Fermentation of fruits, seeds, and other edible substrates is an ancient strategy for obtaining food, involving techniques of biotic resource management and was probably utilized before the advent of agriculture, as suggested by ancient archaeological records in ceramic remains showing the presence of rice ferments in China (about 9000 years BP) and wine in Iran (8000 years BP) and Egypt (5000 years BP) [1
]. Management of fermentation has been crucial for the development of many civilizations, since it allowed for preservation; better digestibility; stable availability; and longer maintenance of the nutritious properties, flavour, and texture, among other features, of food [4
The earliest forms of managing fermentation most likely involved “spontaneous” ferments of fruits and seeds, using strains of microorganisms from the environment [6
]. Later on, people generated specialized methods of fermentation in controlled, lowly variable conditions (in terms of competition, nutrient availability, and the bio-physical environment), which made obtaining desirable products possible [7
]. Management of fermentation has created new niches and contexts in which human selection of microorganisms may operate, consciously or unconsciously [9
]. Specialized strains confer characteristics to ferments like nutrients, flavour, texture, colour, smell, and durability, which are directly “selected” by people by seeking the conditions to ensure them [10
]. In traditional societies, knowledge and practices for preparing fermented products have passed from generation to generation among households and communities, with people sharing some of them but jealously keeping secret others that confer distinctiveness to the producers, thus generating a broad spectrum of product qualities within and among localities [12
Several studies have documented how the diversity and relativeness of microorganism strains are closely related to fermented substrates [5
]. This is the case for Saccharomyces
, a genus of yeasts participating in fermentation of numerous substrates used by humans throughout history, since they produce non-toxic substances, high levels of alcohol, and compounds important for flavour, like esters and phenols [5
]. Another group is the lactic acid bacteria (LAB), which occur in numerous substrates and form part of the microbiota of the digestive tract of numerous animal species, including humans [3
]. The metabolism of these bacteria produce compounds like lactic acid and extracellular polysaccharides (EPS), influencing various qualities of food and beverages [15
Fermented beverages currently have high economic and cultural importance [17
]. Around the world, there is a wide spectrum of substrates used for this purpose, among them economically important plants whose leaves (e.g., tea), sap (palms; palm wine), fruits (grapes; wine), and grains (barley; beer) are used [6
]. In Mexico, numerous cultural groups have used different substrates to prepare fermented beverages, including fruits (e.g., Spondias
spp. and Ananas comosus
for preparing “tepache”, Opuntia
spp. for “colonche”), the sap of plants (Cocos nucifera
for preparing “tuba”, Acrocomia aculeata
for “taberna”, Agave
spp. for “pulque”), grains (Zea mays
for “tesgüino” or “pozol”), and barks (Lonchocarpus longistylus
for “balche”) (Table 1
Pulque is a fermented beverage that has been prepared since pre-Columbian times from sap from 41 taxa of Agave
in Mexico [28
]. The people of Mesoamerica managed fermentation processes; the material evidence of pulque preparation are vessels with remains of this beverage found in residential areas of Teotihuacan, dating back to 1600–1350 BP [29
]. There is also pictographic evidence, for instance in the Vindobonensis Codex (Mixtec) and in the “Matrícula de los tributos”
]. In addition, Sahagún [32
] described in “Historia general de las Cosas de Nueva España”
the methods that the native people of Mexico used to ferment agave sap, the materials and care required, and practices to accelerate the fermentation by adding root plants like the “Ocpatli”
). Such ancient knowledge about the management of fermenting processes has survived in many pulque-producing communities in Mexico.
