Agave juice improves survival and proteolytic activity of Lactobacillus rhamnosus GG during ripening of semi-ripened mexican cheese

During the last decade, the importance of foods added with probiotics has been demonstrated with benefits that range from the activation of the immune system to mechanisms against diarrheas associated with antibiotics (Orsi & Zambrini, 2017). A probiotic is defined as a living microorganism that actively improves the health of consumers, balancing the intestinal microflora when they are consumed viable and in sufficient concentrations (from 106 to 109 colony forming units (CFU / mL) (Vinderola et al., 2019). Recently, new terminology to cover all healthful impacts of live probiotic cells and their derivatives in all possible forms, has been proposed: ‘true probiotic’, ‘pseudoprobiotic’, and ‘ghost probiotic’ (Zendeboodi et al., 2020).


Introduction
During the last decade, the importance of foods added with probiotics has been demonstrated with benefits that range from the activation of the immune system to mechanisms against diarrheas associated with antibiotics (Orsi & Zambrini, 2017). A probiotic is defined as a living microorganism that actively improves the health of consumers, balancing the intestinal microflora when they are consumed viable and in sufficient concentrations (from 10 6 to 10 9 colony forming units (CFU / mL) (Vinderola et al., 2019). Recently, new terminology to cover all healthful impacts of live probiotic cells and their derivatives in all possible forms, has been proposed: 'true probiotic' , 'pseudoprobiotic' , and 'ghost probiotic' (Zendeboodi et al., 2020).
In many cases, oligosaccharides are added to probiotic foods that promote the development and survival of probiotic bacteria (Costa et al., 2019;Jaimez-Ordaz et al., 2018). These oligosaccharides are called prebiotic, which are non-digestible carbohydrates that when are consumed, selectively stimulates the growth of a limited number of bacteria in the colon promoting the survival of probiotic bacteria (Quigley, 2019).
Foods that contain both, probiotics and prebiotics have been defined as symbiotic (Guarner et. al., 2012), this term should be reserved exclusively to products in which symbiosis has been scientifically proven (Sanders et al., 2019) and could be considered as nutraceutical (Sarfraz et al., 2019.
Lactic acid bacteria are among the starter cultures used in cheese processing (for both fresh and ripened cheese). These bacteria may have probiotic characteristics, or antibiotic properties inhibiting the growing of pathogenic strains (Prezzi et al., 2020) or even biological properties such as reducing postprandial glycemia (Grom et al., 2020). However, in the particular case of the use of probiotic lactic bacteria, many of these do not survive due to the stress generated by the presence of salts, storage temperatures, among other factors (Chen et al., 2017). Therefore, to make sure that the amount of probiotic lactic acid bacteria is adequate at the time of its consumption, cheeses have been inoculated with high concentrations of microorganisms (between 10 9 to 10 12 CFU / mL) modifying the manufacturing processes to favoring their survival (Farnworth, 2008, Chung & Kamal-Eldin, 2020. One of the main factors that affect the viability of probiotic lactic acid bacteria is the presence of oxygen, this because they are mostly facultative anaerobic bacteria. That is why, due to the low mobility of oxygen in cheeses, these matrices favor the survival of probiotic lactic acid bacteria, and prebiotics would Agave juice improves survival and proteolytic activity of Lactobacillus rhamnosus GG during ripening of semi-ripened mexican cheese stimulate its selective growth (González-Olivares et al., 2014). That is why; the aim of this work was to determine the effect produced by agave juice on the growth and survival of a probiotic, during ripening of a semi-ripened Mexican cheese.

Agave juice analysis
Agave juice was obtained from the scraping of three agaves (Agave salmiana var. "Ayoteco") from Epazoyucan, Hidalgo (Mexico). It was pasteurized in autoclave at 90 °C for 15 minutes. The carbohydrate concentration was determined according to Jaimez-Ordaz et al. (2018) methodology by High Performance Liquid Chromatography (HPLC) (Lab Alliance, Tokyo, Japan). A 7.8 x 300 mm RNO-oligosaccharide column (Phenomenex, Torrance CA) and a Light Scattering detector (Polymer Laboratories, Amherst, MA). The elution medium was pre-degassed water (at 75 °C) at a flow rate of 0.3 mL/min. The run was carried out at a constant temperature of 75 °C and the detector was maintained at a misting temperature of 110 °C.

