).

## Dietary Fiber Content in Breads

**Table 3** reports total, soluble, and insoluble dietary fiber content in bread samples after baking. The highest level of total dietary fiber of citrus fiber (**Table 2**) had the advantage that small addition to the whole flour did not modify the technological properties of the resulting dough. Furthermore, it resulted in a significant increase in total fiber level in bread. The highest values of total, soluble, and insoluble fiber were recorded in the bread with 2.0% of citrus fibers, even if these thesis did not differ statistically from the others.

All the theses with citrus fiber exceeded 6.0% of total dietary fiber content, which is the minimum required limit to indicate that a food is "high in fibre," as required by Regulation (EC) No 1924/2006 of the European Parliament and of the Council on nutrition and health claims made on foods (35).

Therefore, addition of lemon and/or blood orange citrus fibers used in this study as ingredients to produce fiber-enriched bread can be helpful and interesting in integrating the whole flour with valuable and balanced sources of both soluble and insoluble dietary fiber fractions, including pectins.

## Quality Parameters of Breads and the Evolution During Storage

**Table 4** reports the p-values for all physico-chemical parameters studied on the breads with respect to storage time x thesis factor, TABLE 3 | Dietary fiber content after baking in bread samples.

1.5LF, 2.0LF sample codes for breads with 1.5 or 2% of lemon fiber, respectively; 1.5OF 2.0OF sample codes for breads with 1.5 or 2% of blood orange fiber, respectively; 1.0 + 1.0OLF sample code for bread with 1 + 1% of lemon and blood orange fiber. Data expressed as the mean ± standard deviation. Different letter in the same column indicates significant difference (p < 0.01).

while **Tables 5**, **6** showed the results of the physical and chemical properties of the industrial breads in the MAP conditions during 120 days of storage, respectively. The specific volumes and weights of the loaves were not significant for any of the 3 factors of variability (thesis (A), storage time (B), and their interaction (A × B) (**Table 4**). No significant differences in the specific volumes and weights were shown among the bread samples and during the storage time, whatever the type and level of citrus fibers, probably due to the use of wheat whole durum flour. These findings are not in agreement with those already reported (12). Wheat white bread is bulkier and less heavy than that made with durum wheat whole semolina, and this fact strongly and negatively impacted the contributions of the orange fiber. The ratio between the height and diameter of the loaves is used in the baking industry to parameterize any failure of the dough (**Table 5**). The h/d ratio was significant respect to all the 3 factors of variability, even if with different p levels (p ≤ 0.001 for theses, p ≤ 0.01 for storage time and their interaction) (**Table 4**). During the storage, the control showed the greatest h/d ratio (approximately 3.4) due to the absence of citrus flour that leads to a high dough tenacity. The other bread samples, as expected, exhibited a lower ratio, even if they are not statistically different from the control, with the exception of sample 1 + 1OLF after 30 days of storage (**Table 5**).

After baking (t0), the 50% of the thesis showed a better developed crumb porosity. Starting from 30 days of storage, the porosity performance of 1.5 and 2.0 OF bread samples decreased (value 7), whereas the 2.0 LF sample maintained good characteristics until 90 days of storage (**Table 5**).

As regards the bread internal structure was significant both respect to the theses factor (p ≤ 0.05) and for the other two factors of variability (p ≤ 0.01) (**Table 4**). Only the samples 1.5 LF and 1.0 + 1.0OLF showed an irregular structure both after baking and during storage. The addition of blood orange fiber decreased the loaf volume but did not deteriorate the crumb characteristics (43) (**Table 5**).

Regarding the crust thickness, this parameter was significant respect to theses (p ≤ 0.001), times (p ≤ 0.01) and their interaction (p ≤ 0.05) (**Table 4**). Only the sample 2.0 OF showed a minor crust thickness after 60 days of storage (**Table 5**).

Concerning to the external firmness it was significant respect to the all factors of variability (**Table 4**). The storage time significantly affected the external firmness. After baking (t0), all bread samples showed values ranging from 2.08 to 2.42 kg/cm 2 . At 30 days of storage and up to 60 days, a slight reduction was observed in all samples. Then, the values of the external firmness increased in all bread samples. From 30 days after baking and up to 60 days, the moisture in the loaf moved centripetally, causing a softening of the crust and underlying the crumb, producing a decrease in the firmness of the loaves. Then, the moisture balanced again in different parts of the loaves and the texture gradually increased, reaching a high value after 120 days (**Table 5**).

**Table 6** reports the moisture content, pH, TA and HMF content of the bread samples during the entire storage time. The moisture content was significant respect to the all factors of variability (**Table 4**). The moisture content ranged from approximately 38.5–41.6% at the beginning, but no significant differences were found between the control bread and the other bread samples. At 60 days of storage, the moisture content decreased up to approximately 30% in the control and in the bread with lemon fiber. Bread samples produced with orange fiber and with the mixed fiber highlight the lowest moisture content (about 20%) but only 2.0 OF and 1.0 +1.0OLF were significantly different from the control bread. At 90 and 120 days of storage, no remarkable differences were evident between the bread samples, with the exception of 120 days in the 2.0 LF sample, which had the lowest moisture level (**Table 6**).

The effect of the addition of citrus fiber in bread samples on pH and on titratable acidity seems to be more related to the storage time rather than the recipe. No significant differences in pH were found between the control bread and other bread samples, except for 2.0LF at 90 and 120 days of storage. The pH of this bread sample remains constant throughout the storage time.

