Area of study

The research was developed in the Centro Multidisciplinarios en Estudios de Biotecnología (CMEB)-UMSNH of the Facultad de Medicina Veterinaria y Zootecnia-UMSNH, located at kilometer 9.5 on the Morelia-Zinapecuaro road. 6.5 L of raw milk were supplied from a small-scale dairy herd in Cuitzillo village located at the Tarimbaro municipality, Michoacán, México. The climate in this region is warm and humid in summer with mild winter. Annual precipitation is 609.0 mm and temperatures range from 2.5 to 25.1 ^oC (^{INEGI 2005}).

Mucilage extraction

Mucilage was extracted from 4.5 kg of each species of Opuntia ficus-indica and O. atropes, with a yield of 6.2 and 12.7 g of mucilage kg^-1, respectively. The dried mucilage was obtained according to the methodology described by ^{Rodríguez-González et al. (2014, 286, 287)}. Briefly, fresh cladodes were mixed in a blender for 1 min with water in a ratio of 1:8 (w/v). Then, the suspension was maintained at 83 °C for 2 h and centrifuged at 3,500 rpm for 15 min. The supernatant was added with ethanol 96% in a ratio of 1:4 (v:v). Mucilage mass was separated by centrifugation at 3,500 rpm for 15 min. Mucilage mass was dried and stored at 4°C until use. For the liquid mucilage, another methodology was proposed. Fresh cladodes were disinfected with a 2% solution of sodium hypochlorite. Cladodes were washed with distilled water. After that, cladodes were peeled, cut into slices, mixed in a blender and pressed in cheesecloth, until obtaining the mucilage in liquid form. Mucilage was stored until use in test tubes previously sterilized at 4°C.

Raw milk added with mucilage

The raw milk samples were collected from a pool of ten mature Holstein grade cows permanently confined feed with a strategy of cut and carried fresh alfalfa and supplemented with maize grain plus stover or commercial concentrated. According with the data available; the cows are mechanically milked twice per day averaging 12.5 ± 3.1 L and 3.7 ± 1.8 lactations. During the milking, the only hygienic practice utilized by the farmer was to wash the udder with warm water, the general hygienic conditions of the herd barn was considered poor.

Raw milk was collected every morning from the collecting container; previous homogenization milk was poured into sterilized flasks of 300 mL each and 16 batches of 100 mL of raw milk were obtained at the end. This procedure was repeated for 5 days, obtaining 80 samples with three replicates/samples (n=240). In each flask dried mucilage or liquid mucilage was added in the following concentrations: 0.0 (control), 0.5, 1.0 and 2.0% (w/v) with respect to the 100 mL of raw milk. The mucilage was maintained in contact with raw milk for 2 h.

Microbial enumeration

Each sample of raw milk added with dried mucilage or liquid mucilage was analyzed using the Official Norms of Mexico (^{SSA 1995a, 1-5}; ^{SSA 1995b, 1-13}; ^{SSA 1995c, 203-210}; ^{SSA 1995d, 1-7}). One mL from each sample was transferred to a sterile tube containing 9 mL of sterile 0.85% NaCl solution. Total coliforms were determined by the pour-plate method. Dilutions up to 10^-6 were plated on standard plate count agar and violet red bile agar and incubated at 37^oC for 24 h. For mesophilic aerobic bacteria, samples were pour-plated in plate count agar and incubated at 37^oC for 48 h. and the bacteria counting colonies were done by using the Quebec colony digital counter.

Statistical analysis

A complete block experimental design was used in a 2x2x4 factorial arrangement with three replicates. That is, mucilage of two species of Opuntia was evaluated (Ficus-indica and atropes) in two forms (liquid and dried) and added to raw milk at four levels: 0.0, 0.5, 1.0 and 2.0 % based on 100 mL of raw milk. Microbial counts were changed to Log10 values for statistical analysis, and data were analyzed by using a fixed effects model (^{Littell et al., 2006, 733-735}). Differences between groups were obtained by the method of least square means (^{Littell et al., 2002, 191-194}). The model used was as follows:

Where:

Y_ijkl = log₁₀ CFU mL^-1 of the Mesophilic aerobic or Total coliforms bacteria

μ = General mean

S_i = Fixed effect of the i_ésimo Specie with _i= ficus-indica and atropes

(S*M)_ij = Fixed effect of the i_ésimo Specie with interaction j_esimo form of Mucilage with _i= Dred and Liquid

