Integrity of Probiotic Biotherapy

Abstract
Milk contains high amounts of lactose and protein which are utilized by lactic acid bacteria (LAB) for growth. LAB also ferment milk to produce products such as yoghurt and probiotic health drinks. In this experiment, we used the dilution method to enumerate the number of bacteria in yoghurt, a probiotic liquid and a probiotic capsule to compare the number of bacteria claimed by the manufacturer. The results obtained from the yoghurt and probiotic capsule and probiotic liquid dilution substantiated the manufacturers claim regarding bacterial numbers. Creation of functional foods containing sufficient consumed probiotic contents has been difficult, as processing and storage issues can affect microorganism viability. Challenges exist concerning predatory market positioning of probiotics and claims made by some probiotic manufacturers.
Abbreviations used in this paper: CFU, colony-forming units; EFSA, European Food Safety Authority; FDA, Food and Drug Administration, Food and Agriculture Organization; FAO, World Health Organization; WHO. GIT, gastro-intestinal tract. GRAS, generally regarded as safe
Introduction
In 2013, the global probiotic market was worth US$36 billion (de Simone 2019; Tripathi & Giri 2014).
Probiotics are defined as live microbial food supplements which beneficially affect the host, either directly or indirectly by improving its intestinal microbial balance and increase the resistance against pathogen invasion. The alterations and improvements are mainly in the colon, which harbors a rich flora of more than five hundred different bacterial species (1014 organisms), with a variety of health functions. It is thought that probiotic organisms can correct imbalance between the beneficial and harmful actions. Lab book
Milk fermentation began in 2500 BC and was originally used as preservation (Sarao & Arora 2017).
Circa 1900, Nobel laureate Elie Metchnikoff advanced the concept of probiotics after the discovery that ingestion of fermented milk products by Bulgarian peasants conferred a health benefit to the gastrointestinal tract (GIT) (Tripathi & Giri 2014).
Subsequently, it was found that fermenting milk can improve the bioavailability of its nutrients and additionally, that the lactic acid produced by Streptococcus, Lactococcus and Lactobacillus spp. during fermentation has an antimicrobial effect, thus sustaining a healthy balance of GIT microbiota (Rahmawati & Suntornsuk 2016).
Probiotic food should contain the required minimum viable microorganism count –  ≥105 CFU/gram at time of consumption and confer a health benefit to the host (including resistance to disease), when taken in adequate amounts. Depending on amount ingested and effects of storage, a daily intake of 10-8-10-9 probiotic microorganisms are required to create a probiotic benefit to the human organism (Tripathi & Giri 2014).
There are a number of qualities that define a quality non-pathogenic and non-toxic
probiotic. It must exhibit a beneficial effect on the host, for example disease resistance. Probiotics should contain a large number of viable cells and should be able to survive and metabolize in the GIT. Finally, probiotics must retain stability and capable of remaining viable for prolonged periods under storage conditions (Ray & Bhunia 2014).
LAB consist of a heterogeneous group of fermentative Gram-positive bacteria producing lactic acid. Status as GRAS. The fermentative and health promoting benefits claimed for lactic acid bacteria (LAB) make them essential to the food industry, having a diverse range of industrial applications (Temmerman, Huys & Swings, 2004). Although phylogenetically different to LAB, Bifidobacterium, Propionibacterium and Brevibacterium are also used in the food industry, as some strains of these taxa display LAB characteristics (Temmerman, Huys & Swings, 2004). Correct species identification is vital from a technological, ecological and safety point of view (Temmerman, Huys & Swings, 2004). Milk contains high amounts of lactose and protein which are utilised by microorganisms for growth. Bacteria can be used for fermentation of milk to produce products such as yoghurt and probiotic health drinks.
In this experiment, we used the dilution method to enumerate the actual number of bacteria in yoghurt, a probiotic liquid and a probiotic capsule to compare against the number of bacteria claimed by the manufacturer.
Fermentation of milk enhances its nutritional value through improved bioavailability of nutrients and production of substances which have a biological function. Fermented dairy products, are excellent sources of bioactive peptides. They provide numerous peptides with bioactive properties and form lactic acid and flavour compounds during fermentation and storage.
Bioactive peptides are short chains of amino acids that are produced during gastrointestinal digestion or food processing. These peptides have shown a wide range of biological activities such as anti-hypertension, anti-oxidant, anti-microbial, anti-angiotensin converting enzyme (anti-ACE) and anti-carcinogenic activities.
Lactic acid bacteria (LAB) are commonly used to ferment milk into yoghurt and other fermented milk products. The types of LAB usually used in the dairy industries are thermophilic and mesophilic strains of Streptococcus, Lactococcus, and Lactobacillus species (6). During fermentation of milk, the cell wall associated proteinase of LAB hydrolyses caseins into large peptides, which are taken up into their cells, then broken down by intracellular peptidases, resulting in a range of bioactive peptides showing, for example hypertensive or angiotensin-I- converting enzyme (ACE) inhibitory activity (Rhamawati.