Concerning to the TA it was significant at the highest level respect to the all 3 factors of variability (**Table 4**). The TA ranged from approximately 7.4–8.8 at the beginning. After it decreased gradually up to 90 days of storage while increasing up to the initial levels, afterwards (**Table 6**).

For the first time the HMF level in durum wheat whole semolina bread was reported. In control bread the HMF was higher (about 65.4 mg/kg dry matter) than those determined in durum wheat bread (39.7 ± 2.22) (31). Differences both in recipe and in chemical characteristics of durum wheat whole semolina are responsible for the different HMF level (44). The HMF content was significant for all 3 factors of variability (**Table 4**).

The HMF content (**Table 5**) ranged from approximately 44.8–85.9 mg/kg at the beginning, and significant differences were found between the control bread and the 1.5 LF and 2.0 OF bread. Storage caused significant differences in the HMF levels in all bread samples. HMF decreased with storage up to 60 days, increasing up to 120 days of storage, showing a similar trend respect to TA. This trend, is the result of formation and degradation reactions of HMF (45 ) influenced by the pH, the temperature and the presence and concentration of sugars and aminoacids (26 , 46 , 47). The HMF behavior was similar to those observed for TA and

TABLE 5 | Evaluation of properties of bread samples produced using different levels of lemon (LF) and blood orange (OF) fibers, during storage.

1.5LF, 2.0LF sample codes for breads with 1.5 or 2% of lemon fiber, respectively; 1.5OF 2.0OF sample codes for breads with 1.5 or 2% of blood orange fiber, respectively; 1.0 + 1.0OLF sample code for bread with 1% +1 % of lemon and blood orange fiber. Data expressed as the mean ± standard deviation. Different letter in the same column indicates significant difference (p < 0.01).

a1, most porous; 8, least porous.

b1, regular; 2, irregular.

the two parameters shows a good correlation (r 2 = 0.5200). It is known that during the Maillard reaction several acids (i.e., formic, acetic) were formed as degradation products (48). Similar result was highlighted during storage of durum wheat bread (31).

**Table 7** reports the color parameters of the crust and crumb of bread samples produced using various doses of blood orange and lemon citrus fibers. The crust lightness was significant respect to the all factors of variability (**Table 4**). At time 0, the 1.0+1.0OLF bread samples had the darkest crust values, whereas the other bread samples had the greatest L∗ values. At times 30 and 60, the L ∗ crust values were lower for all bread samples. A similar trend was reported for durum wheat bread fortified with pectins and flavonoids (49). At time 90, the crust lightness values remained equal, whereas after 120 days, all of the bread samples showed the greatest L∗ values.

The crust redness it was significant for storage time and the interaction between theses and storage time, instead crust yellowness was significant respect to all the factors of variability (**Table 4**).

No significant differences were found in the a∗ and b∗ parameters after baking or at time 30 between the crust color of the control and other bread samples. Therefore, low values were shown in the 1.0+1.0OLF crust samples. At times 60 and 90, the a ∗ and b∗ parameters demonstrated a lowering trend for all of the bread samples. After 120 days of storage, an increase in the a∗ and b ∗ values was observed, with intermediate values between 0 and 30, 60 and 90 days.

TABLE 6 | Evaluation of the chemical characteristics of the bread samples produced using different levels of citrus fiber during storage.

1.5LF, 2.0LF sample codes for breads with 1.5 or 2% of lemon fiber, respectively; 1.5OF 2.0OF sample codes for breads with 1.5 or 2% of blood orange fiber, respectively; 1.0 + 1.0OLF sample code for bread with 1 +1% of lemon and blood orange fiber. Data expressed as the mean ± standard deviation. Different letter in the same column indicates significant difference (p < 0.01).

aTitratable acidity expressed on dry matter.

The crumb lightness was not significant respect to one factor of variability (theses), but was significant for storage time and their interaction (**Table 4**). The effect of the addition of citrus fibers on the L∗ parameter of crumbs after baking and after 30 days of storage were not significant. At time 60, the L∗ value was reduced. The control and 1.5LF bread sample had the greatest values.

The a∗ and b∗ crumb color parameters were significant respect to the all factors of variability (**Table 4**). After time 60 the a ∗ values decreased and then increased again to 120 days of storage (**Table 7**).

As browning index parameter has been considered 100–L∗ (22). In this study, no linear correlation was found between HMF and 100–L∗ (r 2= 0.348) according to results reported for durum wheat bread (31), but in contrast with those reported for dough baked at various times even if product with the same colors can have different HMF levels (23).

## Microbiological Assay

**Table 8** reports microbial dynamics of the different groups throughout the storage period. The values of CO2 and O2 in the ATM packaged samples remained unchanged for the entire storage period (data not shown). The yeast, mold and total viable count were significant respect to all the factors of variability (**Table 4**).

No mold growth was visible in well-sealed packages. At the end of storage, the greatest total viable count (TVC) and yeast counts were achieved. The yeast and mold counts exhibited values of approximately 1 log10 cfu/g for all of the bread samples prior to 30 days of storage. From 30 through 120 days of storage, a gradual increase in the yeast and mold counts was detected in all samples, achieving the greatest values of 3.79 log10 cfu/g for sample 1.0+1.0 OLF, whereas the control sample had the lowest at 2.47 log10 cfu/g. A similar trend was reported for TVC, with values of 4.51 and 4.46 log10cfu/g for samples 2.0 OF and 1.0+1.0

1.5LF, 2.0LF sample codes for breads with 1.5 or 2% of lemon fiber, respectively; 1.5OF 2.0OF sample codes for breads with 1.5 or 2% of blood orange fiber, respectively; 1.0 + 1.0OLF sample code for bread with 1 +1% of lemon and blood orange fiber. Data expressed as the mean ± standard deviation. Different letters in the same column indicates significant difference (p < 0.01).