Mexican semi-ripened cheese processing
Lactobacillus rhamnosus GG was used as starter microorganism to produce the cheese. It was obtained from Food Biotechnology Laboratory collection of Metropolitan Autonomus University (Iztapalapa campus). The cheese was prepared according to González-Olivares et al. (2014) with some modifications. Twenty-four cheeses of 250 g were made in the School of Agricultural Sciences (ICAp) of the Universidad Autónoma del Estado de Hidalgo from pasteurized and standardized cow's milk (3.6% of fat content). Twelve cheeses were elaborated with probiotic and agave juice and other twelve without agave juice (control experiment). Milk was heated to a temperature of 36 °C to add 1% of probiotic (Lb. rhamnosus GG) and 2% of agave juice. Pre-mature step was performed at 37 °C for a period of 40 minutes until reaching 18.5 °D. It was added 1 mL of rennet (CUAMEX) was added. It was curdled for 40 minutes and the curd was cut. It was stirred without heat for 40 minutes and the whey was removed. Curd was divided in molds in a Retamoso-type press (5 kg / kg cheese) during 24 hours. Cheese was vacuum packed (Mukltivac C-100) and it was stored at 14 °C and 80% of relative humidity (RH). It was sampled every 7 days during five weeks in aseptic conditions and all experiments were realized in duplicate with independent units.

Viability determination
Viability of Lactobacillus was performed according to González-Olivares et al. (2014). Using plat-count technique. After samples were collected in sterile bags, 10 g of cheese were dissolved in 90 mL of sterile peptone water (1%). Samples were mixed in a Stomacher at 260 rpm during 5 min, obtaining a homogeneous suspension. Then 1 mL of this suspension was diluted in peptone water (1%) previously sterilized, considering a final dilution of 1x10 6 and 100 µL of each dilution were placed in Petri dishes with MRS agar (Difco) adjusted to pH 5.20 with 0.1 N acetic acid, so that only Lb. rhamnosus GG grew. Plates were incubated at 37 °C, under anaerobic conditions for 72 h and the colony forming colonies were reported.

Proteolytic profile analysis
Two different studies of proteolytic profile were carried out to observe the metabolic activity of Lb. rhamnosus GG during cheese ripening following techniques applied in cheese by González-Olivares et al. (2014). The first study was the determination of free amino groups in order to know the concentration of the peptides produced by the TNBS technique. The second study consisted in the separation of peptides by molecular weight by means of polyacrylamide gel electrophoresis.

Free amino groups determination
Free amino groups were determined by the 2, 4, 6-trinitrobenzenesulfonic acid method (TNBS). The concentration was determined according to a glycine concentration curve (0.05 to 0.25 mg/mL).

Peptide separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
Denaturing SDS-PAGE at 15% (w/v) was carried out in order to separate low molecular weight peptides. A broad range (Bio-Rad, USA) molecular weight standard was used. Gels were dyed with Comassie G-250 (Bio-Rad, USA). The molecular weight was estimated and the electrophoretic profiles were analyzed using GelDoc software by BioRad.

Sensory analysis
To find out the effect of the prebiotic (agave juice) addition on cheese flavor and texture, a discrimination test (triangle test) was carried out using a panel of thirty semi-trained panelists (Meilgaard et al., 1999). The samples used for sensory analysis were collected at the end of the ripening process. Two samples (control cheese and cheese prepared with agave juice) were presented to each participant, anonymously and coded with a 3-digit random number. Additionally, an affective test (preference test) was performed in order to determine the influence of such addition on consumers' opinion (Meilgaard et al.,1999). A total of sixty (34 women and 26 men) regular cheese consumers between 30 and 60 years old participated in the preference test. Two samples of cheese (one elaborated with probiotic and agave juice and the other, a commercial semi-ripened Mexican cheese) were presented to the participants. The test was developed in a supermarket near to the dairy section. Participants were selected considering their frequency of consumption of semi and ripened cheeses.
The samples for both sensory tests were served at 5 °C, in cubes of approximately 1 cm 3 . Unsalted crackers and water were available for mouth rinsing between samples. Results of sensory tests were analyzed at a significance level of p ≤ 0.01).