Agave sap contains sugars (sucrose, fructose and glucose), vitamins, minerals, and amino acids, so it is a source of nutrition [33
]. Agave sap has a high diversity of microorganisms, which determine the attributes of pulque [26
]. It is rich in different compounds favourable for microorganisms naturally associated with agaves and that are transported to the scraped cavities of the stems through the air or the action of scrapers and other utensils used for collecting agave sap [26
Viscosity, acidity, and alcoholic content are the main attributes determining pulque qualities, and these attributes are conferred by microorganisms participating in the fermentation process. Viscosity is mainly associated with the metabolism of bacteria of the genus Leuconostoc
, which acidifies the sap using sucrose, producing the CO2
and EPS responsible for viscosity [26
spp. produce lactic acid, thus increasing acidity and also producing aromatic compounds influencing flavour [3
]. In addition, these bacteria compete with and inhibit growth of pathogenic microbes by producing organic acids and protein toxins called bacteriocins, which inhibit the growth of non-related bacteria [40
]. Alcoholic production results from yeasts, which in addition produce vitamins and amino acids, as well as volatile compounds that also influence flavour and the aromatic profile of the pulque [6
Communities of microorganisms are managed by pulque producers based on their experience and knowledge derived from fermenting agave sap throughout many generations, passing down recipes, ingredients, conditions, and techniques of preparation, which are an important biocultural heritage. Historically, pulque preparation has been artisanal, and in such a context, a great variation of qualities have been recorded, associated with the species and varieties of agave used; environmental conditions of sites where the pulque is prepared; the assemblages of microorganisms available in the producing areas; the cultural contexts guiding preferences for viscosity, acidity, alcoholic content, flavour, and other attributes; and the preparation techniques, including other ingredients added to the sap and the utensils used for collecting, storing, and fermenting the agave sap, among other aspects [26
The state of Michoacán, in central-western Mexico, has been a pulque producing area since Pre-Hispanic times. Agave cultivation in this region is associated with other crops such as maize, squash and beans, in systems that have barely been studied compared with other pulque production systems in Mexico. Álvarez–Ríos et al. [27
] documented two pulque producing communities in Michoacán, in which differentiated management techniques are practiced. In this study, we explored the hypothesis that the different management techniques influence the different physical and chemical attributes and microbiological compositions, determining the different features of the produced pulques. This study aimed to document how the techniques of management of agave sap affect the physical and chemical characteristics of the sap and pulque, and the consequences in the structure and dynamics of the consortiums of microorganisms participating in fermentation of this beverage in two communities of Michoacán.
2. Materials and Methods
2.1. Study Site
Our study was conducted in two localities of the state of Michoacán, central Mexico. One was Tarímbaro (T), north of the city of Morelia, at an elevation of 1860 m, where the annual mean temperature and rainfall are 22 °C and 600–800 mm, respectively. Tarímbaro is part of the suburban area of the city of Morelia, and the main economic activities are agriculture of maize, vegetables, and agave [42
]. The other site is Santiago Undameo (SU), southeast of the city of Morelia, at an elevation of 2004 m, with an annual mean temperature and rainfall of 17 °C and 800–1000 mm, respectively. Economic activities in Santiago Undameo are predominantly agriculture of maize, cultivation of agave for preparation of pulque, and cattle raising [42
] (Figure 1
2.2. Ethnobiological Fieldwork
A total of 12 in-depth interviews were conducted with households of pulque producers (six in each village) to document management practices of agave sap and preparation of pulque.
2.3. Evaluation of Physical and Chemical Characteristics of Fermentation Phases
We collected samples of the main phases of pulque preparation. In Santiago Undameo, we collected six samples of each of the following fermentation phases of the beverage: l) fresh sap (FS) or “aguamiel”
, 2) boiled sap (BS), 3) pulque (P), and 4) inoculum for pulque (IP) or “foot of pulque”. Similarly, in Tarímbaro, we collected six samples of the mentioned phases, except the boiled sap since in that community people do not engage in this practice. In SU we collected fresh sap of A. salmiana
while in T the samples were of A. mapisaga
. The pulque and inoculum for pulque in both sites were a mixture of sap of three species of Agave
, as reported by Álvarez-Ríos et al. [27
] (A. salmiana
var. salmiana, A. mapisaga
and A. americana
). In total, we collected and analysed 42 samples; 24 from Santiago Undameo and 18 from Tarímbaro.
