Research Article,  J Biochem Physiol Vol: 7 Issue: 4

Juice-Based Living Probiotics Survive Stomach Acid Significantly better than Dry Powder Live Probiotics

Ahmad Reza Kamarei^1*, Howard F. Robins² and Eric Finkelstein³

¹Department of Research and Development, Doctor’s Biome, Hauppauge, NY, USA

²Department of Health Education and Communication, Doctor’s Biome, Hauppauge, NY, USA

³Department of Operations, Doctor’s Biome, Hauppauge, NY, USA

*Corresponding Author: A. Reza Kamarei,
Department of Research and Development, Doctor’s Biome, Hauppauge, NY, USA
E-mail: mantonikolaidi@gmail.com

Received date: 05 November, 2024, Manuscript No. JBPY-24-151761;

Editor assigned date: 07 November, 2024, PreQC No. JBPY-24-151761 (PQ);

Reviewed date: 22 November, 2024, QC No. JBPY-24-151761;

Revised date: 29 November, 2024, Manuscript No. JBPY-24-151761 (R);

Published date: 06 December, 2024, DOI: 10.4172/jbpy.1000176.

Citation: Kamarei AR, Robins HF and Finkelstein E (2024) Juice-Based Living Probiotics Survive Stomach Acid Significantly better than Dry Powder Live Probiotics. J Biochem Physiol 7:4.

Abstract

Keywords:

Juice-Based Probiotics; Dry Powder Probiotics; Pre-hydrated Probiotics; Probiotic Survival in HCl solution; Probiotic Resistance in Stomach

Introduction

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. The particular health benefits of probiotics include helping reduce the incidence of conditions such as antibiotic-associated diarrhea, digestive discomfort (including in irritable bowel syndrome), colic symptoms in breastfed babies, atopic issues such as eczema in infants, symptoms of lactose maldigestion, acute pediatric infectious diarrhea, upper respiratory tract infections (such as the common cold) and gut infections [1,2].

Nonetheless, there are investigators and organizations that remain skeptical about the benefits of probiotics. For example, the American Gastroenterological Association does not recommend the use of probiotics for most digestive conditions [3]. Some critics argue that the scientific evidence supporting probiotic benefits is not sufficiently robust, citing that many studies are small, short-term or yield conflicting results. Additionally, probiotics vary in dosage and bacterial strains and not all strains have the same effects. Probiotic dietary supplements are typically consumed as dry powder encapsulated in capsules and to a lesser extent in the form of tablets or gummies. Once the gelatin capsule or gummy is digested by stomach pepsin, the dry powder probiotics are exposed to stomach acid (HCl at a pH of ~1.5). Existing literature from clinical trials demonstrates either the ineffectiveness or limited effectiveness of various dry powder probiotics following ingestion.

In a randomized controlled trial, the ineffectiveness of probiotics (Lactobacillus GG) in preventing recurrence after curative resection for Crohn's disease was reported [4]. It was found that Lactobacillus GG was ineffective in preventing nosocomial rotavirus infections, whereas breast-feeding was effective [5]. It was reported that a singlestrain probiotic supplement of Lactobacillus GG changed the ileal pouch intestinal bacterial flora but did not evoke a clinical or endoscopic response and was therefore ineffective as a primary therapy [6]. In a double-blind, placebo-controlled randomized trial, Lactobacillus GG, used as an adjunct to lactulose, was found to be ineffective for the treatment of constipation in children [7].

In a double-blind, placebo-controlled randomized trial, it was determined that Lactobacillus johnsonii LA1 is ineffective for prophylaxis of postoperative recurrence in Crohn's disease [8]. Another double-blind randomized trial conducted in Indonesian children showed probiotics to be ineffective in treating acute diarrhea [9]. A randomized controlled factorial trial found that neither probiotics nor advice to chew xylitol-based gum was effective in managing pharyngitis [10].

