Microbial characterization and diversity of artisanal Ranchero cheese with emphasis in Lactococcus strains

On Mexico, the dairy industry is one of the most important agricultural industries; as such, there is a wide variety of genuine artisan cheeses that represent different regions and their environment of origin. Chihuahua is a Mexican state with one of the highest production of milk and cheeses, where artisanal cheeses are made using traditional production techniques (Servicio de Onformación Agroalimentaria y Pesquera, 2017a). On 2016, the cheese industry produced 342 thousand tons with a market value of 15 thousand 823 million pesos, the main varieties of cheese produced wereI: Fresh (17.0%), Panela (14.0%) and Double cream (13.0%) (Servicio de Onformación Agroalimentaria y Pesquera, 2017b). Because Mexico has a deficit in milk production, it is not considered a cheese exporting country.


Introduction
On Mexico, the dairy industry is one of the most important agricultural industries; as such, there is a wide variety of genuine artisan cheeses that represent different regions and their environment of origin.Chihuahua is a Mexican state with one of the highest production of milk and cheeses, where artisanal cheeses are made using traditional production techniques (Servicio de Onformación Agroalimentaria y Pesquera, 2017a).On 2016, the cheese industry produced 342 thousand tons with a market value of 15 thousand 823 million pesos, the main varieties of cheese produced wereI: Fresh (17.0%),Panela (14.0%) and Double cream (13.0%) (Servicio de Onformación Agroalimentaria y Pesquera, 2017b).Because Mexico has a deficit in milk production, it is not considered a cheese exporting country.
The artisanal Ranchero cheese made in Chihuahua is a semipressed dairy product elaborated from raw cow milk, and it is considered as a fresh cheese of bright, milky, and mild flavor, which is consumed within no more than five days after production (Villegas & Cervantes, 2011).
On cheeses made from raw milk, the contribution of autochthonous fermenting microbiota is prominent (Dagdemir & Dzdemir, 2008;Renye et al., 2008;Tunick et al., 2007); therefore, only specific bacterial combinations of strains isolated are used for cheese making.Artisanal Ranchero cheese is made with raw milk and without the addition of starter cultures; therefore, its organoleptic characteristics rely on the lactic microbiota presents in raw milk, which is dependent to the geographical area of production (Beresford et al., 2001).Also, the presence of yeasts and molds develop and enhance the texture and flavor of the cheese (Lavoie et al., 2012).
Currently, one of the challenges presented by the food industry in Mexico is to provide sufficient safe food for its population.On this context, and until now, artisan cheeses have been given little or no importance, so there is little research related to the quality and characterization of the same as well as a null regulatory regulation in practice.On general, the elaboration of cheese with raw milk is not permitted by Mexican health regulations, which dictate that raw milk can be used only if the cheese is consumed 100 days after its production (Norma Dficial Mexicana, 1994).Nevertheless, artisanal Ranchero cheese is still produced with raw milk because this regulation has not been enforced.An alternative to solve this issue is to develop a specific indigenous starter for use in the manufacture of artisanal Ranchero cheese.Therefore, it is important to know the microbial communities inherent in these cheeses.This knowledge can prove essential in dealing with product quality features, authenticity assurance, and traceability along the entire production chain.
Although researchers have studied the microbiota of Mexican cheese, mainly fresh cheese, there is still a need to study genuine Mexican handmade cheeses, such as Ranchero cheese because, to date, there is a lack of knowledge about many features of its genuineness and typicity, such as microbiological quality and biodiversity of the lactic microbiota.Hence, the aim of this work was to identify and characterize Lactococcus isolates present in artisanal Ranchero cheese elaborate with raw milk.This could be taken into consideration to select proper strains for a suitable starter culture for the manufacturing of this cheese.