The following characteristics were evaluated for all collected samples: (1) Concentration of sugars in sap and pulque, measured through a manual refractometer Vee Gee, ABT-32. (2) Acidity, evaluated through a pH meter Denver Instrument, model 215. (3) Lactic acid: to 10 mL of each sample we added 1 mL of 2% phenolphthalein, then titrated with a solution of 0.1 N NaOH. Based on the amount of NaOH used for neutralizing the acid solution, we estimated the total acidity of the sample in grams of lactic acid using the formula TA= (v × N × 0.09 × 100) / s where TA = total acidity in grams of lactic acid per 100 mL of the sample, v= volume (mL) of NaOH used in titration, N= normality of the NaOH solution, s= volume (mL) of the sample used in the estimation, and 0.09 = lactic acid milliequivalent. (4) Density, measured through a 50 mL pycnometer. (5) Viscosity, estimated through an Ostwald viscosimeter by recording the time the sample took to reach different marks of the viscosimeter. We calculated the viscosity using the formula: n1= (d1 × t1 × n2) / (d2 × t2), where n1 and n2 are the viscosities, t1 and t2 are the times taken by the flow, and d1 and d2 the densities of the liquid studied and water, respectively. (6) Percentage of alcohol: We distilled 200 mL of each sample using a distillation balloon flask and glass beads heated at 80 °C to complete evaporation, recovering 85–90% of the original volume. Then, using the recovered liquid we measured the percentage of alcohol with an alcoholometer.
2.4. Microbiological Characterization of the Beverages through Colony-Forming Units (CFU)
In order to characterize the structure of the microbial community for each fermentation phase (FS, BS, P and IP), we used three media for cultivating CFU: (1) Tryptic Soy Agar, pH 7.3, a general medium that favours the development of a great variety of microorganisms; (2) Man, Rogosa and Sharpe (MRS) Agar, pH 6.5, a selective medium for lactic acid bacteria, with ammonium citrate that prevents the growth of Gram-negative bacteria; and (3) Sabouraud Dextrose (SD) Agar, pH 5.6, a selective medium for cultivation of fungi and yeasts, with antibiotics to inhibit growth of bacteria.
Each sample was diluted through a series of decimal dilutions until reaching l0−6, and 0.1 mL per sample was sown per 10 cm diameter petri dish, with three replicates per sample and culture medium.
Cultures were incubated for 72 h at 26 °C, and then characterization and morphotype counting were conducted. Each morphotype was characterized through attributes such as form, border, elevation, surface, colour, and light reflectance.
2.5. Statistical Analyses
We conducted one-way and two-ways analyses of variance (ANOVA) and Tukey’s multiple range tests to evaluate differences among treatments, and principal component analysis to evaluate differentiation patterns associated with all variables studied. All data analyses were conducted with R software (v. 3.5.0).
3.1. Sap Management for Pulque Production
Pulque producers at both sites collect sap from 8 to 10-year-old agaves, just when the meristem starts producing inflorescence. At that point, the producers cut the meristem and dig a cavity where the sap flows and accumulates. Every day, the producers collect the accumulated sap with a cup and a bucket and scrap the cavity to allow the sap to continue to flow. The producers have 10 to 15 agaves in production and collect on average 32 L of sap in T and 26 L in SU per day (Figure 2
Differences in the way the sap is managed were recorded among sites. In T, after the sap is collected, it is transported to the homes of the producers and stored in spaces specially designed for the creation of pulque. The creation of pulque in T occurs in clean rooms, with little exposure to sunlight, and the people here consider it convenient to maintain low temperatures for a good preservation of the beverage. In these spaces, the producers have 20 L plastic barrels where they prepare the pulque. Additionally, the producers have a little bottle for maintaining the inoculum of the pulque, which is the remaining sediment from pulque prepared the day before. The producers use this to inoculate the fresh sap and, according to the people interviewed, for enhancing “the aguamiel to work fast and making pulque tasty”.