There is also abundant evidence indicating that the survivability of probiotics in the acidic environment of the stomach is a critical determinant of their effectiveness. One study examined the impact of meals on a probiotic during transit through a model of the human upper gastrointestinal tract and showed that bacterial survival was best when given with a meal or 30 minutes before a meal [11]. Another investigation explored the survival of commercial probiotic formulations in biorelevant gastric fluids, with results indicating that liquid-based products generally provided superior survival compared to freeze-dried products [12]. Further analysis examined the effect of glucose on Lactobacillus rhamnosus GG survival after 90 minutes in simulated gastric juice at pH 2.0, finding that glucose concentrations from 1 mM to 19.4 mM enhanced bacterial survival, achieving final bacterial concentrations from 6.4 to 8 log₁₀ CFU/ml^-1. Additional experiments confirmed that glucose alone was responsible for survival in acidic conditions, as sugars that could be efficiently metabolized by the bacteria enabled ATP provision to F₀F₁-ATPase via glycolysis, facilitating proton exclusion and enhancing survival during gastric transit.

In current practice, dry powder probiotics are protected from direct exposure to stomach acid by means of acid-resistant, delayed-release enteric-coated capsules, which help ensure that the probiotics reach the less harsh environment of the small intestine and therefore realize optimal effectiveness [13]. However, these enteric-coated capsules are often more expensive than regular capsules because the coating process incurs additional cost.

We hypothesized that pre-hydrating dry powder probiotics in a fruit and vegetable juice carrier would enhance their resistance to stomach acid on account of the combined effects of cellular hydration, buffering properties and sugar content of the juice.

The aim of this in vitro study was to determine whether a prehydrated Doctor's Biome Signature Probiotic Blend (DBSPB) in a fruit and vegetable juice carrier can better survive the stomach's acidic environment (HCl at pH 1.5) compared to the same probiotic blend in its dry powdered form.

Methodology

Doctor's Biome Signature Probiotic Blend (DBSPB)

Bacteria of the genera Bifidobacterium and Lactobacillus are broadly recognized for their key roles in the human intestinal microflora. We designed a proprietary blend of five strains of Bifidobacterium and ten strains of Lactobacillus obtained from the reputable company Cultures Supporting Life (Table 1).

Table 1: Composition of Doctor's Biome Signature Probiotic Blend (DBSPB) used in this study.

Species identification of these probiotics has been performed by sequence analysis of the 16S rRNA gene and strain identification by pulse field gel electrophoresis. These probiotics have been shown to be sensitive to antibiotics and have passed microbiological assays and heavy metal analyses. They are not genetically modified, are free from allergens, are considered safe with respect to bovine spongiform encephalopathy and do not contain colorants.

Organic Green Juice (OGJ)

As an optimum liquid carrier, we chose a proprietary sterilized blend of 100% organic green fruit and vegetable juices, specifically mint, cucumber, apple, lettuce, kale, celery and lemon juice (pH 4.0).

Study Design

The evaluations used in this study and associated sample counts (Table 2).

Table 2: Summary of the study design.

Evaluation of the pre-hydrated probiotic blend

• Testing performed at room temperature.

• 0.154 g probiotic blend added to 59 ml OGJ, mixed for 1 min.

• Stored for 0 min, 30 min and 60 min at room temperature.

Evaluation of the dry probiotic blend

• Testing performed in a 37°C water bath.

• 0.154 g probiotic blend added to 59 ml HCl at pH 1.5, mixed for 1 minute.

• Stored for 0 min, 30 min and 60 min in a 37°C water bath.

Evaluation of the combined pre-hydrated probiotic blend (in juice) and the dry probiotic blend

• Pre-hydrated probiotics in OGJ and dry probiotics in HCl prepared as above.

• Combined OGJ preparation with HCl preparation in the 37°C water bath.

• Stored for 0 min, 30 min and 60 min at 37°C.

Probiotic enumeration (three replicates)

Probiotic enumeration was performed following published reference methods [14,15]. This evaluation was performed three times.

Baseline evaluation

The pH of the Organic Green Juice (three separate aliquots) was assessed (AOAC 981.12) at baseline and that of the HCl throughout the titration to achieve a final working solution with pH 1.5.

