Innovative Probiotic Lactic Yeast for the pharmaceutical, nutritional and agricultural industries

Candida albicans, significant decrease.

Trial#129: Verification of the presence of Candida albicans in the gastrointestinal tract in healthy subjects, after supplementation of the lactic yeast Kluyveromyces marxianus fragilis B0399, through the examination of the feces.

Prof. Giorgio Mustacchi *1; Dr, Teresa De Monte*2; Dr. Franca Bearzi*3;Dr. Flaviano Collavini*4;Dr. Paolo Valles*5;
Dr. Paola Lovrovich*6.


 
0 - Summary:
 
The object of this study was to verify and evaluate the presence of candida albicans in the feces of 23 patients, before and after treatment with Kluyveromyces marxianus fragilis B0399.
The statistical analysis of the results obtained shows that there is a significant decrease in the presence of Candida albicans ---from those found in the pre-treatment analyses to those found in the post-treatment analyses. This confirmed that Kluyveromyces marxianus B0399 has the capacity to limit the development of Candida albicans  at the intestinal level.
We know that the intestine constitutes the natural receptacle for this fungus and that the importance of this type of trial in-vivo is self-evident.
Since this probiotic may be taken simultaneously with antibiotics thanks to its particular resistance, its presence would inhibit the phenomenon of re-colonization of Candida albicans (spore-forming) after the treatment with fungicides and antibiotics have ended, thus avoiding the impoverishment of the intestinal flora.
 
1 - Structures employed in the experimentation
 
*1 Prof. Giorgio Mustacchi (Oncological Center for Health Services no. 1—triestina, University of Studies of Trieste):
*2 Dr. Teresa De Monte, medical surgeon.
*3 Dr. Franca Bearzi, laboratory analyses of the nursing home “Pineta del Carso” (Aurisina- Trieste), preparation of sample in suspension (attachment B)
*4 Dr Flaviano Collavini, CATAS S.p.a, environmental and agri-dietary (microbiological laboratory) , CCIAA of Udine ( attachment C) microbiological analyses.
*5 Dr. Paolo Valles, microscopic analyses
*6 Dr. Paola Lovrovich, Doctor in biology, statistical analyses.
 
 
 
 
2 - INTRODUCTION:
 
 Candida albicans is a spore-forming fungus which is normally present in the intestine of humans in a non-pathogenic form. The intestine constitutes the principal receptacle for the Candida.
As far back as 1978 it was demonstrated and established that the transition from the spore-forming form to the pathogenic form of the candida was determined by the alteration or destruction of the normal intestinal native probiotic flora. In these conditions, the polymorphic fungus, which  is highly invasive when it has no antagonists (probiotic yeasts and bacteria), colonizes the areas of the body in which the conditions of the environment are favorable. This happens when the beneficial intestinal flora is reduced or destroyed for various reasons causing its lactic acid production to diminish and the pH level to become less acidic and more basic and therefore suitable to the colonization of the fungus. When this happens, the Candida becomes pathogenic a migrates from the intestine to other parts of the body.
The treatments with fungicides and antibiotics in an acute phase eliminate the Candida and the serioussymptomatology. However, these drugs significantly alter the intestinal flora, destroying its equilibrium. It’s not unusual to have a situation in which, once the treatment is finished, the spores of the Candida (which are highly resistant) take advantage of the intestinal flora’s imbalance and once again attack the more sensitive areas. The addition of treatment with probiotics in conjunction with the fungicides and antibiotics is vital in recuperating and maintaining an equilibrium.
Often, Candida is referred to as a “silent epidemic,” given the long series of pathologies which have often been misdiagnosed or not recognized by the anamnesis and are related to infections caused by this fungus.
One of elected sites for migration of Candida in its pathogenic form , is the vaginal mucosa. The resulting pathology, Candida albicans Vaginitis, is a particularly disturbing infection, both for the difficulty in eradicating the pathogenic agent and for the relapses which may occur over time.
 
 
A treatment with selected probiotic yeasts and bacteria, capable of normalizing the beneficial bacterial flora, impeding the transformation of Candida albicans into the pathogenic form and directly blocking its development in the vagina, has gained the recognition today as being a fundamental and vital defense.
Probiotic organisms are defined by the guidelines of the FAW/WHO (Cordoba, Argentina 2001) as living organisms which, when taken in an adequate quantity, create beneficial effects in the subject.
Various studies underline the effectiveness of probiotics:
-          in the modulation of the immune system (Matsuzaki T. 1),
-          in the prevention and treatment of imbalance of microbial intestinal flora which can provoke diarrhea or syndromes caused by the degeneration of inflammatory reactions (e.f. Crohn’s Disease, Irritable Bowel Syndrome) (Castagliuolo M.S. et al.2, Gorbach S.L.3),
-          in the reduction of the development of allergic phenomena like asthma and eczema in children (Benn C. et al.4) if used by the mother when pregnant.
In the reduction of the risk of infections in the genital-urinary tract (Senok A.C. et al. 5, Reid G. et al. 6, Reid G. et al. 7, Reid G. 8). 
 