To prepare the inoculum, the producers collect 3 L of aguamiel and leave it resting in a container covered with a blanket, causing the sap to undergo “spontaneous” fermentation. The liquid is allowed to ferment for 48 to 72 h. Later, this highly fermented aguamiel will be combined with fresh aguamiel in a ratio of 1:2 (fermented aguamiel and fresh aguamiel, respectively) for the first round of pulque production. After selling or consuming the pulque, a whitish sediment remains in the container, which may be perceived as slightly sandy; this is the inoculum, which people use to continue producing pulque on successive days (Figure 2
When adding the inoculum to the fresh sap, the people pour the sap through a mesh in order to remove the remains of the scraped tissue or insects that may fall into it. Once the fresh sap is added, the resulting mixture starts fermenting, producing an effervescence and a white foam. According to the producers, after 3 hours of fermentation, the pulque is ready for consumption or to be sold.
In SU, after the fresh sap is collected, it is strained and placed in a pot over a fire to slightly boil it for no more than 1 minute (Figure 2
C). As soon as the aguamiel begins boiling, the people remove it from the heat and let it cool for 1 hour, then mix it with the inoculum. The effervescence of the beverage takes place after 2 hours, at which point the producers consider the pulque ready to be consumed and commercialized. The process of saving the inoculum in SU is like that practiced in T, but in this village, people leave the sap to ferment for a couple of days, and then mix this “foot of pulque” with the boiled sap. According to the pulque producers of SU, the sap generates an irritation called “carame”
, and by boiling the sap they avoid carame.
3.2. Physical and Chemical Characteristics of Sap and Pulque
shows the mean values and standard errors of the characteristics measured in the sap and pulque at different phases of fermentation at each study site. We identified significant differences in beverages among localities, and highly significant differences were identified based on the processes of sap boiling and inoculation (Table 2
Concentrations of sugars (°Brix) in fresh sap were 9.8 ± 0.89 in SU and 9.57 ± 0.76 in T, whereas in pulque we recorded 8.22 ± 0.49 in SU and 7.48 ± 0.4 in T, and in the inoculum 5.47 ± 0.4 and 5.42 ± 0.29 in SU and T, respectively.
The beverage became progressively more acidic as fermentation advanced. Fresh sap had a pH of 6.23 ± 0.39 and 4.58 ± 0.13 in SU and T, respectively. The boiled sap of SU had a pH of 7.32 ± 0.58. Later, the pulque had a pH of 4.15± 0.14 and 3.94 ± 0.08, whereas the inoculum had a pH of 3.87 ± 0.13 and 3.69 ± 0.05, in SU and T, respectively.
Lactic acid concentration (g/100 mL) in fresh sap was 0.23 ± 0.04 and 0.53 ± 0.06 in SU and T, respectively, increasing during fermentation to 0.82 ± 0.05 and 0.67 ± 0.06 in the pulque and 1.02 ± 0.03 and 0.77 ± 0.06 in the inoculum in SU and T, respectively. The boiled sap of SU had the lowest value of lactic acid (0.05 ± 0.02).
Density (g/cm3) decreased with fermentation from 1.01 ± 0.001 and 1.01 ± 0.001 in fresh sap to 0.99 ± 0.007 and 0.98 ± 0.001 in pulque, and 0.97 ± 0.002 and 0.99 ± 0.003 in inoculum, from SU and T, respectively. Boiled sap had a density similar (1.02 ± 0.006) to non-boiled sap.
The viscosity (cP) of fresh sap was 1.22 ± 0.12 and 1.3 ± 0.09 in SU and T, respectively, and it increased with fermentation to 1.14 ± 0.03 and 1.59 ± 0.019 in pulque and 2.92 ± 0.16 and 2.48 ± 0.09 in the inoculum in SU and T, respectively. The viscosity of the boiled sap was 1.14 ± 0.03, similar to that of the non-boiled sap.
The fresh sap in both localities had an alcohol content of 0%. When boiled, a small amount of alcohol was present, 0.53% ± 0.15, increasing to 3.88% ± 0.47 and 4.92% ± 0.29 in pulque in SU and T, respectively, and even more in the inoculum; 6.73% ± 0.75 in SU and 6.03% ± 0.35 in T.