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Yakult is a fermented dairy drink that contains probiotic cultures rather than yogurt cultures. The main difference between yogurt and probiotic cultures is that probiotics must have scientifically proven health benefits while yogurts do not. Another point of distinction is the type of cultures; probiotics are typically various species and strains of Lactobacilli or Bifidobacterium, while yogurt starter cultures are specifically Lactobacillus bulgaricus and Streptococcus thermophilus, according to the National Yogurt Association (Yakult website).
Aims
To isolate by serial dilution method and enumerate probiotic bacteria from natural yoghurt and two other commercially available probiotic samples for comparison of stated probiotic contents.
Materials and Methods
Study design
Bacterial strains and growth conditions
Bifidobacterium and Lactobacillus reference strains used in this study were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).
Bifidobacterium and Lactobacillus strains were grown in BSM broth (Bifidus Selective Medium, Sigma-Aldrich, St. Louis, MO, USA) and MRS broth (deMan, Rogosa and Sharpe Medium, Sigma-Aldrich) supplemented with 5 g L−1 cysteine respectively. The strains were grown at 37 ◦C for 48 h.
Lactobacillus
The probiotic LGG works by making resident gut bacteria produce anti-inflammatory products. Lactobacillus rhamnosus GG (LGG), one of the most common probiotics available, can improve a variety of digestive disorders, psychiatric disorders, and atopic dermatitis in infants and children (Rewrite with reference).
Bifidobacteria
Both genera are found in the normal intestinal flora of healthy human adults. Adherence is believed necessary for adequate, long term colonization of the gut. Evidence for true colonization and continued excretion without continued ingestion is lacking (Rewrite with reference).
Data and sample collection
Isolates used in the study were collected from various commercial sources. Each product had a slightly different isolation procedure and bacterial composition (see table 1 for examples) and different dilutions were selected for plating out, dependent on the expected final counts.
Table 1: Commonly used species of lactic acid bacteria in probiotic preparations

Bacterial genera

Species

Lactobacillus sp.

L. acidophilus, L. casei, L. delbrueckii ssp.,

 

L. cellobiosus, L. curvatus, L. fermentum, L. lactis,
L. plantarum, L. reuteri, L. brevis,  

Bifidobacterium sp.

B. bifidum, B. adolescentis, B. animalis, B. infantis

 

B. thermophilum, B. longum

Enterococcus sp.

Ent. faecalis, Ent. faecium

Streptococcus sp.