OLF, respectively. In the control bread, the yeast, mold and TVC remained slightly lower during all storage times possibly due to the absence of fiber, although the differences are not significant up to 60 days of storage.

Trials on freshly baked bread enriched with potato flour with 10 days of storage showed similar trends, with mold values of approximately 1–2 log10 cfu/g and bacteria of approximately 5 log10 cfu/g for bread enriched with 10% Irish potato flour (50).

Aerobic plate counts (log10 cfu/g) of bread enriched with plantain flour after baking ranged from 3.20 to 4.50 (51). These counts were minimal and would not constitute health hazards, but are greater compared with the data obtained in this study. This is the first time that microbiological data on industrial bread enriched with fiber have been reported after a long storage period. Confirming the importance of packaging conditions and good practices during industrial production to prevent contamination and microbial proliferation after processing.

## Sensory Evaluation

For the first time the sensory profiles of a durum wheat whole semolina bread and durum wheat whole semolina bread with citrus fiber were determined.

The sensory profile both of control and of the fortified bread with citrus fiber was peculiar due to the ingredients used, such as durum wheat whole semolina and citrus fiber. A previous study (52) reported that durum wheat breads, have peculiar sensory features, different from those of common wheat breads (38).

**Table 9** reports the mean scores of all attributes for t0, while for the other days of storage only the statistically significant attributes. From the ANOVA results of the sensory data, the freshly baked samples were significant different for the attributes humidity, bitter, overall and citrus aroma and flavor.

Panelist of bread samples containing up to 30% of fine bran, with a fiber level ranging from 1.5 to 3% did not find significant differences in crumb color, texture and flavor when

TABLE 8 | Mean log10 of microbial population counts of breads produced using various doses and types of citrus fibres during storage.

1.5LF, 2.0LF sample codes for breads with 1.5 or 2% of lemon fiber, respectively; 1.5OF 2.0OF sample codes for breads with 1.5 or 2% of blood orange fiber, respectively; 1.0 + 1.0OLF sample code for bread with 1 + 1% of lemon and blood orange fiber.Data expressed as the mean of microbial log counts (log10 cfu/g) ± standard deviation. Different letters in the same column indicates significant difference (p < 0.01).

compared with the control (whole wheat flour) (53). Results reported in this study showed that bread samples containing up to about 7.3% of total dietary fiber, due to fortification with citrus fiber, did not show changes in crumb color, alveolar structure, softness and bread flavor, suggesting citrus fiber as a good source of dietary fiber that did not significantly change the main sensory attributes of durum wheat whole semolina bread.

The samples 1.5LF and 1+1OLF had the low intensity of citrus aroma; the sample 2.0OF showed the high intensity of bitter while the addition up to 1.5% of orange fiber improves the overall intensity scores.

At 30 days of storage, the samples were significant different for the attributes crumb color, humidity, softness, overall, citrus aroma, and flavor. Samples with citrus fiber had the same intensity of citrus aroma and flavor. The bread sample 1.5OF had the highest intensity of softness due to its highest humidity intensity score.

At 60 days, the bread samples were significant different for the attributes humidity, sweet, softness, overall, bread flavor, citrus aroma, and flavor. Samples 1.5 LF and 2.0LF showed the high intensity of citrus aroma, while the sample 1.5OF and control had the highest intensity of softness strictly related to their highest humidity intensity score. Moreover, the control bread showed the highest intensity of bread flavor and overall.

At 90 days of storage, the sensory profile of samples changes. In fact, on the nine sensory attributes, four attributes, such as alveolar structure, sour, astringent and yeasty flavor, for the first time were statistically significant. Other significant different attributes were crumb color, humidity, citrus aroma and flavor and overall. The samples with orange fiber had an alveolar structure similar with control bread. Also the astringent intensity was similar in control and 1.5OF bread samples. Bread sample 1.5OF had the highest intensity of yeasty flavor. The overall scores of bread samples were similar.

At 120 days, the samples were significant different only for the attributes citrus aroma and flavor, yeasty flavor and overall, suggesting a leveling of the samples' sensory profile. The bread sample with the highest addition of lemon fiber (2.0LF) showed the highest intensity of citrus flavor and the lowest of yeasty flavor, while the sample 1.5OF had the highest intensity of overall. For the attribute citrus aroma all the bread samples fortified with citrus fiber showed the same intensity. The control sample had the highest intensity of yeasty flavor. The attribute of yeasty flavor differentiated the samples after 90 days of storage. This is probably due to bread aging, ascribed to a reduction of the intensity of citrus aroma and flavor. These changes were more evident in control bread produced without citrus fiber. Moreover, it is important to highlight that bread samples, during storage, did not develop off-odor and off-flavor, and up to 30 days of storage overall scores of bread samples were similar independently to recipe.

## CONCLUSIONS

For the first time in this paper, was studied the possibility to add citrus fiber to produce a wheat whole durum bread fortified in dietary fiber. The addition of citrus fiber up to 2% allowed to produce, generally, wheat whole durum bread with quality parameters, similar to control bread. No deep differences were highlighted between bread samples. Long storage time in MAP conditions significantly affected the bread characteristics independently from the addition of citrus fiber and allowed to maintain the microbiological safety of the bread samples.

