Use of Maldi-Tof MS biosensor in microbial assessment of Brazilian kefir grains¹

Utilização do biossensor Maldi-Tof MS na avaliação de micro-organismos dos grãos de kefir brasileiro

Kefir is a polysaccharide grain containing microorganisms that produce fermented beverages, such as the traditional Turkish beverage also named "kefir", which is from milk, and has low alcohol content (Simova et al., 2002; Guzel-Seydim et al., 2005; Irigoyen et al., 2005; Magalhães et al., 2011a, 2011b, 2011c; Puerari et al., 2012; Cho et al., 2018). Kefir is a mixed culture of various yeasts and bacteria species combined in a matrix of proteins and polysaccharide “kefiran”, which are formed during cell growth under aerobic conditions (Magalhães et al., 2010a, 2010b). The kefir grains are irregularly shaped and hard granules, with yellowish-white colour, which resemble miniature cauliflower blossoms (Corona et al., 2016).

In Brazil, kefir grains are used in private households (Magalhães et al., 2011c) and are added to different types of milk, such as cow, goat, or sheep, coconut, rice, and soy (Irigoyen et al., 2005; Magalhães et al., 2011c). The grains are responsible for the fermentation that produce lactic acid, acetic acid, CO₂, alcohol (ethyl 2 alcohol), and aromatic compounds. These compounds provide the unique sensory characteristics of kefir: fizzy, acid taste, and tart and refreshing flavour (Corona et al., 2016).

The kefir beverage contains vitamins, minerals, and essential amino acids that help the body with healing and maintenance functions and contains easily digestible complete proteins (Roos et al., 2018). In accordance with Medrano et al. (2008), the benefits of consuming kefir in the diet are numerous, such as its antitumoral, (Vinderola et al., 2005), antimicrobial (Rodrigues et al., 2005), antiinflammatory, and antiallergical (Lee et al., 2007) activities. The composition of microbial species is an important factor to characterize the kefir therapeutic benefits (Simova et al., 2002; Guzel-Seydim et al., 2005; Irigoyen et al., 2005; Magalhães et al., 2011a, 2011b, 2011c; Cho et al., 2018). A rapid identification of the microorganisms is necessary in the fermentative process of kefir grains, which facilitates the microbiological control of fermentative processes on a large scale.

An optical technique with potential use to rapidly identify the microorganisms is the Maldi-Tof MS (Matrix Assisted Laser Desorption/Ionisation - Time of Flight Mass Spectrometry) (Microflex-Bruker Daltonics/BioTyper™) (Chang et al., 2016; Pasternak et al., 2012; Gaudreau et al., 2018; Mayoral et al., 2018). In this technique, the sample is uniformly mixed in a large quantity of matrix. The matrix absorbs the ultraviolet light (nitrogen laser light, wavelength 337 nm) and converts it to heat energy. A small part of the matrix (down to 100 nm from the top outer surface of the Analyte in the diagram) heats rapidly (in several nano seconds) and is vaporized, together with the sample. Charged ions of various sizes are generated on the sample spot (Figure 1). A potential difference V0 between the sample slide and ground attracts the ions in the direction shown in the diagram. The velocity of the attracted ions v is determined by the law of conservation of energy. As the potential difference V0 is constant with respect to all ions, ions with smaller m/z value (lighter ions) and more highly charged ions move faster through the drift space until they reach the detector. Consequently, the time of ion flight differs according to the mass-to-charge ratio (m/z) value of the ion. The method of mass spectrometry that exploits this phenomenon is called Time of Flight Mass Spectrometry (Pasternak et al., 2012; Mayoral et al., 2018).

Figure 1:
Brazilian kefir micoorganisms analysis methodology in the Maldi-Tof MS biosensor.

The aim of this study was to evaluate the use of Maldi-ToF biosensor in the microbial assessment of Brazilian kefir grains, as well as to identify the microorganisms associated with them.

Rapid diagnosis of microorganisms is decisive to guarantee adequate identification in biological samples. Biochemical methods are precise and sensitive, but rather slow. New resources are available to enable faster diagnosis, and the most promising is Maldi-Tof MS technology, applied to microbiological diagnosis by rapid method using the microbial colonies.

The microbial population count of the kefir grains performed in this study Found: lactic acid bacteria with a population of 8.10 log CFU mL^-1 (approximately 10⁸ cells); AAB in a low population of 4.01 log CFU mL^-1 (approximately 10⁴ cells); and yeasts in population of 6.01 log CFU mL^-1 (approximately 10⁶ cells). This microbial population is expected in the kefir grains because they are considered probiotic (Simova et al., 2002; Guzel-Seydim et al., 2005; Irigoyen et al., 2005; Magalhães et al., 2011a, 2011b, 2011c; Puerari et al., 2012; Cho et al., 2018).

A total of 358 microorganisms were isolated, 31 yeasts and 327 bacteria (divided into lactic and acetic bacteria). The isolates were identified by Maldi-Tof MS technique (Figure 1). The results showed higher population of Lactobacillus paracasei (10⁶ CFU mL^-1); Saccharomyces cerevisiae, Lactobacillus plantarum, Acetobacter pasteurianus, and Acetobacter syzygii were found in smaller quantities (10⁴ CFU mL^-1) (Figure 2).

Figure 2:
Microbial identification of Brazilian kefir grains by Maldi-Tof MS technique.

Quintilla et al. (2018) evaluated the efficiency of Maldi-Tof MS to identify foodborn yeasts. The identified yeasts were named as Rhodotorula babjevae, Meyerozyma caribbica, Clavispora lusitaniae, Debaryomyces hansenii, Candida oleophila, Pichia membranifaciens, Kazachstania telluris, and Mrakia frigida. Authors showed that Maldi-Tof MS is applicable for routine identification and validation of foodborne yeasts.

Gaudreau et al. (2018) evaluated Maldi-Tof MS to identify bacteria from biofilms. They compared three sample preparation procedures on biofilms grown in vitro. The extended direct transfer method was able to identify 13 isolates out of 18 (72%) at the species level and 15 out of 18 (83%) at the genus level.

Mayoral et al. (2018) reported two new cases involving immunocompetent girls with cervicofacial lymphadenitis due to Mycobacterium mantenii, and the reliability of Maldi-Tof MS for identifying Mycobacterium mantenii was efficient.

The Maldi-Tof MS technology proved to be useful for diagnosis of kefir microorganisms, allowing a fast and safe diagnosis for the scientific environment. The technology can facilitate the microbiological control in a fermentation process using kefir grains as starter cultures. The microorganisms identified in the Brazilian kefir are the same commonly found in kefir grains, with predominance of Lactobacillus paracasei.

The authors thank the Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).