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Probiotics media: significance, challenges, and future perspective - a mini review
Food Production, Processing and Nutrition volume 4, Article number: 17 (2022)
The health benefits associated with probiotics have increased their application in pharmaceutical formulations and functional food development. High production of probiotic biomass requires a cost-effective production method and nutrient media optimization. The biomass production of probiotics can be enhanced by optimizing growth parameters such as substrate, pH, incubation time, etc. For economical industrial production of probiotic biomass, it is required to design a new medium with low cost. Wastes from the food industries are promising components for the development of the low-cost medium. Industrial wastes such as cheese whey and corn steep liquor are excellent examples of reliable sources of nitrogen for the biomass production of probiotic bacteria. The increased yield of biomass reduced the cost of production. This review focuses on the importance of probiotic media for biomass production and its challenges.
When food is insufficient to meet the basic health demands of the body it can be fulfilled through alternative methods using pills, powders, or other supplements. In earlier times, the food quality was improved biologically. The Romans and Greeks were well recognized for the use of fermented products (Gismondo et al. 1999). One of the common examples in this category is curd, which is considered the most important source of probiotics and is globally consumed. It is prepared by using Lactobacillus bacteria. This bacterium not only helps in the formation of curd but also positively affects the health of the gut and is extremely helpful in reducing the risk of diseases. Several scientific findings establish a positive relationship between probiotics and human health (Ranjha et al. 2021). FAO and WHO defined probiotics as “Live microorganisms, when administered in sufficient amounts provide a health benefit to the host” (FAO Joint 2007). Lactic acid bacteria (LAB) are commercially employed as food additives in dairy products and fruit juices. They alter the dynamics of the microbial community in the digestive system of the host by balancing the quantity of good and harmful microbiota (Pereira and Rodrigues 2018; Marco et al. 2021; Plessas 2021; Puntillo et al. 2022; Marchwińska & Gwiazdowska 2022). They also help to manage gastrointestinal diseases such as Crohn’s disease (Liang et al. 2021), urogenital infections (Nader-Macías et al. 2021), and pouchitis (Kuehbacher et al. 2006). These groups of microbes are produced by using fermentation technology (Marco et al. 2021). Traditional batch fermentation with suspended cells is solely used in industrial operations for food culture production, including probiotics. Continuous fermentation with probiotics has received little attention until now, even though Cha et al. (2018) examined the benefits of this technique for Bifidobacterium longum. Continuous culture, under carefully selected conditions, can result in high cell yield and process volumetric productivity, as well as a reduction in the requirement for downstream processing capacity (Cha et al. 2018; Doleyres & Lacroix 2005).
The term ‘probiotic’ was initially proposed by Lilly and Stilwell which in Greek meant ‘for life’. The term was created in contrast to the word ‘anti-biotic’ which means a substance produced by one microbe to kill another. Probiotics are a group of microbes associated with food to enhance their nutritional value and maintain gut health (Ailioaie & Litscher 2021; Milner et al. 2021). They are highly promoted for their ability to support gastrointestinal health and strengthen the immune system (Palanivelu et al. 2022). Currently, the consumption of probiotic cells via food products is in high demand. Probiotics are also considered functional foods. Functional foods are defined as foods that look like traditional foods yet have established physiological benefits. Functional food components include probiotics, prebiotics, vitamins, and minerals, which are utilized in fermented milk and yoghurts, sports drinks, infant meals, sugar-free sweets, and chewing gum (Al-Sheraji et al. 2013). Apart from the ongoing dispute over whether live probiotics are safe to take, most research papers continue to investigate the beneficial benefits of living probiotic cells in the gastrointestinal tract. So far, the focus has been on the immediate consequences of gastrointestinal problems (Mishra et al. 2018). Lactobacillus reuteri, Lactobacillus rhamnosus, Bifidobacterium, Bacillus coagulans, Lactobacillus casei, Lactobacillus acidophilus-group, Escherichia coli strain Nissle 1917, various enterococci, and the yeast Saccharomyces boulardii are some of the most prevalent probiotic bacteria (Mishra et al. 2018). According to Euromonitor, global sales of fortified/functional foods reached $292 billion in 2021, up from $274 billion in 2020. According to a Kerry poll of consumers in 16 countries, four out of ten (42%) bought more functional foods last year than they were in 2020 (Elizabeth Sloan 2022). It creates a tremendous impact on the global economy. The major products that contributed to the boost in the economy of functional foods are dairy products containing probiotic bacteria such as cheese, buttermilk, ice cream, flavoured milk, fermented milk, infant food, and whey-based beverages (Granato et al. 2010).