This study showed that the addition of citrus industry byproduct fibers in durum wheat whole semolina is a possible strategy and a good prospect to produce "high fibre" wholemeal durum wheat bread to increase the nutritional value and the dietary fiber intake. These results, moreover, should be interesting for the possibility to produce a "high in fibre" durum wheat bread with a long shelf-life.

TABLE 9 | Mean scores of the sensory attributes at the different days of storage.

1.5LF, 2.0LF sample codes for breads with 1.5 or 2% of lemon fiber, respectively; 1.5OF 2.0OF sample codes for breads with 1.5 or 2% of blood orange fiber, respectively; 1.0 + 1.0OLF sample code for bread with 1 + 1% of lemon and blood orange fiber.

aFor t0 all attributes are shown, for the other days of storage only the statistically significant attributes.

Data expressed as the mean ± standard deviation. Values marked with different letters in the same row are significant difference (p ≤ 0.05).

Finally, the use of citrus fibers in bread making can be considered as an environmental-friendly alternative for the reuse and valorisation of citrus processing wastes and by-products.

## AUTHOR CONTRIBUTIONS

AS and EA planned the experiments, surveyed laboratories, interpreted and discussed the results, and co-wrote the manuscript. SM performed laboratory flour analysis, acquired bread data during storage, performed the microbiological assay, and the statistical analyses. AM performed sensorial analysis, interpreted, and discussed sensory results. SB performed laboratory chemical analysis and acquired bread data during

## REFERENCES

storage. SF performed laboratory citrus fiber analysis and participated in the critical revision of manuscript. BF and PR conceived the idea, designed the study and participated in the critical revision of manuscript.

## FUNDING

This work was supported by the Regional Operational Program of Regione Siciliana PO FESR 2007-2013—Asse IV, Obiettivo 4.1.1, Linea di Intervento <IP_ADDRESS> with the title of the research program Impiego e valutazione di fibre e sostanze nutraceutiche per l'ottenimento di prodotti da forno salutistici [grant number 5787/3 of the 14/12/2011].

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Spina, Brighina, Muccilli, Mazzaglia, Fabroni, Fallico, Rapisarda and Arena. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Hydroxytyrosol Improves Obesity and Insulin Resistance by Modulating Gut Microbiota in High-Fat Diet-Induced Obese Mice

Zhuoqun Liu1 , Ningning Wang1 , Yanan Ma2 and Deliang Wen3 \*

1 School of Public Health, Dalian Medical University, Dalian, China, 2 School of Public Health, China Medical University, Shenyang, China, 3 The Institute of Health Science, China Medical University, Shenyang, China

Obesity is a common chronic metabolic disease that is harmful to human health and predisposes the affected individuals to a cluster of pathologies. Insulin resistance (IR) is one of the most frequent complications of obesity. Hydroxytyrosol (HT) may reduce obesity and IR in high-fat diet (HFD)-fed mice; however, the mechanism underlying is still unknown. Systemic low-grade inflammation and intestinal dysfunction are thought to be associated with obesity and IR. In this study, we found that HFD feeding for 8 weeks altered the intestinal microbiota, injured intestinal barrier function, increased endotoxin release into the blood, enhanced the expression of inflammatory factors (TNF-α, IL-1β, IL-6) and lipid accumulation in liver, caused obesity, and aggravated IR via the JNK/IRS (Ser 307) pathway in HFD mice. We also found that HT gavage could reverse those effects and the beneficial effects of HT were transferable through fecal microbiota transplantation. Our data indicate that HT can improve obesity and IR by altering the composition of the intestinal microbiota and improving integrity of the intestinal wall. We propose that HT replenishment may be used as a dietary intervention strategy to prevent obesity and IR.

Keywords: hydroxytyrosol, fecal microbiota transplantation, obesity, inflammation, insulin resistance, liver steatosis

## INTRODUCTION

Childhood obesity is a major public health problem and is becoming increasingly prevalent in most regions of the world. The worldwide prevalence of childhood obesity has risen from 4% in 1975 to 18% in 2016, and recent studies have shown that more than 340 million children and adolescents are overweight or obese (Orlando et al., 2018). Obesity is often accompanied by chronic low-grade inflammation, disorders of glucose and lipid homeostasis, and metabolic diseases such as type 2 diabetes, cardiovascular disease, and hepatic steatosis (Hotamisligil, 2017). Childhood obesity may increase the likelihood of obesity and metabolic disorders in adulthood, greatly decreasing the individual's quality of life (Bass and Eneli, 2015).

#### Edited by:

Cinzia Caggia, Università degli Studi di Catania, Italy

#### Reviewed by:

Alex Galanis, Democritus University of Thrace, Greece Qingping Zhong, South China Agricultural University, China

\*Correspondence:

Deliang Wen<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

Received: 30 October 2018 Accepted: 13 February 2019 Published: 04 March 2019

#### Citation:

Liu Z, Wang N, Ma Y and Wen D (2019) Hydroxytyrosol Improves Obesity and Insulin Resistance by Modulating Gut Microbiota in High-Fat Diet-Induced Obese Mice. Front.

It is widely accepted that unhealthy diets, sedentary lifestyles, and sleep deprivation are associated with an increased risk of obesity (Baranowski and Taveras, 2018). In addition, a growing number of studies have shown that changes in gut microbiota in early life may also play a role in the development of obesity. There are trillions of microorganisms in the human body (Tanaka et al., 2009; Dominguez-Bello et al., 2010), which undergo dynamic changes during an individual's life. Environmental factors (Zhang et al., 2010), including diets (Siddharth et al., 2013), are the main contributors to the composition of microorganisms rather than genetic factors. Most studies have shown that the fetus's intestinal tract is sterile during pregnancy (Makino et al., 2013), and the initial bacterial colonization occurs during birth. It has been suggested that the methods of birth and feeding determine the type of bacteria which are colonized into the baby's gastrointestinal tract (Munyaka et al., 2014). Moreover, the timing of supplementary food addition also affects the composition of the gut microbiota, after which the gut microbiota remains stable until old age (Villanueva-Millan et al., 2015).

