Bacterial diversity in organic and conventional Minas Frescal cheese production using targeted 16S rRNA sequencing

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Abstract

Molecular identification and the study of bacteria in processed food products can be challenging or suffer limitations when using conventional methods. However, with Next Generation Sequencing (NGS), one can identify bacterial species with a significantly higher detection sensitivity using a 16S rRNA target gene. This study aimed to characterise the bacterial diversity of the organic and conventional production of Minas Frescal cheese, through the NGS analysis of the 16S rRNA gene. DNA was extracted from 96 samples, 48 from organic production and 48 from conventional production. The bacterial families and genera with the highest prevalence were identified. The most prevalent families were Enterobacteriaceae, Planococcaceae and Moraxellaceae. The NGS 16S rRNA tool proved to be very useful for bacterial characterisation, but it is still not possible to say whether a product contains more or less bacteria based on organic production, as there are several factors that contribute to contamination.

Introduction

Over the past decades, the search for health foods has increased worldwide (Britwum, Bernard, & Albrecht, 2020; González, Marquès, Nadal, & Domingo, 2019; Katt & Meixner, 2020; Kushwah, Dhir, Sagar, & Gupta, 2019); among health food, organic milk production generates important benefits for the environment, animal welfare and human health (Brito & Silva, 2020; Schwendel et al., 2015). According to the Brazilian Normative Instruction Nº. 46/2011 (Brasil, 2011): to obtain and maintain the health of animals in an organic system, the principle of prevention must be used; such as adequate food (from the production unit itself or from another source under organic management), preferably live in a free regime, practice regular exercise and have access to pasture to promote immune defences.

In the case of diseases or injuries in which the use of natural substances is not having an effect, and for which reason the animal is suffering or is at risk of death, synthetic antibiotic products may be used (Brasil, 2011); as opposed to conventional production where the use of synthetic antibiotics is allowed.

Among the consumed dairy products in Brazil, Minas Frescal cheese is the third most produced type of cheese (Lollo et al., 2015), with both organic and conventional milk used for its production (Cândido et al., 2020). According to Normative Instruction Nº. 4/2004: Minas Frescal cheese is produced by enzymatic coagulation of milk with rennet and/or other suitable coagulating enzymes, complemented or not with the action of specific lactic bacteria; it is an unripened, semi-fat cheese of very high humidity, to be consumed fresh, and which must be stored at temperatures lower than 8.0 °C (Brasil, 2004).

Primarily, Minas Frescal cheese production is comprised of the following stages: raw milk filtration; pasteurisation that can be slow (65 °C, 30 min) or fast (75 °C, 15 s); a coagulation preparation step, which consists of calcium chloride addition (this salt is required for increasing the content of soluble calcium in milk) and rennet to promote milk coagulation, thus converting liquid milk into cheese curd; treatment of the paste after coagulation, which consists of cutting the paste into cubes; stirring the cubes for 1 min and resting for 3 min, and then repeating this cycle for 30 min; hanging, which is necessary to give the cheese its characteristic shape; salting, both dry salting, which is adding salt to the surface of the paste, or salt application to the paste mixture; packaging into plastic bags; and storage under refrigeration (Silva, 2005).

Raw milk and Minas Frescal cheese both present physico-chemical characteristics that contain various nutrients, such as carbohydrates, lipids, proteins, essential amino acids, enzymes, vitamins and minerals; making them an excellent culture media for several microorganisms (Delorme et al., 2020; Prezzi et al., 2020; Verraes et al., 2015).

The identification of microorganisms has always been a challenge for the food industry, especially in the dairy sector, since contamination can occur at different stages of production. Therefore, monitoring the bacterial diversity is important and can provide relevant information regarding product quality (Delorme et al., 2020).

Thus, compared with traditional molecular methods, Next Generation Sequencing (NGS) platforms offer significantly higher detection sensitivity when it comes to studying bacterial communities (Ercolini, 2013; Fuka et al., 2013; O'Sullivan et al., 2015). NGS of the bacterial 16S rRNA amplicons showed that the set of microorganisms can vary depending on the milk source, processing (using raw or pasteurised milk), and with the addition of several ingredients. It is also possible to identify genera that were not previously associated with cheese ecosystems (Prevotella and Helcococcus), and to identify specific microorganisms in certain types of cheese (Arthrobacter in goats' milk cheese) (O'Sullivan et al., 2015; Quigley et al., 2012). Based on NGS, the microbial communities of different types of cheese can be characterised. Therefore, it can provide information on the sensory properties of the product, as well as its overall quality. Additionally, one can identify the microbial communities throughout the production facilities (Bokulich & Mills, 2013; O'Sullivan et al., 2015).

Previously, the applicability and advantages of NGS have been outlined for the study of microbial ecology in several food products (De Filippis, Parente, E., & Ercolini, 2017). These included microorganism monitoring during curd fermentation, cheese ripening and investigation of the spatial distribution of microbes in different parts of the same cheese.

Although many studies have investigated the contribution of a single or a few microorganisms in dairy products, information on microbial communities is still lacking. Therefore, understanding the dynamics of the milk microbiota is of great importance to control the qualitative, sensory and biosafety characteristics of dairy products (Lessard, Viel, Boyle, St-Gelais, & Labrie, 2014; Marino et al., 2019; Tilocca et al., 2020).

Based on the lack of information on microbial communities associated with the production of Brazilian Minas Frescal cheese; this research aims to identify and characterise the bacterial diversity in organic and conventional Minas Frescal cheese production, raw material such as raw milk, pasteurised milk, swabs from food handlers and swabs from surfaces in the processing environment, through Next Generation Sequencing of the 16S rRNA gene.

Section snippets

Sample origins

The samples were obtained from three organic (A, B and C) and three conventional dairy producers (D, E and F), in different cities of the state of São Paulo. Sampling was conducted twice in each dairy company, with an interval of about one month. Samples were collected during each visit, from Minas Frescal cheese, pasteurised milk, raw milk, swabs of food handlers, environment and equipment (lira, floor, table, cold chamber, shape mold, drains, boards, tanks). The swabs were chosen randomly

Native microbiota assessment in organic and conventional Minas Frescal cheese production

This study evaluated the bacterial diversity of three organic and three conventional Minas Frescal cheese producers in the state of São Paulo, Brazil, including its processing line, food handler swabs, surface swabs, equipment, and samples of raw milk, pasteurised milk and Minas Frescal cheese.

Using NGS data, we were able to verify great bacterial diversity. We identified 51 different bacterial families; 40 in raw milk, 32 in pasteurised milk, 28 in Minas Frescal cheese and 42 from swabs (

Conclusion

It was possible to identify a large taxonomic variety of families and genera in samples of organic and conventional dairy products, from raw milk to the final product, Minas Frescal cheese. This study provided a broader view of the bacterial diversity of dairy products. Thus, the NGS analysis of the 16S rRNA gene allows a complex and complete analysis of the bacterial diversity of the sample. It can help to point out new pathogenic or deteriorating bacteria in products due to its ability to

Funding

This study was funded by the São Paulo Research Foundation (FAPESP) [grant numbers 2016/02118-7 and 2018/00986-7]; Programa de Excelência Acadêmica (PROEX) [grant numbers 23038.000795/2018–61]; and Coordenação de Aperfeiçoamento Pessoal de Nível Superior – Brasil (CAPES) - Finance Code 001. The authors thank Espaço da Escrita – Pró-Reitoria de Pesquisa – UNICAMP - for the language services provided. This research used resources from Brazilian Biorenewables National Laboratory (LNBR), an open

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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