Health benefits of probiotics
Probiotic bacteria have gained popularity over the past two decades due to growing scientific data pointing to their positive benefits on human health. As a result, they have been used in a wide variety of products, with the food sector particularly active in researching and promoting them (Kechagia et al. 2013). Probiotics have come into action as medical remedies for gastrointestinal and non-gastrointestinal ailments such as diarrhoea, irregular bowel movements, inflammatory reactions, etc. (Depoorter & Vandenplas 2022). The maintenance of health using probiotics is based on the principle of competitive interaction of probiotics with pathogens surviving in the intestinal medium by inhibiting their harmful activities (Bermudez-Brito et al. 2012). Probiotics are safe, cheap, and capable to fight microbial infections, hence are recognized as the secondary immune system by the World Health Organisation (Zhou et al. 2005). Diarrhoea, constipation, irritable bowel syndrome, inflammatory bowel syndrome, asthma, atopic dermatitis, peptic ulcer, colon cancer, coronary heart disease, and urinary tract infections are among the gastrointestinal and non-gastrointestinal diseases for which probiotics have emerged as a promising source of medical therapy (Doleyres & Lacroix 2005). Probiotics are also used for the management of Crohn’s disease as well as vulvovaginal candidiasis in females (Prantera 2006; Xie et al. 2017). Lactose intolerance, Helicobacter pylori infection, microscopic colitis, diverticulitis prevention and treatment, and colon cancer can all be avoided by taking probiotics (Verna & Lucak 2010). Some babies, diagnosed with colic are found to provide better results after treatment with some probiotics (Zermiani et al. 2021). Escherichia coli is one of the most abundant bacteria in the large intestine of humans responsible to produce vitamin K 95 and B (LeBlanc et al. 2013). According to recent reviews, many probiotics are effective in acute viral gastroenteritis and antibiotic-associated diarrhoea (such as Clostridioides difficile toxin-induced diarrhoea). According to one study, probiotics can prevent C. difficile infections by 50% in high-risk individuals (Mills et al. 2018). Another systematic review revealed that probiotics reduced the incidence of streptococcal pharyngitis (Wilcox et al. 2019). Biological detoxification of chemical food contaminants by probiotics is another important aspect of the health benefits. Industries and agricultural practices that produce various chemical pollutants that intentionally or unintentionally enter our food are called food contaminants and have long-term negative effects on human health. Probiotics are a beneficial strategy in this situation for preventing dysbiosis caused by external pollutants and alleviating toxicity (Srednicka et al. 2021).
The most common group of probiotics are Lactobacillus and Bifidobacterium (Song et al. 2012). Other genera that are critical for obtaining effective probiotic strains are Enterococcus, Saccharomyces, Pediococcus, Streptococcus, Streptococcus salivarius, Lacticaseibacillus, and Streptococcus thermophilus, and Leuconostoc (Li et al.2022; Ranjha et al. 2021). These groups of bacteria are generally regarded as safe (GRAS), making them applicable as a food additive (EFSA 2017; Nasrollahzadeh et al. 2022). The Lactobacillus plantarum is usually used to produce fermented foods (Behera et al. 2018). The most often utilized probiotics in food and feed are Lactobacillus and Bifidobacterium, which are also added to fermented foods to boost their health benefits (Abdou et al. 2018). Lactobacillus acidophilus, Bifidobacterium spp., and Lactobacillus casei species are utilized in dairy products and have been shown to improve human health. Lactobacillus rhamnosus is a common probiotic found in the production of yogurts (Kamal et al. 2018), commercial fruit drinks (Champagne & Gardner 2008), and soy beverages (Daliri et al. 2022). Probiotics for animals have been tried using Lactobacillus acidophilus, Streptococcus faecalis, and other lactic acid bacteria (Abe et al. 1995).