The "Mediterranean diet" (MD), a diet rich in fruits and vegetables, fish, whole grains, beans, and olive oil, is considered to be one of the healthiest diets (Del Chierico et al., 2014). Numerous studies have shown that people in the Mediterranean region are far less likely to suffer from cardiovascular disease, diabetes, colon cancer, and rectal cancer than people in other European and American countries. Studies have shown that the MD increases Bacteroides and Clostridium, reduces Proteus and Bacillus populations in the gut microbiota (Marlow et al., 2013). A recent study showed that the MD could increase the gut microbiota diversity in cynomolgus monkeys (Nagpal et al., 2018), increase the abundance of Lactobacillus and Clostridium, and reduce the abundance of Ruminococcus and Coprococcus. The current literature on the role of the MD in childhood is lacking. Olive oil, an important component of the MD, is beneficial due to its abundance of monounsaturated fatty acids and antioxidants (Hamden et al., 2009). HT, the main component of olive oil, has been shown to have no toxic effects on cells and animals within a certain dose range (Auñon-Calles et al., 2013a,b; D'Angelo et al., 2001). Previous experiments have shown that HT prevents obesity, inflammation, hyperglycemia, and IR induced by a HFD (Carito et al., 2013; Cao et al., 2014; Voigt et al., 2015; Wang et al., 2018). HT may decrease lipid deposits by downregulating the SREBP-1c/FAS pathway in the liver and skeletal muscle tissues in HFD-fed mice (Cao et al., 2014). Additionally, HT regulates Toll-like receptor 4 (TLR-4)-dependent inflammation through a NF-κB-independent pathway to relieve oxidative stress (Takeda et al., 2014). Our recent research has revealed that HT improves inflammation, IR, and hepatic steatosis by reducing endoplasmic reticulum (ER) stress and by regulating the JNK/IRS pathway in HFD-induced obese mice (Wang et al., 2018). However, little research has been done on the effect of HT on the gut microbiota. In the present study, we hypothesized that HT may prevent obesity and IR by altering the composition of the intestinal microbes and alleviating inflammatory status. Therefore, we examined the effect of HT supplementation on HFDinduced obese mice in early life and explored the mechanism of action of the gut microorganisms through bacterial fecal transplantation experiments.

## MATERIALS AND METHODS

## Animal and Dietary Intervention

Twenty-eight three-week-old male C57BL/6J mice were raised at the Specific Pathogen Free Animal Experimental Center of Dalian Medical University (The Ethical approval number of C57BL/6J mice is AEE17057) and were divided into 4 groups: control group (chow), HFD group, HT group, and HTF. Seven mice per cage were housed in the following conditions: 12/12 hour light/dark cycle, maintained temperature of 23◦C ± 2 ◦C, and a humidity of 60%, with free access to food and water.  The intragastric interventions were performed for eight weeks as follows: the chow group and HFT group were gavaged with distilled water (10 ml/kg/day), the HT group was gavaged with HT (50 mg/kg/day, dissolved in distilled water, purity ≥ 98%, APP-ChemBio, China), the HTF group was subjected to fecal transplantation (fecal transplant donor from the HT group, 100 mg/1 mL sterile saline).

## Fecal Collection and Transplantation

One hundred milligrams of fresh feces (collected during the experiment every day) from the HT group were dissolved in 1 mL of sterile saline in an autoclaved tube, vortexed with a turbine for 10 s, and then centrifuged at a speed of 800 g for 3 min (Biofuge Primo R, Thermo). The prepared solution was used within 10 min as described previously (Chang et al., 2017).

## Oral Glucose Tolerance Tests and Insulin Tolerance Tests

After 8 weeks of feeding, the mice were fasted for 10 h overnight without restrictions on drinking water and gavaged with glucose solution [2.0 g/kg body weight (bw)] for OGTT. For ITT, the mice were fasted for 5 h and injected intraperitoneally with insulin (0.75 unit/kg bw, FosunPharm). Tail vein blood was collected, blood glucose was measured using a glucometer (Yue Zhun Type II 560, Yuyue), and the area under the curve (AUC) was calculated as described previously (Wang N. et al., 2017).

## Sample Collection and Preservation

After the mice were sacrificed, the liver, scWAT, BAT, rWAT, epididymal white adipose tissue (eWAT), and ileum were removed, weighed, and wrapped in aluminum foil. The blood samples were divided into two parts: one part was centrifuged (4◦C, 3000 rpm, 15 min) for serum, and another part was centrifuged (4◦C, 3000 rpm, 15 min) for plasma in a test tube containing heparin. All samples were placed in liquid nitrogen for quick freezing and stored at −80◦C for further use.

## Hematoxylin and Eosin Staining

A portion of the scWAT was fixed in 10% formalin overnight and was then dehydrated and embedded in paraffin. Sections (thickness of 4−5 µm) were stained with hematoxylin and eosin and were then visualized with a microscope (Olympus BX72, Japan). Cell size was analyzed using Image J software (National Institutes of Health).