RESULTS

Visual observation

As can be observed in the representative images of enumeration plates, 30 minutes storage at 37 °C, when mixed in OGJ (“baseline”), colonies of the probiotic Bifidobacteria (1B) and lactic acid bacteria (1L) happily survive and grow. However, when the corresponding dry powder formulations (2B and 2L) are mixed with HCl solution (pH 1.5), the bacteria do not survive to form colonies. Interestingly, when the probiotic bacteria pre-hydrated in OGJ are combined with the HCl solution (3B and 3L), a substantial number survive to form colonies far more than with direct mixing of the dry powder probiotic and HCl solution (Figure 1).

Enumerative observation

The enumerations of probiotic Bifidobacteria, lactic acid bacteria and their total counts were assessed in OGJ, HCl at pH 1.5, and the combined treatment (Table 3).

Table 3: Survival of Juice-Based living probiotics and dry powder live probiotics upon exposure to stomach acid.

For probiotics hydrated in OGJ, both individual and total bacterial counts remained high and relatively constant (from 36 million CFU/g to 28 million CFU/g) for up to 60 minutes, with 36 million CFU/g serving as the baseline. For the dry powder formulation, the total bacterial count was reduced to 64,000 CFU/g upon mixing with HCl (pH 1.5), further declined to 7 CFU/g after 30 minutes, and reached a low of 2 CFU/g after 60 minutes, effectively eliminating the bacteria.

In contrast, when pre-hydrated probiotics were mixed with HCl (pH 1.5), the baseline count was immediately reduced to 3.2 million CFU/g, then decreased to 25,900 CFU/g after 30 minutes, and finally reached 5,100 CFU/g after 60 minutes, showing impressive resistance to elimination.

Graphical observation

The enumeration data in graphical form, vividly that illustrating the greatly superior survival of probiotics exposed to stomach acid (HCl, pH 1.5) when delivered in a juice carrier rather than as a dry powder (Figure 2).

DISCUSSION

From the averages of our enumerative observations, we can calculate the "Survival Ratio" at each time point of CFUs in HCl for OGJ pre-hydrated DBSPB and for dry DBSPB. In short, the prehydrated probiotics survived stomach acidity 50 times better at time zero, 3,700 times better after 30 minutes and 2,188 times better after 60 minutes (Table 4).

Table 4: Survival ratio of Juice-Based living probiotics and dry powder live probiotics upon exposure to stomach acid.

These results confirm our hypothesis that incorporating dry powder probiotics like DBSPB into a fruit and vegetable juice carrier enhances their resistance and survivability when exposed to stomach acid.

We believe this enhanced survivability is due to the combined effects of hydrating the probiotic cells, the buffering capacity of the juices and the presence of glucose in the juices. In dry powder form, probiotic cells quickly absorb proton ions from HCl, leading to their inactivation, whereas in pre-hydrated form, the cells absorb proton ions more gradually. Additionally, the juices provide buffering capacity, meaning the solution can resist pH changes when acids or bases are introduced. Finally, glucose metabolism has been shown to improve probiotic survival during exposure to gastric acid.

In light of the above, it is appropriate to designate a blend of optimal probiotics (such as DBSPB) delivered in fruit and vegetable juices as a "Next Generation Probiotic."

Conclusion

Our study demonstrates that pre-hydrating dry powder probiotics in a fruit and vegetable juice carrier significantly enhances their resistance to stomach acid, improving their survivability. The combined effects of cellular hydration, the buffering capacity of the juice, and the presence of glucose contribute to this improved survival. These findings suggest that using a juice carrier may be an effective method to optimize probiotic efficacy by protecting them from harsh gastric conditions, offering a potential advantage over traditional dry powder formulations.

Declarations

Acknowledgments

The authors acknowledge the valuable contributions of Wendy Reid (Senior Research Operations Manager) and Ben Howard (Laboratory Director) from the accredited Certified Laboratories in performing this study.

Author contributions

A. Reza Kamarei proposed, designed and oversaw this study and prepared the first draft of the manuscript. Howard F. Robins had observed the efficacy of juice-based probiotics in his patients and reviewed the draft of the manuscript. Eric Finkelstein assisted in the preparation of the samples, contractual arrangements with Certified Laboratories, conversion of tabulated values to graphs and reviewed the draft of the manuscript.