Manufacturing of probiotics biomass
Lactic Acid Bacteria (LAB) and Bifidobacterium are manufactured on a commercial level to compensate for the demand of customers for probiotic dietary supplements. The probiotic supplement that is being produced commercially must have the highest possible yield, stability, and consistent performance for the intended application. It should be stable with the environmental conditions such as humidity, temperature, and pressure with rapid action without any significant delay (Fenster et al. 2019). The commercial production of probiotic cells biomass is carried out in bioreactors (Aguirre-Ezkauriatza et al. 2010). Traditional batch and fed-batch fermentation with suspended cells are almost solely used in industrial operations for food culture production, including probiotics. In batch and re-alkalized fed-batch fermentation in diluted whey (DW) media supplemented with de Man, Rogosa, and Sharpe (MRS) broth nutrients (except glucose and Tween 80), the production of a highly concentrated probiotic preparation of Lactococcus lactis CECT 539 was investigated by Malvido et al. (2019). The maximum concentrations of probiotic biomass (5.98 g/L) and nisin (258.47 BU/mL) were achieved in the fed-batch culture using DW100 medium, which was obtained at lower production costs than those projected for the fed-batch culture in DW medium. Fed-batch fermentation of Pediococcus acidilactici using a lactic acid removal system employing IR A 67 resin improved maximum viable cell concentration by 55.5 and 9.1 times, respectively, as compared to batch and fed-batch fermentation without resin (Othman et al. 2017). Cell density might be improved by modified continuous fermentation or fed-batch fermentation with cell recycling through a membrane to eliminate lactic acid. Furthermore, additional fed-batch techniques based on exponential feeding or with feedback control, such as DO stat, might boost cell density and biomass production even more (Hwang et al. 2011). Continuous fermentation with probiotics has received relatively little attention until now, although Doleyres and Lacroix (2005) recently examined the benefits of this technique for Bifidobacterium. Continuous culture, under properly selected conditions, can result in high cell yield and process volumetric productivity, as well as a reduction in the requirement for downstream processing capacity. The basic flow chart diagram to produce probiotics is given in Fig. 1. The basic requirement to produce LAB and bifidobacterial is frozen seed culture to act as mother culture consisting of a single pure strain (De Vuyst & Leroy 2007). The pure strain is checked by the Quality and Control department to counter any contamination present in the colony so that the cells are not at a risk for genetic drift. This colony of pure strain is transferred to a fermentation vessel for growth. The major ingredients for fermentation are water, nitrogen sources, carbohydrates, salts, and micronutrients that are necessary for growth (Fenster et al. 2019). The fermentations are carefully demonstrated and after its completion in the main tank, the cells are made to concentrate through the process of centrifugation so that the medium separates from it. Before the freezing process, some stabilizers are added to the medium that maintains the stability of cells. The two major stabilizers that have their different roles are, cryoprotectants and lyoprotectant (Yuste et al. 2021). Cryoprotectants protect cells from injury during freezing and lyoprotectants protect cells from freezing-drying. Cryoprotectants slow the formation of ice by raising the viscosity of the solution and retaining the amorphous structure of ice close to the cells. Lyoprotectants work by stabilizing the cell membrane's lipid bilayer structure in the absence of water (Santivarangkna et al. 2007). After blending the cells with stabilizers, further freezing processes can be carried out. The freezing can be performed by filling the solution into the cans and then immersing them in the nitrogen bath. These frozen cans are capable of being transported to different companies where they have their uses such as in beverages or foods.
Low-cost substrates and media optimization
The industrial production of probiotic bacteria at low cost is important to produce functional food incorporated with probiotic biomass. The substrates used in probiotic production must meet the rigorous nutritional needs of the strain of probiotics. According to the origin from which the bacterium was isolated, Lactobacilli and Bifidobacteria have complicated nutritional needs since they might be auxotrophic for roughly 20 amino acids. Lactobacillus plantarum isolated from plants contains fewer auxotrophies than Lactobacillus johnsonii isolated from the human digestive system (Fenster et al. 2019) MRS broth is the most extensively used medium for the culture of LAB and is the principal propagation medium at the laboratory level. However, various synthetic or natural fermentation media have been reported in recent years (Fenester et al. 2019). Low-cost substrates such as whey, maize starch, cane molasses, whole milk, fruit juices, and agro-industrial leftovers have recently been proposed by various authors based on Lactobacilli biomass production. The heat-treated cells, cytoplasmic fraction, and EPS produced from Lactobacillus acidophilus BCRC 14,079, which was cultivated on taro waste, displayed an antiproliferative effect on HT 29 and CaCo-2 cell lines, which is an intriguing example (Hsieh et al. 2016). After applying statistical experimental design to develop antioxidant-rich beverages that would aid in the prevention of chronic illnesses, the growth of dairy probiotics on djulis, a traditional Taiwanese drink prepared by the fermentation of Chenopodium formosanum, was accomplished (Kuo et al. 2021). The evaluation of media is critical for lowering costs, which may be up to 30 