## Oil Red O Staining

fmicb-10-00390 March 4, 2019 Time: 11:35 # 3

The liver tissue was successively placed in 10, 20, and 30% sucrose solutions for gradient dehydration. The dehydrated liver was processed into frozen sections (thickness of 6 µm), stained with oil red (WLA055a, Wanleibio) for 10 min, and observed using a microscope. The size of the lipid droplets was analyzed using Image J software.

## Biochemical Analysis

Plasma lipopolysaccharide (LPS) levels were quantified using a Chromogenic Endpoint Tachypleus Amebocyte Lysate (CE TAL, Xiamen Bioendo Technology Co., Ltd., China) assay according to the manufacturer's instructions. The concentrations of TNF-α, IL-1β, and IL-6 in liver were analyzed using commercial kit (MB-2868A, MB-2776A, MB-5737A, Meibiao, China). Fasting serum insulin concentrations were determined using a commercial ELISA kit (ER1113, FineTest, China). IR was assessed using the index of HOMA-IR: fasting blood glucose (mmol/L) × fasting blood insulin (mU/L) / 22.5.

## Gut Microbiota Analysis

Before the mice were sacrificed, stool samples were collected for gut microbiota analysis using an autoclaved tube. Fecal DNA was extracted from the stool samples using the E.Z.N.A Soil DNA Kit (Omega Bio-tek, United States) according to the manufacturer's protocol. The hypervariant region V3–V4 of the bacterial 16S rRNA gene was amplified with the primers 338F (50 -ACTCCTACGGGAGGCAGCA-30 ) and 806R (50 -ACTCCTACGGGAGGCAGCA-30 ) by PCR. The PCR product was recovered using a 2% agarose gel, purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, United States), eluted with Tris-HCl, and detected by 2% agarose electrophoresis. Quantification detection was performed using Quantifluor-ST (Promega, United States). Sequencing was performed using Illumina's MiSeq PE300 platform (Shanghai Meiji Biomedical Technology Co., Ltd., China). UPARSE software was used to perform operational taxonomic unit (OTU) clustering of sequences based on 97% similarity (version 7.11 ). The OTU was subsampled for further analysis. Chimeras were eliminated using UCHIME software. Each sequence was compared to the Silva database (SSU123) for species classification annotation using the RDP classifier2 , with the alignment threshold set to 70%. The Wilcoxon rank-sum test was used to compare differences between the two groups.

The R language tool was used to make the graph of the Rank-Abundance curve, the Venn diagram, and the community histogram/heatmap. Rarefaction curve and alphadiversity indexes were analyzed by Mothur. Sample hierarchical clustering and principal coordinates analysis (PCoA) were performed using the Bray-Curtis distance algorithm, and the graphics were created with R language. Community comparison was evaluated using the UniFrac Server followed by a Wilcoxon rank sum-test.

## Western Blotting

Total proteins were extracted from the liver and ileum using total protein extraction kits (KGP1100, KeyGEN BioTECH). The concentration of the extracted proteins was quantified using a Pierce BCA Protein Assay Kit (Thermo). Equal amounts of protein from each sample were transferred to a polyvinylidene fluoride membrane after polyacrylamide gel electrophoresis. After blocking with 10% skim milk for 1 h at 37◦C in a water bath, the blot was incubated with primary antibody (a suitable concentration diluted with TBST) overnight at 4◦C. After being washed with TBST (5 times, 5 min at a time), the blot was incubated with secondary antibody for 2 h at room temperature. We used a Bio-Rad ChemiDoc MP imaging system to detect the bands of target proteins, and the relative density of the bands was analyzed using Image J software.

 Secondary antibodies: peroxidase-conjugated goat anti-mouse IgG (1:5000; ZB-2305, ZSGB-BIO) or peroxidase-conjugated goat anti-rabbit IgG (1:5000; ZB-2301, ZSGB-BIO).

## Statistical Analysis

The data are expressed as the means ± standard error of mean (SEM) and were analyzed and plotted with GraphPad Prism 6 (GraphPad Software, United States). Data sets that involved more than two groups were assessed by one-way ANOVA followed by Tukey's honest significant difference post hoctests. P-value ≤ 0.05 was considered statistically significant.

## RESULTS

## HT and HTF Reversed HFD-Induced Obesity in Mice

To determine whether HT supplementation or fecal transplant from HT-supplemented mice would reduce obesity in HFD mice, we recorded the body weights of the mice and the excised adipose tissue. HFD induced significant increases in final body weight, body weight gain, liver weight, and tissue mass of the

1http://drive5.com/uparse/

2http://rdp.cme.msu.edu/

content was assessed using the Image J software. Scale bar, 50 µm. In (I), adipocyte size was estimated using the Image J software (lower panel). Scale bar, 50 µm. Graph bars with statistically significant results (P ≤ 0.05) based on one-way ANOVA analysis followed by Tukey's honest significant difference post hoc tests. Data are shown as mean ± s.e.m (∗P ≤ 0.05, ∗∗P ≤ 0.01).

BAT, perirenal adipose tissue (rWAT), epididymal white adipose tissue (eWAT), and the scWAT compared with the chow group. HT supplementation significantly reduced the final body weight, mass of the rWAT, eWAT mass, and liver mass compared with the HFD group. The body weight gain and the weights of the BAT and scWAT tended to decrease, but there were no statistically significant differences (**Figures 1A–G**). All of the above parameters also declined in the HFT group compared to the HFD group, but none reached significance (**Figures 1A–G**). Consistently, we observed evident lipid deposition in the hepatocytes and increases in adipocyte size in the HFD group compared to the chow group, which were reversed by HT supplementation and HFT (**Figures 1H–I**). Interestingly, HFD mice consumed less energy than the chow group, and the HFT mice consumed more energy than the HFD mice (**Figure 1J**).