Materials and methods
Ten traditional Ranchero cheeses made with raw milk were purchased from different local producers located in different geographical areas in the state of Chihuahua, Mexico (n = 10).Ot is known that the microbiological flora of milk changes when the type of food and climate changes.Chihuahua is the largest state of Mexico, it has mountains, plains and deserts, where temperatures and milk production systems vary considerably.Because of this, the ten samples of cheese were purchased from producers located in ten different geographic areas of the state.The cheeses were transported in sterile bags in a container at 4 °C, and they were examined before 24 h of chilled storage (4 °C).
PCR amplification and DNA sequence analysis were performed on randomly chosen isolates from the most significant genera.Genomic DNA was isolated from 1 mL of the overnight cultures using the DNeasy  Blood & Tissue Kit (Qiagen, Hilden, Germany), according to the instructions of manufacturers.The obtained genomic DNA was used as template for PCR with the following oligonucleotidesI: forward primer G17I: 5'GTGAAGTCGTAACAAGG'3 (Pangallo et al., 2008), and reverse primer GplusRI: 5'GTGAAGTCGTAACAAGG'3 (Chebeňová-Turcovská et al., 2011) for amplification of 16S rRNA genes.The PCR mixture contained 12.5 µL Go Taq  Green Master Mix (Promega Corporation, USA), 1 mM each primer, 250 ng template DNA and HPLC-quality water up to the volume of 25µL.The PCR conditions consisted in an initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min and polymerization at 72 °C for 3 min, followed by a final polymerization at 72 °C for 10 min.The PCR products were purified with the Wizard  SV Gel and PCR Clean-Up System (Promega Corporation, USA), according to the manufacturer's specifications, and sequenced.Sequencing was performed in the Onstitute of Biotechnology, UNAM in Cuernavaca, Morelos, Mexico.Sequences were analyzed using Sequencher 5.3 (Gene Codes Corp., Ann Arbor, MO) and compared with sequences available in GeneBank using the BLASTn algorithm (Altschul et al., 1997).
A multiple alignment of artisanal cheese clone sequences, identified as 16S rDNA, and 16S sequences obtained from GenBank database was performed using the PHYLOP (Felsenstein, 1989) and Clustal W 1.6 program (Thompson et al., 1994).On addition to the strains isolated in this study, 16S rRNA gene sequences from five closely related strains were included in the analysisI: Lactococcus chunganensis (HM2419.1);Lactococcus raffinolactis (HM241918.1);Lactococcus lactis subsp.lactis (KC768838.1);and Lactococcus garvieae (HM241915.1).A phylogenetic tree was generated using the two parameters of Kimura for genetic distances (Kimura, 1980) and the neighbor-joining method (Saitou & Nei, 1987) with 1000 replications.To avoid any potential bias introduced as a result of sequence addition, the taxon addition order was randomized in the neighbor joining analysis.
Microorganisms genera were analyzed using basic statisticsI: y = μ + ε, where μ = general average, and ε = random error.The significance level used for all statistical analyses was 5%.The analyzes were performed using the statistical package SAS version 9. 1.3 (Statistical Analysis System, 2006).

Results
The microbial logarithmic colony forming units (log 10 CFU) mean counts for lactic and non lactic microorganisms of artisanal ranchero cheese, are summarized in Tables 1 and 2.