(S*C)_ik = Fixed effect of the i_ésimo Specie with interaction k_esimo mucilage Concentration with _i= 0, 0.5, 1 and 2

(S*M*C)_ijk = Fixed effect of the i_ésimo Specie with interaction j_esimo form of Mucilage and k_esimo mucilage Concentration

ε_ijkl = Error (NID ~ 0, δ²)

Effect on the growth of mesophilic aerobic bacteria

Significant effects was observed in microbiological loads at different form of mucilage and levels of concentration of mucilage in both species of Opuntia (Table 1), however, a slight decreasing trend can be observed on microbiological count when mucilage concentration increases (Figure 1). Effect of interactions on the growth of mesophilic aerobic was found: in the interactions Concentration of mucilage*Species of Opuntia (P=0.0006), Form of Mucilage*Species of Opuntia (P=0.0378) and Concentration of mucilage*Species of Opuntia*Form of Mucilage (P=0.020). In general, the addition of mucilage (Figure 1), independently of the physical form added to the raw milk (dried or liquid) (Figure 2), from different species of Opuntia decreased significantly (P < 0.05) the growth of mesophilic aerobic bacteria. The effect was more pronounced when concentration of mucilage O. ficus indica was 1 and 2% (2.8 log₁₀ CFU mL^-1) and 2% in O. atropes (2.8 log₁₀ CFU mL^-1) (Figure 1). Although, the counts of mesophilic aerobic bacteria were not different significantly (P > 0.05), log₁₀ 2.9 and log₁₀ 3.1 CFU mL^-1, when mucilage from O. atropes was added to raw milk at concentrations of 0.5% and 1%, respectively; and, counts were different (P < 0.05) from the control (0% mucilage: 3.6 log₁₀ CFU mL^-1). Low growth of aerobic mesophilic bacteria was observed when raw milk liquid mucilage O. ficus-indica in 1% and 2% ( 2.7 log₁₀ and 2.6 log₁₀ CFU mL^-1, respectively ) was added and 2% of dry mucilage O. atropes ( 2.7 log₁₀ CFU mL^-1). Representing a reduction of 3.4x10^-3 FCU mL^-1 aerobic mesophilic bacteria, with respect to the control (3.6 Log₁₀ = 3.9x10^-3 FCU mL^-1) when raw milk was added 2% mucilage liquid (2.7 Log₁₀ = 5.0x10² FCU mL^-1) or dried of ficus-indica or atropes respectively (Figure 3).

Table 1. Analysis of variance for aerobic mesophilic (Log₁₀ CFU/mL) in raw milk according to the treatment 

Literals a, b, c = differences between treatments (P<0.05)

Figure 1. Least square means for mesophilic aerobic bacterias (log₁₀ CFU/mL) in raw milk post addition of mucilage according to the concentration interaction*species of Opuntia 

The decreased growth of mesophilic bacteria by adding of mucilage was also reported by ^{Ortiz-Rodriguez et al. (2013, 3677-3679)} when mucilage from O. ficus-indica was added to the raw milk (log₁₀ 6.1 CFU mL^-1) as compared to the control with no adding mucilage (log₁₀ 7.3 CFU mL^-1). Evidence exists that species of Opuntia has antibacterial properties, attributed mainly to the presence of pectic-derived oligosaccharides as mentioned by ^{Guevara-Arauza et al. (2012, 997)}, which may explain the reduction of mesophilic aerobic bacteria and total coliforms when mucilage from Opuntia was added to raw milk. In our study, the values obtained of mesophilic aerobic bacteria are lower than that established by the Mexican Official Standard as safe (COFOCALEC 2004, 8). The Mexican Official Standard establishes that values of mesophilic aerobic bacteria in raw milk must be lower than log₁₀ 6.1 CFU mL^-1.

Effect on the growth of total coliforms

In the case of growth of total coliform bacteria was only affected by the interaction of Form of Mucilage*Species of Opuntia (P = 0.0225), while interactions Concentration of mucilage*Species of Opuntia and Concentration of mucilage*Species of Opuntia*Form of Mucilage were not significant (P = 0.0702 and P = 0.7342, respectively).