## HT and HTF Promoted Intestinal Integrity and Alleviated Inflammation in HFD Mice

To examine whether HT and HTF can improve intestinal integrity, we measured the concentration of lipopolysaccharide (LPS) in the plasma and the protein expressions of ZO-1 and

occludin in the ileum. As shown in **Figures 2A–D**, HFD increased the plasma LPS levels and disrupted the intestinal barrier, whereas HT and HTF were able to reduce the plasma LPS concentration and prevent intestinal barrier damage. Thus, HT and HTF may reduce the release of LPS into the blood by promoting intestinal barrier integrity. Additionally, intestinal integrity can also affect inflammation levels. As expected, the protein expressions of TLR-4, TNF-α, IL-1β, IL-6, and p-JNK in the mouse livers increased significantly in the HFD group compared to the chow group (**Figures 2E–J**). We further discovered that the concentration of the liver's IL-1β and IL-6 declined significantly after HT and HTF intervention, the concentration of TNF-α have only a downward trend (**Figures 2G–I**). Simultaneously, the expression of IκB-α, significantly decreased compared to the chow group (**Figure 2K**). The expression of TLR-4, TNF-α, IL-1β, IL-6, and p-JNK were significantly decreased in the HT and HTF groups (**Figures 2E–J**). The expression of IκB-α was significantly decreased in the HT group, but did not change significantly in HTF group (**Figure 2K**). Previous studies have shown that the TLR4 signaling pathway promotes the production of pro-inflammatory cytokines through the activation of JNK and NF-κB. Increased phosphorylation of JNK and NF-κB ultimately triggers IR via phosphorylation of IRS-1 on serine-307 and deactivation of AKT.

## HT and HTF Decreased IR in HFD-Fed Mice

To evaluate insulin sensitivity, we performed oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT), tested fasting blood glucose and insulin, and calculated the index of HOMA-IR (**Figures 3A–G**). Specifically, after 8 weeks of a HFD, the OGTT area under the curve (OGGT-AUC) (**Figure 3A**), fasting blood glucose concentration (**Figure 3E**), fasting insulin concentration (**Figure 3F**), and HOMA-IR index (**Figure 3G**) were enhanced significantly in the HFD group compared to the chow group. In addition, the ITT-AUC (**Figure 3D**) tended to increase. The fasting blood glucose concentration, fasting insulin concentration, and HOMA-IR index were decreased significantly in HT group; and the OGTT-AUC, fasting insulin concentration, and HOMA-IR index were decreased significantly in HTF group. Although the other indicators also decreased with HT and HTF, no significant differences were found. These results indicate that HT supplementation and HTF alleviated IR induced by HFD. Furthermore, we measured the expression of key proteins related to IR (**Figure 3H**). As shown in **Figures 3I–J**, after 8 weeks of HFD, the expression of p-IRS (**Figure 3I**) was elevated significantly compared to the chow group, and the expression of p-AKT (**Figure 3J**) was significantly decreased. The expression of p-IRS and p-AKT (**Figures 3I–J**) significantly decreased in HT and HTF groups.

## HT and HTF Modulated Gut Microbiota Composition and Reversed HFD-Induced Gut Dysbiosis

To explore changes to the gut microbiota after HFD, HT, and HTF, we performed 16S rRNA gene analysis of fecal samples

FIGURE 2 | HT and HTF protected intestinal integrity, decreased concentration of plasma LPS and alleviated inflammation in HFD mice. Effects of HT and HTF treatment on plasma endotoxin using a Chromogenic End-point Tachypleus Amebocyte Lysate (A) The expression of Occludin, ZO-1 and GAPDH in ileum was assessed using western blot (B). Bar graphs represent normalized data of Occludin /GAPDH (C) and ZO-1 /GAPDH (D) n = 3. The expression of Occludin, TLR-4, TNF-α, IL-1β, IL-6, p-JNK, IκB-α and GAPDH in liver was assessed using western blot (E). Bar graphs represent normalized data of TLR-4/GAPDH (F), TNF-α/ GAPDH and the concentration of TNF-α in liver (G), IL-1β/GAPDH and the concentration of IL-1β in live (H), IL-6/GAPDH and the concentration of IL-6 in live (I), p-JNK/JNK (J), IκB-α/GAPDH (K) n = 3. Molecular weight markers were indicated as kilodaltons (kDa), Graph bars with statistically significant results (P ≤ 0.05) based on one-way ANOVA analysis followed by Tukey's honest significant difference post hoc tests. Data are shown as mean ± s.e.m ( ∗P ≤ 0.05, ∗∗P ≤ 0.01).

from the mice. High-throughput sequencing yielded 1574700∗ 2 original sequences from 24 samples (**Supplementary Table S1**), of which 1,090,437 were valid sequences (33,690 ± 4,147 per sample) (**Supplementary Table S2**). The coverage index was 1 ± 0.00001, indicating adequate species coverage during sequencing (**Supplementary Table S3**).

We found no significant differences in Firmicutes between the four groups (**Figure 4A**). However, the number of Bacteroides in the chow group was significantly higher than the HFDfed groups (**Figure 4B**). The Chow group had the lowest ratio of Firmicutes to Bacteroides (F/B) compared to the other three groups (**Figure 4C**). Neither the number of Bacteroides nor the ratio of F/B were altered after HFD, HT, and HTF (**Figures 4B–C**). Notably, taxonomic profiling showed that supplementation with HT and HTF reduced the numbers of Proteobacteria and Deferribacteres in HFD-fed mice (**Figure 4D**).