Statistical analysis
All experiments, except for the sensory analysis, were performed by triplicate and the experimental data were subjected to an analysis of variance (ANOVA), the means comparison was done by the Tuckey's method with a significance level of 0.05. The program used for the analysis was NCSS-2007 (version: 07-1-15).

Agave juice analysis
Carbohydrates present in agave juice were determined and the identification of its compounds was performed by comparing the retention times obtained with the ones of carbohydrates standards. Peaks corresponding to polysaccharides of more than two units were observed at minute 14, which presented the lowest concentration. At 18 minutes the presence of sucrose was observed, this peak represented the highest concentration of all separated carbohydrates. The total carbohydrate concentration was 7.2% (w/v). Fructose and glucose reaching concentrations of 20.27% and 20.10% respectively, while di-and poly-saccharides concentration was of 47.45% for sucrose and 12.17% for oligosaccharides. Moreno-Vilet et al. (2017) mentioned that sucrose is the most abundant carbohydrate in agave juice, and glucose and fructose are found in smaller amounts. Additionally, it is known that agave juice is rich in carbohydrates such as inulin, sucrose and fructose in addition to containing small amounts of amino acids and vitamins . On the other hand, agave juice contains a considerable amount of polysaccharides of more than two units, so it can be considered as a prebiotic (Rodríguez-Huezo et al., 2007).

Viability
The probiotic concentration in control cheeses decreased from 9.56 log CFU/g to 8.00 logs, which represented a difference of 1.5 logarithm cycle. An increase of probiotic amount (two logarithmic cycles) was observed during the first week of ripping in the enriched cheese, maintaining this concentration up to the end of the study (Table 1). In both experiments, the final concentration is higher than that recommended to obtain a beneficial effect (7-8 log CFU/g) due to its consumption (Karimi et al., 2011). But, in several studies it has been observed that the decrease in the viability of probiotics depends on the strain used and can vary up to 3 cycles (Dinkçi et al., 2019;Sadaghdar et al., 2012).
The death of probiotic in dairy products, like cheeses, is related to the carbon source (oligosaccharides) that stimulate their growth (Farnworth, 2008), which are important promoters of bifidobacteria growth and other kind of probiotics, acting as prebiotic (Kuerman et al., 2020).
Adding prebiotics or potential prebiotic ingredients constitutes a strategy to increase the development and survival of probiotic bacteria in products to which they are incorporated, especially when the development of this kind of microorganisms is difficult (Lourens-Hattingh & Viljoen, 2001, Siang et al., 2019, Lucatto et al., 2020, Pivetta et al., 2020. On the other hand, non-prebiotic ingredients such as fruit pulp, have been proven in order to increase the probiotic bacteria viability in dairy products (Reeta et al., 2018).

Free amino groups determination
The results of free amino groups determination are observed in Figure 1. In the initial time there was a generation of free amino groups in both analyzed samples. Different amounts of free amino groups were observed in day zero; this may be due to the addition of prebiotics and probiotics before cheese coagulation (pre-ripening process) moment in which proteolysis starts. An increase of amine groups was observed from day 0 to week 2 in both samples, maintaining the concentration until week 5 of storage, getting higher concentration in the added cheese (8.63 mg/mL) than in the not added (7 mg/mL).
Peptide fractions are transformed to smaller peptides or even free amino acids by the proteolytic system of the inoculum (Rodríguez-Serrano et al., 2018). In fact, some strains of lactobacilli used as starters modify the free amino acid profile (Di Cagno et al., 2006). This protein degradation sustains the growth of bacteria, since the amount of free amino acids found mainly in milk is insufficient for the optimal growth of the microorganism (Rodríguez-Serrano et al., 2018). Also, due to agave juice composition (sucrose and fructooligosaccharides concentration) probiotic viability was promoted because there is proteolytic enzymes generation (proteinases and peptidases) involved in peptidic fractions release (Lim et al., 2019). This can explain the higher concentration of amine groups in added cheese.