Discussion
The average count of aerobic mesophilic bacteria (7.90 ± 0.69 log 10 CFU/g) was similar to that reported by Renye et al. (2008) and Caro et al. (2013) in fresh cheese (8.02 log CFU/g) and Daxaca cheese (7.63 log CFU/g), respectively, highlighting that both cheeses were made with raw milk.Mexican legislations do not have a regulation for this parameter (Norma Dficial Mexicana, 1994).
Count of Staphylococcus spp.was, on average, 7.32 ± 0.73 log 10 CFU/g.Staphylococcus spp. is considered as microbiological safety only when it exceeds the 4 log 10 CFU/g or when it produces enterotoxins (Yoon et al., 2016).Moreover, Mexican official regulations stipulate a maximum permissible level of 1000 UFC/g (3.00 log10 CFU/g) in the specific case of S. aureus in fresh cheese.
Coliform counts were 5.90 ± 1.06 log 10 CFU/g on average.Similar values were reported in fresh cheese elaborated with raw milk.Renye et al. (2008) observed counts of 5.90 log CFU/g; while Torres-Llanez et al. ( 2006) registered elevated counts not only of total coliforms (around 4.25 log CFU/g) but also fecal coliforms (around 4.5 log CFU/g).The Mexican official regulations established a maximum permissible level for total coliform bacteria of 2.00 log 10 CFU/g.High numbers of coliforms could be associated with low hygiene and production process of the artisanal cheese (Solís et al., 2009).
Molds and yeast had an average count of 4.12 log 10 CFU/g.Higher values have been found in fresh cheeses elaborated from raw milk (Renye et al., 2008;Torres-Llanez et al., 2006).
Coliforms, molds, and yeast can deteriorate sensorial characteristics and even became a real public health risk.Ot has been stablished that counts of coliforms, molds, and yeast in artisanal cheeses decrease within the maturation process (Alejo-Martínez et al., 2015).Nonetheless, the artisanal Ranchero cheese is commonly consumed fresh, within five days after processing, representing a risk for the health of the consumers.
As is shown in Table 2, Lactococci was the most abundant LAB isolated from artisanal ranchero cheese (8.14 log 10 CFU/g), followed by mesophilic Streptococci (7.79 log 10 CFU/g), thermophilic Streptococci (7.65 log 10 CFU/g), and Lactobacilli microorganisms (7.65 log 10 CFU/g), which could indicate that these microorganisms have important roles in odor and flavor characteristics of artisanal and fresh cheeses.
Lactococcus are an important part of the nonstarter microorganisms because they contribute to cheese flavor and texture (Tabak et al., 2012).Lactococcus from dairy products can be generally considered safe because they are consumed daily by humans, and only low incidence of human infections has been reported (Casalta & Montel, 2008).Lactococcus are also responsible for the initial acidification during the cheese-making process, which determines curd formation and whey expulsion and prevents the growth of undesirable bacteria due of the resulting low pH concomitant with the production of lactic acid (Karakuş & Alperden, 1995;Molloy et al., 2011;Renye et al., 2008).Therefore, in the present research, it has been proposed that the isolated Lactococcus could be used as part of starter culture for artisanal Ranchero cheese elaboration, preserving the sensorial characteristics and, according to official regulations, offer an innocuous product to consumers.On this study, 19 randomly chosen presumptive Lactococcus isolates were further analyzed.Thirteen (68.42%) isolates were identified as L. lactis subsp.lactis and six (31.57%) as L. garvieae.
The majority of the isolates evaluated were recognized as Lactococcus lactis subsp.Lactis (68.42%).This LAB is the main component of dairy starter cultures used for the production of diverse fermented products (Cavanagh et al., 2015).However, it is a limited component of the starter cultures to produce cheddar cheese (Wouters et al., 2002).Ot also may serve as one of the principal acidifying agents for the production of fresh cheese because L. lactis subsp.lactis has been previously identified as part of its natural microbiota (Renye et al., 2008;Torres-Llanez et al., 2006).
Meanwhile, six (31.57%) isolates were identified as garvieae.Ot has been isolated from diverse food sources such as fishes, crustaceans, raw milk, and raw milk cheeses (Foschino et al., 2008).At the same time, it has been identified as a fish pathogen and as the etiological agent of mastitis in cows and buffalos (Teixeira et al., 1996).This last may be the reason for its presence in cheeses made with raw milk and the fact that being recognized as a component of the native microflora of dairy products elaborated with raw milk (Fortina et al., 2007;Flórez et al., 2012), such as Ranchero cheese (Renye et al., 2008).Further, it has been linked to human infections (Foschino et al., 2008); however, no association between raw milk cheeses and human infections has been established (Flórez et al., 2012;Foschino et al., 2008).
Even though scarce information is available on phenotypic and biochemical properties of L. garvieae isolated from dairy sources, it has been found that strains from dairy sources are lactose fermenters (Fortina et al., 2007;Teixeira et al., 1996), showing a slow rate of acidificacion, but still comparable with that presented by some Lactococcus isolates (Wouters et al., 2002).And have low proteolitic activity (Fortina et al., 2007), which make their use as starter or adjunct cultures feasible; however, technological characterization should be conducted on the isolates found in this study because some L. garvieae strains are found tetracicline resistance (Fernández et al., 2010).

Conclusions
Results obtained in the present work showed counts of total coliforms, molds, and yeasts that exceeded the maximum levels allowed by Mexican official regulations; in addition, high counts of aerobic mesophilic bacteria, psychotrophs, and Staphylococcus spp.were observed.These results shows hygienic deficiencies in the production process, which translates into a potential risk factor for consumers' health.Likewise, no differences were found between counts of Lactococcus, Streptococcus mesophilus, Streptococcus thermophilus, and Lactobacillus.Both the GenBank database and the phylogenetic analysis achieved the identification of two Lactococcus speciesI: Lactococcus lactis subsp.lactis and Lactococcus garvieae.

Figure 1 .
Figure 1.Phylogenetic tree of partial 16S rDNA from strains isolated from artisanal Ranchero cheese.The sequences were aligned by the Clustal W program.The distance matrix and phylogenetic tree were calculated with the neighbour-joining algorithms and the method of two parameters of Kimura.Artisanal ranchero cheese 16Sr rDNA sequences are represented by the strain number.The sequence of Streptococcus parasanguinis served as outgroup sequence.The number of 1000 bootstrap resamplings that support each topological element in the neighbour-joining analysis is indicated.

Table 1 .
Microbial logarithmic counts (mean ± SD) of non lactic acid bacteria of artisanal Ranchero cheese.

Table 2 .
Microbial logarithmic counts (mean ± SD) of lactic acid bacteria of artisanal Ranchero cheese.

Table 3 .
Bacterial species identified in artisanal Ranchero cheese.
*Percent Odentity and Accession Number of closest related species in GeneBank.