3.3. Fermenting Microorganisms
Lactic acid bacteria (LAB) were recorded in the different phases of the beverage at both sites. In fresh sap of SU, we recorded 2.98 ±1.18 × 108
colony-forming units per 1 mL (CFU), while 3.53 ± 0.65 × 108
CFU were recorded in T. When the sap was boiled in SU, this decreased to 7.15 ± 4.55 × 106
CFU, but in the pulque it increased markedly to 3.16 ± 1.68 ×108
CFU in SU, while it increased to 1.9 ± 0.84 × 108
CFU in T, where the fresh sap was not boiled. The highest abundance was recorded in the inoculum (4.91 ± 1.44 × 108
CFU in SU and 3.80 ± 0.92 × 108
CFU in T) (Table 3
, Figure 3
The pattern of yeast abundance was similar to that of LAB: 3.06 ± 1.33 × 108
and 3.39 ± 0.88 × 108
CFU in fresh sap from SU and T, respectively; a notable decrease was seen in boiled sap (8.72 ± 6 × 106
CFU), then the levels recovered in pulque (2.52 ± 1.44 × 108
CFU in SU and 1.47 ± 0.72 × 108
CFU in T), and reached a maximum in the inoculum (6.27 ± 1.33 × 108
CFU in SU and 4.32 ± 0.83 × 108
CFU in T) (Table 3
, Figure 3
In the general culture medium, we recorded the following abundances of yeast: 5.24 ± 1.17 × 108 CFU in fresh sap of SU, and 8.49 ± 2.28 ×108 in T, which decreased after boiling (16.56 ±10.04 × 106 CFU). For pulque, we recorded 8.97 ± 4.79 ×108 CFU in SU and 5.21 ± 1.51 ×108 CFU in T, while for the inoculum we recorded 16.34 ± 6.11 ×108 CFU for SU and 17.17 ± 3.92 ×108 CFU for T.
Although a clear pattern of CFU increase was seen during fermentation, with the greatest abundance of LAB and yeast found in the inoculum, the ANOVA showed no significant differences among most treatments or sites, except for boiled sap.
In SU, LAB and yeasts are present in the fresh sap, but their abundance decreases in boiled sap. Then, when the boiled sap is inoculated, the inoculation promotes pulque production.
In beverages from T, where the fresh sap is inoculated with the inoculum to produce pulque, it is noted that the CFU of LAB and yeasts decreased in the pulque, which suggests that the colonies that were inoculated competed with those already occurring in the sweet sap. After fermentation reached the phase of pulque, the microorganisms continued growing, thus forming the inoculum that was used for the next inoculation (Figure 3
The richness of LAB morphotypes recorded in fresh sap was 4.3 ± 0.7 (and 0.6 ± 0.2 after boiling) in SU and 4.3 ± 0.8 in T. In the pulque, we recorded 3 ± 0.5 in SU and 4.2 ± 0.9 morphotypes in T, as well as in the inoculum (2 ± 0.5 and 3.8 ± 0.5 morphotypes in SU and T, respectively). The richness of yeast morphotypes in fresh sap was 4.5 ± 0.6 (1 ± 0.4 after boiling) in SU and 4.5 ± 0.6 in T. For the pulque, we recorded 1.8 ± 0.3 in SU and 3 ± 0.6 morphotypes in T, whereas in the inoculum for the pulque we found 1.3 ± 0.3 and 2.3 ± 0.5 morphotypes in SU and T, respectively (Table 3
As fermentation progressed, a decrease in the morphotypes of both LAB and yeast was observed, with more occurring at the beginning of the process and less at the end. The lowest number of morphotypes was reported in boiled sap, because the abundance and richness of the microbial communities decrease with the effect of the temperature increase.
Regarding diversity, the reported values were H´
= 1.22 ± 0.11 for fresh sap of SU, and H´
= 1.17 ± 0.12 for T. For the pulque, it was H´
= 0.98 ± 0.11 in SU, and H´
= 1.33 ± 0.06 in T. Finally, in the inoculum, the diversity was H´
= 0.73 ± 0.09 in SU and H´
= 1.21 ± 0.14 in T. The lowest diversity was reported in the boiled sap, at H´
= 0.48 ± 0.22 (Table 3
, Figure 3
Pulque from T, from sap that had not been boiled, had a higher diversity than those from SU, apparently because the absence of boiling allows more morphotypes to be added from the inoculum to those already existing in fresh sap.