times cheaper than MRS, and for producing the precise metabolites required for each strain (Boontun et al. 2020). Strains have different growth circumstances and dietary needs. When the active metabolites or postbiotics are identified, culture conditions may be tuned to obtain high levels of synthesis of the molecules of interest, such as EPS, where fermentation duration, nitrogen quantity and source, and temperature have all been shown to be important (Amiri et al. 2019). For this various statistical tool are used to optimize the diverse cultural and nutritional factors to get an increased yield of probiotic biomass which reduce the cost of production (Manzoor et al. 2017). Plackett–Burman's design was used to optimize various cultural parameters by Pandey (2016) for biomass production of Bacillus coagulans and reported that the glucose concentration, C/N ratio, and agitation speed significantly affected factors however mineral concentration and pH had negligible effects (Pandey 2016). Taguchi's experimental design was applied to find the most significant variables from the eleven factors on the growth of Lactobacillus casei ATCC 334. Three factors such as carbon and nitrogen source i.e., palm date powder and tryptone, and agitation rate were found to be the most significant variable. The optimum conditions of the three significant variables were obtained by the response surface methodology of Box-Behnken which include date powder, 38 g/L; tryptone, 30 g/L; and an agitation rate of 320 rpm (Eyahmalay et al. 2020). Increased 171 biomass production of L. plantarum LP02 and L. plantarum Pi06 by optimizing the medium using a combination of the Taguchi array design and Box- Behnken design. Hwang et al. (2012) have been recently reported. The factors such as lactose, inulin, yeast extract concentration, and culture pH were optimized by using response surface methodology to maximize the growth of Bifidobacterium animalis subsp. Lactis. The concentration of yeast extract is most significantly affecting variables along with inulin, concentration, and culture pH (Hwang et al. 2012). Taguchi design and Box-Behnken design (RSM) were used for the determination of the most significant variables among the culture parameters including cost-effective carbon source cheese whey with corn steep liquor in all possible combinations for enhanced biomass production of Lactobacillus plantarum AS-I4 (Anvari et al. 2014). The conventional method i.e., “one factor at a time” was replaced by response surface methodology (RSM) for quick and effective optimization of the cultural and physical condition of probiotic biomass production (Abdulrazzaq et al. 2022; Manzoor et al. 2017). Response surface methodology with a central composite design has often been used for the optimization of biomass yield of Lactobacillus rhamnosus (Ridwan et al. 2021), Bacillus coagulans (Wang et al. 2020), and Bifidobacterium longum (Sen & Babu 2005).
Challenges regarding the high yield of biomass of probiotics
Probiotic lactobacilli are nutritionally fastidious organisms. Therefore, their viability and growth activity are commonly influenced by growth factors such as medium formulations, pH, temperature, and others (Chang & Liew 2013; Terpou et al. 2017; Dang et al. 2021). The less cell mass production of lactic acid bacteria during its industrial production using a bioreactor is attributed to the reduced growth rate of cells and high production of lactate (Du Toit et al. 2013). During the production of probiotic cell mass using a bioreactor, it is important to maintain conditions such as optimum temperatures, pressures, and pH levels essentially inside the bioreactor, as these conditions are different for the growth of different types of probiotics. Probiotic, Lactococcus lactis gives the highest yield of biomass i.e., about 20 g/ L after 30 h of incubation on mono-glucose feeding under uncontrolled pH and static dissolved oxygen of 30% (Elmarzugi et al.2010). The freezing or lyophilization process damages the probiotic cells and reduces their viability which can be prevented by using cryoprotectants and lyoprotectants (Martin et al. 2015). Rehydration of dried cells is also essential for maximum productivity. When the cells are not provided with proper conditions, they are at the risk of losing their viability. Several studies have shown that depending upon the applied re-healing conditions such as a buffer (Abe et al. 2009), pH, duration, sugar content (Muller et al. 2010), and rehydration temperature (Jankovic et al. 2010) and the difference in the final concentration can even lead to the difference of 1 log cycle. These observations suggest that a large proportion of probiotic cells may be killed or made uncultivable depending on the rehydration conditions. Hence, the conditions of rehydration play a very important role in the productivity of biomass production at the commercial levels.
Another challenge that is faced in the biomass production of probiotic cells is the conditions that can affect the functional properties of probiotic cells (Jankovic et al. 2010). Moreover, the time of harvesting also influences the functional properties of the cells (Fayol-Messaoudi et al. 2005). Last but not least, the challenge of biomass production of probiotic cells includes its economic perspective which is the backbone of any industrial or commercial production (Kolacek et al. 2017). If the production is attained at a cheap cost, the sale is high, and therefore, consumers will be high in number. The cost in the market makes it comfortable for the users or consumers to buy probiotics for their consumption. This entire perspective is very essential for the growth of the nation as well, because of the good health of the people and more contribution in the exports. The large export claims to be the larger holder of the Gross Domestic Product (GDP) which plays an important role in increasing the economy of a nation.