Peptide separation by SDS-PAGE
There was an accumulation of low molecular weight peptides in both types of cheese, which were not observed at the beginning of ripening. Some high molecular weight peptides could be degraded to small peptides and amino acids by the action of proteases and peptidases (Pérez-Escalante et al., 2018). These kinds of peptides were presented from the first week of ripening and were preserved in subsequent weeks, especially in the cheese with agave juice (Figure 2 and 3). The presence of low molecular weight peptides involves the fractionation of caseins. Similarly, peptides between 8.8 and 9.5 kDa could be observed throughout the ripening process, but their concentration decreased during this time. These results showed that the accumulation of low molecular weight peptides is related to the ripening time and At the end of ripening it was observed the accumulation of low molecular weight peptides in both cheeses ( Figure 4).
However, clearly the most observable concentration was that in cheese with agave juice. The production of this kind of peptides is related to both, probiotic survival and agave juice presence, because the proteolytic activity is favored by this potential prebiotic.
The number of microorganisms that survived during the ripening of cheese maintains a high protease activity (Mane & McSweeney, 2020). Viability results confirmed the presence of probiotic that are related to the metabolic activity. Results of this research are relevant because accumulation of this kind of peptides is related to biological activity, such as antimicrobial and antihypertensive (Moreno-Montoro et al., 2017).
At the same time, accumulation of peptides in dairy products enriched with agave juice has been demonstrated (Martínez-Gutié rrez et al., 2017;Jaimez-Ordaz et al., 2018). However, any study is related with ripening of cheeses and its effect in the addition of agave juice and its relation with prebiotic effect over the proteolytic capacity of probiotics.

Triangle test
The results showed that the addition of agave juice as prebiotic in the manufacturing of semi-ripened Mexican cheese did not produce any noticeable sensory change. Only 11 panelists out of 30 were able to identify the cheese sample that was different. Thus, according to the results, there were no significant (p> 0.01) differences between the control and the experimental cheese.

Preference Test
Twenty-eight out of the sixty consumers who evaluated the cheese samples preferred the cheese elaborated with prebiotic. Figure 1. Amine groups concentration during cheese ripening with and without agave juice.   This result indicated that there was no significant preference (p> 0.01) between the experimental and the commercial cheese among the participants. Indeed, it can be assumed that using agave juice, as ingredient for the elaboration of semi-ripened cheese did not had any negative effect. From the point of view of the group of regular semi-ripened cheese consumers, the sensory properties of both cheeses were similar. It is remarkable due to the impact of sensory characteristics such as the taste and aroma of foods on consumer acceptance (Simmering & Blaut, 2001;Stanton et al., 2001). The result observed is in agreement with those reported by Azambuja et al. (2013) and Zamora-Vega et al. (2013) in preference tests carried out for fresh and ripened cheeses, respectively, with the addition of probiotic and prebiotic ingredients. Those authors observed no effect on the taste, color and odor of the experimental cheeses. Nevertheless, it has also been reported positive changes in sensory properties derived by the addition of probiotics (Akarca, 2020) as well as the use of fruit juices to improve the acceptability of dairy products containing probiotics (Ryan et al., 2020).
The use of agave juice as a prebiotic ingredient allowed the elaboration of a semi-ripened Mexican cheese, improving survival of the probiotic used as starter and activating its proteolytic system without negatively affecting the sensory characteristics of the final product. However, in the same way of Ryan et al. (2020) recommendation, sensory evaluations using trained panelists would be highly beneficial in the commercialization process of probiotic dairy products.

Conclusion
Agave juice is a promising raw material to be used in the manufacturing of semi-ripened Mexican cheese. This carbon source promotes the survival of Lactobacillus rhamnosus GG having a direct influence on their proteolytic activity and generating a higher concentration of free amino groups. Moreover, this probiotic releases a higher amount of low molecular weight peptides throughout the ripening process in the presence of agave juice, which allowed the elaboration of a product similar to a commercial one.
Our results highlight that dairy matrices as semi-ripened cheese, represent an opportunity in food science and technology, especially to develop foods with nutritional benefits for the health of consumers by adding pre-and probiotics. Nevertheless, since there are few reports similar to our experiments, further research would be necessary to analyze the effect of the addition of agave juice in semi-ripened cheese using a bigger group of consumers.