S. diacetylactis, S. intermedius

Adapted from Tripathi & Giri 2014.
Lactic acid bacteria in yoghurt
A sample of Jalna ‘ Bio-Live’ Organic Biodynamic Whole Milk Yoghurt (Lactobacillus acidophilus, Bifidobacterium & Lactobacillus casei >300,000,000 probiotic count)
(9.83g) was aseptically transferred into a tube and homogenized in the Griffin Shaker for 5 min with sterile deionised water and made up to a final volume of 40mL. The sample was then serially diluted with the same diluent. Aliquots (100µL) of 10-2 through to 10-4 dilutions were spread onto separate MRS agar plates which were then incubated at 37ᴼC. Plates containing 30 to 300 colonies were used to determine CFU/gram.
Lactic acid bacteria in probiotics (capsule)
The content of one capsule of Inner Health Plus probiotic (Lactobacillus acidophilus (NCFM) 12.5 billion, Bifidobacterium lactis (Bi-07) 12.5 billion, Bovine colostrum powder 67mg) was rehydrated (15 min) in sterile deionized water (final volume of 10mL) and then vortexed. The sample was serially diluted with the same diluent. Aliquots (100µL) of 10-6 through to 10-8 dilutions were spread onto separate MRS agar plates which were then incubated at 37ᴼC. Plates containing 30 to 300 colonies were used to determine CFU/ capsule. 
Lactic acid bacteria in probiotics (liquid vial)
Yakult probiotic (6.5 billion live Lactobacillus casei spp. shirota strain, Bifidobacterium breve spp. Yaku strain and small amounts of naturally-occurring milk sugar (lactose) per 65mL (1mL) was aseptically transferred into a tube containing sterile deionized water (9mL). The sample was then serially diluted with the same diluent. Aliquots (100µL) of 10-4 through to 10-6 dilutions were spread onto separate MRS agar plates which were then incubated at 37ᴼC. Plates containing 30 to 300 colonies were used to determine CFU/ per vial.
Results
The results obtained for the yoghurt were within the reference range provided by the manufacturer. The Probiotic product 1 (capsule) results showed a slightly higher than stated CFU yield.  Whereas, results for probiotic product 2 showed a far higher than stated CFU/g value than claimed by the manufacturer (Table 2).
Table 2: Number of bacteria claimed compared to number of bacteria in samples
 

Product

Number of bacteria claimed (CFU)

Actual number of bacteria in sample (CFU)

yoghurt (per 1g)

> 3 x 103/ 100g

5.12 x 105/100g

probiotic product 1 (per capsule)

12.5 x 109/ capsule

11.2 x 109/capsule

probiotic product 2 (per vial)

6.5 x 109/ 65mL

71.5 x 109/ 65 mL

Figure 1: CFU grown on MRS agar after 24hrs incubation at 37ᴼC for 48 hrs. Yoghurt sample (10-2 dilution) and Probiotic product 2 sample (10-6 dilution).
Discussion
In this practical, we compared the actual number of bacteria against the claimed number of bacteria in fermented milk products. The actual number of bacteria in the yoghurt sample and Probiotic product 2 could not be obtained due to the colonies being too numerous to count. However, not a negative result as the results do indicate the abundance of viable cells which may be cultured in these products. The experiment would need to be repeated with either a shorter incubation time or greater dilution to procure results.
The results for Probiotic product 1 indicate the actual number of bacteria in the sample is within an allowable range compared to the claimed amount by the manufacturer. The product is also 109 CFU/gram, which would confer a health benefit to the host if taken in adequate amounts.
Results from the bacterial colonies from the serial dilution of yoghurt and probiotic capsule were too numerous to count and would need to be repeated to ascertain reportable results. However, the results obtained from the Yakult dilution substantiated the manufacturers claimed number of bacteria.
The clinically proven health benefits of viable lactic acid bacteria include, balance of the intestinal microbiota via antimicrobial activity, a reduction of lactose intolerance and food allergies and an enhancement of immune cells (Sarao & Arora 2017). 
A study conducted on two groups, a placebo group and a Yakult group, of Japanese women showed that the Yakult group had enhanced keratinocyte differentiation and less drying of the stratum corneum compared to the placebo group (Kano et al 2013). This was thought to be due to the lower pH caused by the lactic acid bacteria which gave rise to lower serum phenol levels (Kano et al 2013).
Further testing on species by molecular methods and careful attention to safety aspects in all facets of the production, packaging and labelling of probiotics can only improve the current uncertainty regarding manufacturers stated product health benefits, especially with regard to probiotics used as treatment in established medical conditions.
Consumers of probiotics should be aware that not all of the health benefits claimed by manufacturers have been scientifically proven and that information regarding health benefits of probiotics must be obtained through clinical and independent scientific research (de Simone 2019).
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