Potential Health-Promoting Benefits of Paraprobiotics, Inactivated Probiotic Cells

Viability plays an important role in the beneficial microbes (probiotics) to produce health benefits. However, this idea has been changed after the invention of the term "paraprobiotics," indicating that non-viable microbes could produce health benefits similar to those produced by live probiotics. Occasionally, it might be dangerous to administer live probiotics to people with weak immunity. In such cases, ingestion of paraprobiotics could be a potential alternative. The definition of paraprobiotics refers to the use of inactivated (non-viable) microbial cells or cell fractions to provide health benefits to the consumer. Paraprobiotics have attracted much attention because of their long shelf life, safety, and beneficial effects, such as modulation of immunity, modification of biological responses, reduction of cholesterol, anti-inflammatory, and antiproliferative properties. These features indicate that paraprobiotics may play a vital role in improving the health of the consumer by enhancing particular physiological functions, even though the exact underlying mechanisms have not yet been completely elucidated. In this mini-review, we briefly discuss the historical backgrounds of paraprobiotics and evidence of their health-promoting effects, prophylactic, and therapeutic properties.

been previously mentioned in literature as "inactivated probiotics" and "ghost probiotics" [2]. Paraprobiotics are non-viable cells of microbes, which could be intact or broken and/or crude extracts of cells, and could produce a positive effect on the consumer when administered in sufficient amounts [1]. Recent results suggest that probiotics may exert health benefits even when they are dead (Fig. 1). This phenomenon has been labeled "the probiotic paradox" [4] and is likely explained by bioactive compounds that are released when bacterial cells dissolve in the digestive system. Preparing a probiotic product with live probiotic microbes is still challenging. The food products first undergo thermal processing, following which the live microbes are added to the products. However, adding the live microbes to the heat sterile products can unexpectedly induce contamination [2]. From the viewpoint of technological feasibilities, the advantage of using inactivated probiotic microbes is that there is no chance of such contamination, as the inactivated microbes can be added to the food products before thermal processing. Nonviable materials of microbial origin exert marked benefits compared to probiotics for safer and more stable product development [4,8].

Health-Promoting Benefits, Prophylactic, and Therapeutic Properties of Paraprobiotics
Various beneficial effects, which are originated from the living cells of probiotics can also be derived from the dead cells; indeed many reports have confirmed a variety of biological responses obtained from the consumption of heat-inactivated probiotic strains. The biological response-modifying activity of dead probiotic cells is clearly  similar to the oral administration of an immunization vaccine. In calves, the use of live Salmonella typhimurium vaccine conferred excellent protection against a challenge infection by a virulent S. typhimurium. Enterotoxigenic E. coli cells were killed by formalin treatment, and the killed cells were able to induce a strong immune response when they were used as a vaccine in human subjects. Similarly, it was observed that oral administration of inactivated whole-cell Pseudomonas aeruginosa vaccine was safe for human participants.
Research has shown that products composed of both viable and non-viable cells can produce beneficial biological responses [9,10]. To enhance immune responses (immunomodulatory activities), paraprobiotics which are made from heat-inactivated cells can be used. The components of dead cells display an antiinflammatory response in the gastrointestinal tract (GIT). Notably, specific actions are exerted by both live and dead probiotics. Although the function-mechanisms of paraprobiotics are not properly clear, some literature has suggested possible mechanisms of action for their beneficial effects. The health-promoting benefits, prophylactic, and therapeutic properties of paraprobiotics along with their possible mechanisms are shown in Fig. 2 [2,4,11].

Bioactive Substances of Paraprobiotics
Scientific evidence has demonstrated that the biogenic and paraprobiotic functions of inactivated cells of microbes, microbial fractions, or cell lysates could sustain human as well as animal health [1,12]. On the other hand, the inactivating method would be critical to correctly interpret these results. The inactivating method (Fig.  3) can disrupt the bacterial cells, and allow for the potential interaction of intracellular bioactive compounds with the host cells on administration. Different mechanistic studies have been conducted, and their results revealed that a specific chemical compound is required to induce a specific immune response, and this compound has been isolated from microbes. Dried or fragmented cells of yeast probiotics could exert probiotic activity, and produce a beneficial effect [13]. These products are primarily composed of cell wall fragments, which contain β-(1,3)-Dglucans, β-(1,6)-D-glucans, chitin, and mannoproteins [1,6]. Several other studies suggested that different components of the microbial cell, for instance, β-glucans, teichoic and lipoteichoic acids [14], cell homogenates [15], peptidoglycans (PGN), lipopolysaccharides (LPS), and DNA [16,17] can produce immunomodulating effects [18][19][20][21]. The functions of bioactive compounds from inactivated probiotic cells are summarized in Table 1.

Current Situation and Emerging Future Prospects of Paraprobiotics
The development of paraprobiotics as supplements and their application in foods and beverages comprise an important alternative to specific cases where probiotics are damaged, and not alive during processing and/or shelf life. Thus, paraprobiotics will have several applications where the addition of probiotics should involve a technological challenge. Indeed, pararpobiotic products allow for the generation of safer and more stable products over those of the live probiotic products. Therefore, the popularity of paraprobiotics is fast increasing, and in the near future, paraprobiotics will be extensively used in food, medicine, supplements, and fodder [1].
Recent studies have demonstrated that the biogenic and paraprobiotic function of dead cells, microbial fractions, or cell lysates can conserve host health [1,12,31,32]. Probiotic products prepared by using dead cells could be stored without a refrigerator, and could reduce microbial translocation risk, increased inflammatory response or infection risk in consumers [33]. Serin and Andruskiene [34] reviewed a new perspective on the functional component of foods, or so-called paraprobiotics. Nevertheless, more clinical and epidemiological studies are required to understand the role of paraprobiotics on human health.
The selection of probiotic species and strains to be used for paraprobiotic production [2], the use of appropriate methods for inactivation and delivery, the evaluation of their stability and activity in foods during shelf life, and the use of adequate methods to assess their biological effects are especially important [2]. The discovery of clear potentially beneficial immunological effects of inactivated bacteria suggests that further investigation of their mechanisms of action is required. The effects of these cell wall components, after heat treatment, on the immunostimulatory activity of LAB still needs further study. Furthermore, studies should be conducted to understand the mechanism of action used by dead cells to produce beneficial effects, and whether that mechanism is similar to that of viable cells or not. Specific binding efficiencies of probiotics, as well as paraprobiotics against infectious agents in vivo, should be analyzed.