The Simpson Index was proposed by Edward Hugh Simpson in 1949 to quantify the biodiversity of a region and serves as an indicator of microbial diversity. Larger Simpson Index values indicate lower diversity in the community. The Simpson index of HFD group was significantly higher than that of the chow group and was decreased after HT and HTF treatment, albeit no statistical significance was observed in the HT group (**Figure 4E**).

All sequences were classified into 553 operational taxonomic units (OTUs) at 97% similarity after subsampling. The Venn diagrams in **Figure 4** showed that the OTU of the chow group was the highest, reaching 482, followed by 440 in the HTF group, 421 in the HFD group, and 412 in the HT group. 553 OTUs were found in all of the groups. In this study, there were 80 OTUs unique to the chow group, 3 OTUs unique to the HFD group, 2 OTUs unique to the HT group, and 5 OTUs unique to the HFT group (**Figure 4F**).

We used principal coordinates analysis (PCoA) plots (**Figure 4G**) and sample-level clustering analysis (**Figure 4H**) to reflect the similarities and differences in the composition of the microbial community. In the PCoA plots, the chow group is distributed on the left side, the HFD group is mostly distributed in the lower right corner, the HTF group is mostly distributed in the upper right corner, and the HT group is evenly distributed on the right side. The results showed a clear difference in the gut microbiota composition between the chow group and the other three groups, and the microbiota composition of HT and HTF groups were similar.

To further explore bacterial species changes in obese mice undergoing HT or HTF treatment, we performed a significant difference comparison between the groups. Since a large number of bacteria were found in this study, statistical differences at the species, genus and family levels were calculated. At the species level, we found that 2 species were altered by HT and/or HTF. Lactobacillus johnsonii was significantly increased after HT supplementation and HTF, while Anaerotruncus sp. G3 (2012) was significantly decreased after HTF supplementation (**Figure 5A**). At the genus level, four representative genera were altered by HT and/or HTF treatment. Lactobacillus significantly increased after HT and HTF, while Rikenella was significantly decreased after HT and HTF. Desulfovibrio and Ruminiclostridium were decreased after HT and HTF, but the decreases were not statistically significant (**Figure 5B**). At the family level, Ruminococcueae and Christensenellaceae were decreased after HT and HTF, but the decreases were not statistically significant (**Figure 5C**).

## DISCUSSION

Previous studies have shown that HT prevents inflammation, hyperglycemia, hyperlipidemia, IR, and obesity. Some studies have shown that HT supplementation is beneficial to HFDinduced obese mice, while other studies have shown that HT has no effect on obesity (Cao et al., 2014; Voigt et al., 2015; Wang et al., 2018). Specifically, Voigt et al. (2015) found that HT (20 mg/kg/day, 21 days) reduced weight gain in HFD mice, but there have statistical difference only on the 15th and 18th days. A previous study has showed (Jemai et al., 2015) that HT treatment (8 mg/kg, 16 mg/kg, 4 weeks) has no effect on the body weight of diabetic rats, but can improve plasma glucose, liver glycogen and total cholesterol levels. At the same time, high doses significantly improved plasma glucose and total cholesterol levels compared to low doses. In addition, Zheng et al. (2015) found that the neurons factor of the cerebral cortex were lost in db/db mice compared with the control mice. HT supplements (10 mg/kg, 50 mg/kg, 8 weeks) improved the neuronal survival of brain in db/db mice. Of note, high doses significantly improved neuronal survival compared to the low doses. The differences in these results may be due to a difference in HT concentration and intervention time. In the present study, HT gavage at a concentration of 50 mg/kg/day for 8 weeks was able to maintain the integrity of intestinal barrier, alter the composition of gut microbiota, reduce the release of endotoxin into the blood, and reduce inflammation, ultimately preventing HFD-induced obesity and IR.

Jumpertz et al. (2011) found that the gut microbiota is associated with energy metabolism (Bäckhed et al., 2004; Fleissner et al., 2010; Velagapudi et al., 2010). Our results demonstrated that mice fed regular chow consumed more energy than HFD-fed mice, but the body weight of the chow group was lower than that of the HFD mice, indicating that HFD induces body weight increase by regulating energy utilization. In addition, we observed that the body weight of the HTF mice was lower and the energy intake was higher than that of the HFD mice, suggesting that changes in the gut microbiota caused by HT may affect energy absorption and metabolism. Interestingly, the energy intake of the HT mice was lower than that of the HTF group, and more metabolic benefits were seen in the HT group. It is possible that the concentration used for the fecal transplantation was not enough, or that HT may benefit HFD mice through a mechanism which isn't dependent upon gut microbiota.

Previous studies have highlighted that HFDs reduce microbial diversity (Turnbaugh et al., 2008; Turnbaugh et al., 2009). In contrast, Kasai et al. (2015) found that the bacterial diversity in obese individuals was significantly higher than that in lean individuals in clinical trials. We observed that HT and HTF restored bacterial diversity which had been decreased by HFD. Our results suggest that bacterial diversity is inversely proportional to obesity. Moreover, one of the characteristics of the gut microbiota in obese individuals is a high ratio of F/B (Ley et al., 2005; Ley et al., 2006; Turnbaugh et al., 2006). We found that HFD mice had a significantly increased F/B, which was not altered by HT or HTF intervention. This indicates that HT may be beneficial for metabolism but does not regulate F/B.