Challenges interfering with the present scope
On ingestion of the probiotic should tolerate the condition provided by the stomach and intestine and maintain its cellular integrity and functional properties. The bile juices inhibit lactic acid bacteria as compared to other probiotic cells. S. Thermophilus was found to be most sensitive to it. Hydrophobicity or the ability of the probiotic cells to adhere to hydrocarbons less for the lactic acid bacteria as compared to other probiotics (Tarique et al. 2022). The lack of scientific proof for the benefits of probiotics and some associated harmful effects reduces its application in product formulation (Pohjanheimo & Sandell 2009). The product should have negligible harmful effects to build up its market and authorities’ approval. Any product that must be sold in the market for the consumer’s use needs the essential approval from their related authorities (Foligné et al. 2013). This is done to make the selling of the product legal so that no other companies or consumers can put any questions about the quality of the product. If the probiotics are being used to manufacture a food product in India, then, the approval can be issued by the agencies like FSSAI (Food Safety and Standards Authority of India) (Singh et al. 2013). An individual becomes a consumer of the product when able to use it as per their ideas and views. It is very essential to spread awareness about the benefits and uses of the product that we manufacture to induce self-inspiration in an individual to buy that product. The quality and safety of the product decide the market demand for the product (Bei & Chiao 2001). The growth of a company is maintained only if the quality of the product is always better than the consumer’s expectation. Role of culture media on the biomass production of probiotics. A culture medium is a special type of medium or environment that is used in microbiological laboratories to grow different kinds of microorganisms. These media are also used for the growth of probiotic cells. A culture medium is a very essential part of the production of the colony of any kind of microorganism (Neidhardt et al. 1974). The optimized media for different probiotics is given in Table 1. The basic elements of a culture medium comprise a source of carbon, nitrogen, minerals, vitamins, growth factors, and water. The medium in which the biomass is to be produced plays a very important role in its production. For larger production of biomass require optimum cultural and physical conditions of production. These conditions help for the better growth of the cells with the required output. Media conditions need to be maintained periodically because they act as the life-supporting mechanism for probiotic cell growth and contribute to most of the high yield of biomass production (Marova et al. 2012).
Probiotics are a group of microorganisms that have health benefits. These are never taken as medicine, but as a food supplement (Jankovic et al. 2010). Since the major probiotics are unicellular bacteria; They can be easily cultured or grown by providing necessary media and essential conditions like optimum temperature, pH, nutrients, and minerals. Probiotics can be produced on a large scale as per the requirement of the cells to be used by functional foods. The conditions mandatory for their growth can be easily created in a medium without any huge investment. In industries their production achieved in large tanks called the Bioreactors (Brinques et al. 2010). Different cells consume different materials; Therefore, different cells are cultured using different bioreactors designed accordingly. Probiotics efficiency can be increased if it multiplies in the medium where it is being used. This is possible only when the optimum conditions are continuously provided for these cultures. (Jangra et al. 2016). Along with the number of health benefits, probiotics also have some ambiguities (Obafemi et al. 2022). It has been observed that in the case of young children with a weakened immune system or severe illness, probiotic cells can enter the bloodstream by a process called bacteraemia leading to sepsis. In this condition, the body produces an incredibly significant immune response, including heavy breathing, which can be fatal in most cases. Since the immune system is already weak or fighting the illness, further immune responses are just responses with no more significant production of antibodies in response to bacterial activity (Singhi & Kumar 2016).
It can be concluded from the above review that the optimization of cultural and physical variables plays a critical role to obtain a significant yield of probiotic biomass on an industrial scale. The utilization of cheap agro-waste like whey, corn steep liquor, date powder, etc. can reduce the cost of biomass production for probiotics. Application of statistical tools to optimize medium composition, pH temperature, agitation, etc. also helps in improving the yield of probiotic biomass.
Availability of data and materials
The datasets used and analyzed during the present study are available from the corresponding author on reasonable request.
Food and Agriculture Organization of the United Nations
World health organization
De Man, Rogosa and Sharpe
Revolution per minute
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Kumar, V., Naik, B., Kumar, A. et al. Probiotics media: significance, challenges, and future perspective - a mini review. Food Prod Process and Nutr 4, 17 (2022). https://doi.org/10.1186/s43014-022-00098-w
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