Counts of total coliforms when different Form of mucilage (dried or liquid) were added to raw milk ranged from 3.2 to Log10 2.9 CFU mL^-1) and were significantly lower (P < 0.05) than control (3.8 Log10 CFU mL^-1) (Figure 5). However, the treatment of dried mucilage of O. ficus-indica observed higher coliform counts (3.2 Log₁₀ CFU mL^-1; P < 0.05) in comparison to mucilage liquid O. ficus-indica and dried mucilage and liquid of O. atropes (2.9 Log₁₀ CFU mL^-1) (Figure 5).

Although, when triple interaction (Concentration of mucilage*Species of Opuntia*Form of Mucilage) was not significant (P > 0.05) on total coliforms counts, it can be observed when mucilage dried of O. ficus-indica is added, less variability is observed considering total coliforms (2.8 log10) in comparison to the rest of treatments (Figure 6). Finally the reduction of total coliforms in such treatment was 5.6x10³ UFC mL^-1 with respect to Control, then, if UFC Log10 values were transformed that means 6.3x10² CFU mL^-1 (2.8 Log₁₀) of total coliforms in raw milk added with dried mucilage of O. ficus-indica, in comparison to 6.3x10³ CFU mL^-1 (3.8 Log10) of total coliforms of Control treatment (Figure 6).

The results of total coliforms observed in our study are higher than those found by Ortiz- Rodriguez et al. (2013, 3678), who reported lower counts of total coliforms in raw milk from cows fed with Opuntia used as supplement of the diet. Few studies have reported the antimicrobial activity of extracts of Opuntia ficus-indica; ^{Shafiei et al. (2013, 907-910)} studied the antimicrobial effect of methanol extracts of O. ficus-indica fruits, they found an inhibitory effect on Staphylococcus aureus, Escherichia coli and Candida albicans. ^{Rabhi et al. (2013, 5875-5885)} when evaluating the antimicrobial activity of extracts of O. ficus-indica they found a strong inhibiting activity against Enterococcus facium and Candida albicans growth using methanolic extracts and associated such effect to phenolic compounds. ^{Ennouri et al. (2014, 1-7)} evaluated the antibacterial activity of hexane extracts of Opuntia ficus- indica flowers at full and post-flowering stages against Gram positive (S. aureous and B. subtibilis) and Gram negative (P. aeruginosa and E. coli), they found an activity against Gram negative growth associated to camphor and aromatic compounds content.

In general, our results for total coliforms (log₁₀ 3.8 CFU mL^-1) were lower than that found by ^{Ortiz-Rodriguez et al. (2013, 3677)}, who reported initial counts of total coliforms of log₁₀ 6.7 CFU mL^-1 when they evaluated the effect of adding nopal cactus fractions (mucilage, epidermis and ground nopal) on aerobic mesophilic bacteria and coliform growth in raw milk. However, these authors observed similar results when raw milk was added with mucilage from O. ficus-indica at concentrations of 0.5%, 1% and 2%, obtaining values from log₁₀ 6.1 to log₁₀ 6.0 CFU mL^-1. It is possible that the difference in values found in this study as compared with our results was due to the different sanitary and management of raw milk; however, the effect shown was the same.

^{Sánchez et al. (2014, 659)} reported antimicrobial effect of extracts of different species of Opuntia on Campilobacter jejuni, Vibrio cholera and Clostridium perfringens, in which these authors associated the bactericide activity with the phenolic and flavonoid compounds found in solvent extracts from Opuntia. Also, ^{Kim et al. (2002, 71-78)} found inhibition on the activity of Salmonella spp. and E. coli from extracts of O. ficus-indica var. saboten, in studies done in vitro. In studies in vivo testing extracts of O. ficus-indica var. saboten, not observed the presence of Salmonella typhimurium DT104 in feces and in the intestinal tract of mice previously challenged with the microorganism. According to the Mexican Official Standard (^{COFOCALEC 2005, 8}), raw milk is considered safe when having a value of total coliforms lower than log₁₀ 2.0 CFU mL^-1. The values found in this research were higher than those established by the NOM. However, we can observe that mucilage from O. ficus-indica and O. atropes promotes the reduction of total coliforms.