**Agnieszka Szparaga 1, Sylwester Tabor 2, Sławomir Kocira 3,\*, Ewa Czerwi ´nska 4, Maciej Kubo ´n 2, Bartosz Płóciennik <sup>1</sup> and Pavol Findura <sup>5</sup>**


Received: 14 August 2019; Accepted: 27 October 2019; Published: 1 November 2019

**Abstract:** This study aimed at determining the survivability of probiotic bacteria cultures in model non-dairy beverages subjected or not to the fermentation and storage processes, representing milk substitutes. The experimental material included milks produced from desiccated coconut and non-dehulled seeds of hemp (*Cannabis sativa* L.). The plant milks were subjected to chemical and microbiological evaluation immediately after preparation as well as on day 7, 14, and 21 of their cold storage. Study results proved that the produced and modified plant non-dairy beverages could be the matrix for probiotic bacteria. The fermentation process contributed to increased survivability of *Lactobacillus casei* subsp. *rhamnosus* in both coconut and hemp milk. During 21-day storage of inoculated milk substitutes, the best survivability of *Lactobacillus casei* was determined in the fermented coconut milk. On day 21 of cold storage, the number of viable *Lactobacillus casei* cells in the fermented coconut and hemp milks ensured meeting the therapeutic criterion. Due to their nutritional composition and cell count of bacteria having a beneficial effect on the human body, the analyzed groceries—offering an alternative to milk—represent a category of novel food products and their manufacture will contribute to the sustainable development of food production and to food security assurance.

**Keywords:** probiotic; non-dairy beverages; survivability; fermentation; bacteria; coconut; hemp; sustainable food production

#### **1. Introduction**

Sustainable food production should be considered through the perspective of a better understanding of food security. In recent years, many researchers and policy makers have focused only on the physical availability of food, owing to the sufficient agricultural production [1,2]. This has partly been driven by widespread claims that we need to boost the global food production to feed the world in 2050 [3]. However according to the FAO (Food and Agriculture Organization of the United Nations) definition [4]: "food security exists when all people, at all times, have physical and economic access to sufficient, safe and nutritious food that meets their dietary needs and food preferences for an active and healthy life". Hence, it needs to be emphasized that the sustainable food production is inevitably related to the food security in its three aspects: food security, food safety, and food quality, without which the development of the food industry sector would not be possible [5,6]. Due to the current dynamic development of sciences related to food and human nutrition, a correlation has been confirmed between the health status and nutritional patterns. A well-balanced diet is the key factor in diseases prevention and treatment. The growing nutritional interests and awareness of consumers have prompted many producers to manufacture functional food [7–9]. A functional food definition covers certain strains of microorganisms being constituents of food of plant and animal origin that contain physiologically active compounds. These compounds are beneficial for human health and help minimizing the risk of chronic diseases development [10]. One of the multiple examples of functional food products are these containing microorganisms endogenous to the human gastrointestinal tract and exhibiting a positive effect on human health [11]. So far, the greatest part of probiotic products has been offered by fermented beverages made of animal milk. Currently, research is underway into other products that may be matrix for probiotic bacteria [12,13]. Consumers avoiding milk because of allergies or lactose intolerance, and consumers following a vegan diet can replace milk with other plant-based substitutes. Beverages derived from soybeans have for many years been the predominant equivalents of milk. Today, coconut, almonds, hemp, and various cereals (e.g., oats, buckwheat, and rice) are also used to produce plant-based beverages [14,15]. A drawback of these products is however their specific taste that does not suit to everyone. A solution to this problem is offered by lactic acid fermentation, which imparts a characteristic, pleasant after-taste to these products and contributes to the improvement of the digestibility [13,16]. Numerous attempts have recently been undertaken to ferment vegan beverages serving as milk substitutes using various strains of probiotic bacteria, which was expected to additionally increase their health value [16,17]. However, most of the study results reported in literature concern the feasibility of producing fermented soybean milk [13–16]. This is related to the fact, that manufacture of high quality plant-based beverages containing probiotic bacteria poses a serious challenge [18,19]. According to Yuliana et al. [20], the production of coconut-based beverages is difficult because of the suppressed growth and survivability of these probiotic microorganisms, compared to dairy beverages. Difficulties in the manufacture and fortification of hemp milk were encountered by Batkiene et al. [21]. The first ones were related to the stability of produced emulsions, whereas the latter ones to the survivability of probiotic bacteria during storage. Worthy of notice is that the production of hemp-based products has increased in recent years due to the confirmed nutritional value and low allergenicity of seeds of this plant [22]. This has been feasible owing to new varieties characterized by a low concentration of a psychoactive compound delta-9-tetrahydrocannabinol (THC) [23] and to cultivations with the use of elite category sowing material [24].

The current definition of a probiotic means those microbial strains that positively affect consumer health when taken in the right amount [25]. Accordingly to FAO/WHO guidelines, the count of probiotic bacteria cannot be less than the value corresponding to 10<sup>6</sup> cfu per 1 mL of a product through the entire period of its storage till the end of its shelf life. This value has been deemed the therapeutic minimum [26–28].

The main problems associated with the fermentation of plant beverages are related to the sensory quality of the final product and to the resistance of probiotic microorganisms. Producers encounter difficulties with the physical stability caused by milk coagulation (it occurs at the beginning or in the course of storage). The appearance of these products resembles that of low-fat yoghurt [29–32]. Additional problems concern the survivability of probiotic bacteria, which is dependent on multiple factors, including e.g., presence of other microorganisms in the product, time and conditions of strains culture and product storage, product processing technology or pH value [13,26,33,34].

Considering the above, the major objective of this study was to determine the survivability of probiotic bacterial cultures in model fermented and non-fermented stored plant beverages being milk substitutes, because today the plant-based alternative milks provide a huge perspective for the sustainable development of the healthy food market and should therefore be widely scrutinized. Evaluation of the effect of production and processing techniques, and also of fortification techniques, of plant-based beverages may serve to develop a nutritionally complete beverage with a high overall acceptability and health values.