Data availability statement

The dataset (final report from Certified Laboratories) used and or analyzed during the current study is available from the corresponding author upon reasonable request.

References

1. Swanson KS, Gibson GR, Hutkins R, Reimer RA, Reid G, et al. (2020) The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of synbiotics. Nat Rev Gastroenterol Hepatol 17(11):687-701.

   [Crossref] [Google Scholar] [PubMed]

2. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, et al. (2014) Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 11(8):506-514.

   [Crossref] [Google Scholar] [PubMed]

3. Su GL, Ko CW, Bercik P, Falck-Ytter Y, Sultan S, et al. (2020) AGA clinical practice guidelines on the role of probiotics in the management of gastrointestinal disorders. Gastroenterology 159(2):697-705.

   [Crossref] [Google Scholar] [PubMed]

4. Prantera C, Scribano ML, Falasco G, Andreoli A, Luzi C (2002) Ineffectiveness of probiotics in preventing recurrence after curative resection for Crohn's disease: A randomised controlled trial with Lactobacillus GG. Gut 51(3):405-409.

   [Crossref] [Google Scholar] [PubMed]

5. Mastretta E, Longo P, Laccisaglia A, Balbo L, Russo R, et al. (2002) Effect of Lactobacillus GG and breast-feeding in the prevention of rotavirus nosocomial infection. J Pediatr Gastroenterol Nutr 35(4):527-531.

   [Crossref] [Google Scholar] [PubMed]

6. Kuisma J, Mentula S, Jarvinen H, Kahri A, Saxelin M, et al. (2003) Effect of Lactobacillus rhamnosus GG on ileal pouch inflammation and microbial flora. Aliment Pharmacol Ther 17(4):509-515.

   [Crossref] [Google Scholar] [PubMed]

7. Banaszkiewicz A, Szajewska H (2005) Ineffectiveness of Lactobacillus GG as an adjunct to lactulose for the treatment of constipation in children: a double-blind, placebo-controlled randomized trial. J Pediatr 146(3):364-369.

   [Crossref] [Google Scholar] [PubMed]

8. Marteau P, Lémann M, Seksik P, Laharie D, Colombel JF, et al. (2006) Ineffectiveness of Lactobacillus johnsonii LA1 for prophylaxis of postoperative recurrence in Crohn’s disease: A randomised, double blind, placebo controlled GETAID trial. Gut 55(6):842-847.

   [Crossref] [Google Scholar] [PubMed]

9. Hegar B, Waspada IM, Gunardi H, Vandenplas Y (2015) A double blind randomized trial showing probiotics to be ineffective in acute diarrhea in Indonesian children. Indian J Pediatr 82:410-414.

   [Crossref] [Google Scholar] [PubMed]

10. Little P, Stuart B, Wingrove Z, Mullee M, Thomas T, et al. (2017) Probiotic capsules and xylitol chewing gum to manage symptoms of pharyngitis: A randomized controlled factorial trial. CMAJ 189(50):1543-1550.

    [Crossref] [Google Scholar] [PubMed]

11. Tompkins TA, Mainville I, Arcand Y (2011) The impact of meals on a probiotic during transit through a model of the human upper gastrointestinal tract. Benef Microbes 2(4):295-304.

    [Crossref] [Google Scholar] [PubMed]

12. Fredua-Agyeman M, Gaisford S (2015) Comparative survival of commercial probiotic formulations: Tests in biorelevant gastric fluids and real-time measurements using microcalorimetry. Benef Microbes 6(1):141-151.

    [Crossref] [Google Scholar] [PubMed]

13. Corcoran BM, Stanton C, Fitzgerald GF, Ross R (2005) Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Appl Environ Microbiol 71(6):3060-3067.

    [Crossref] [Google Scholar] [PubMed]

14. Da Silva N, Taniwaki MH, Junqueira VC, Silveira N, Okazaki MM, et al. (2018) Microbiological Examination Methods Of Food And Water: A Laboratory Manual. CRC Press;

    [Crossref] [Google Scholar]

15. Roy D (2001) Media for the isolation and enumeration of bifidobacteria in dairy products. Int J Food Microbiol  69(3):167-182.

    [Crossref] [Google Scholar] [PubMed]