 
It has furthermore been demonstrated that many yeasts show a significant killer-type activity against pathogenic fungi of clinical importance (Sugisaki Y. et al. 9, Walzer G.M. et al. 10,Cerikcioglu N. 11). This activity was attributed to the production of proteinic-type toxins.
In particular, a possible anti-fungus action of di Kluyveromyces marxianus B0399 has been reported: it is a homo-fermenting yeast of human diet origin, used as a probiotic for persons affected by intestinal problems stemming from a imbalance of microbial intestinal flora (meteorism, constipation alternating with diarrhea, difficulty in assimilating, etc.) and/or lactose intolerance. The yeast, in fact, produces the enzyme βgalactosidase.
 
Objective of the study
 
 
The objective of this study was to verify and evaluate the presence of Candida albicans in human feces before and after taking Kluyveromyces marxianus fragilis B0399 at the dosages recommended by the distributing company.
 
A few notes regarding the active ingredient in the trial.
 Kluyveromyces marxianus fragilis B0399 is a lactic yeast with characteristics which differ from those of bifidobacter and the yeast Saccharomyces. It has been used for some time now as a probiotic in the zootechnical field as well (Commission Regulation EC 773/06). It is a eukaryotic cell (Lachance M.A   12)   equipped with an elevataed lactasic enzymatic activity ((β-galactosidase). It therefore ferments lactose, producing lactic acid. The enzymatic activity, in anaerobic conditions typical of the intestine, is homo-fermenting, in that it transforms all the resulting glucose into lactic acid, without the production of gas (CO2) (ex.
Saccharomyces) (Vananuvat-Kinsella13 ,Wasserman-Hopkins-Porges14). This contributes in modulating the intestinal environment, reducing the pH. It has also proven to be particularly resistant to antibiotic action (Voughan 15 )
 
 Kluyveromyces marxianus fragilis B0399 is able to resist gastric shock. This ability was first tested in vitro (Susmel and Stefanon17 ) through the measurement of the fermentative capacity before and after gastrointestinal digestion. The resistance to digestion proved to be elevated.
 
The capability of intestinal colonization after treatment with Kluyveromyces B0399 was later demonstrated  through the examination of human feces ( trial 130 Mustacchi at all,16) .
The ability of bypassing the gastric barrier was also confirmed in in vivo trials on monogastric animals such as piglets (which have a digestive system very similar to that of humans) (Lovrovich P. 18 ) and horses, by testing the presence of Kluyveromyces B0399 in the feces and the modification of the pH in the colon ( Lowell R. S. 19. Susmel-Stefanon  20,Bosi P. 21.)
 
The benefits of Kluyveromyces B0399 have also been demonstrated by the various studies on cases of colon disorders. (Andreoli S.22,   Bottona-Parisi-Zilli 23).


3 – MATERIALS AND METHODS
 As reported by the producing company (Turval Laboratories srl of Udine) the strain utilized is Kluyveromyces fragiilis marxianus B0399, deposited at the BCCM-Belgium Coordinated Collections of Microrganism, Culture Collection Mycoteque de l’Université Catholique de Lovain (Belgium) with the trade-mark B0399, (Attachments A, A2)
 
 
 
 
 
3.3.4 – Product object of the study and dosage
 
 The product in capsule form, which is normally found on the market in pharmacies and
has been notarized by the Italian Ministry of Health, was utilized.
 
The active ingredient Kluyveromyces marxianus fragilis B0399 was administered at a dosage of one capsule a day (20*106 cfu per caps.), for a total daily dosage of 20*106 CFU of Kluyveromyces B0399, for 14 days.
 
3.1 – Subjects enrolled
 
23 healthy subjects were enrolled in the same period and in different geographic areas, all over the age of 18 ( ages 23 to 72, with average age of 49), 8 males and 15 females.
 
17 of the 23 subjects were from the areas of Udine and Trieste and were overseen by the team of Prof. Giorgio Mustacchi (Oncology Center for Health Services #1 – Triestina, Università degli Studi di Trieste) and were responsible for the recruiting and diagnostic phases.
 
The remaining 6 subjects were soldiers in the military at the barracks of Venzone (Udine), overseen by Dr. Teresa De Monte.
 
The evaluation of the state of health was made through medical consultation for the collection of all the necessary case-history data. In particular, the subjects were asked which, if any, drugs they had taken during the six months prior to testing; which, if any, recent illnesses they had had; which, if any, symptoms they experienced, and what was the typology of their routine, everyday diet.
 
Criteria for exclusion
 
Subjects were excluded who had: taken antibiotics or probiotics in the three months prior to testing, a severe chronic illness and/or other disorder of the colon which causes fragility of the mucosa, celiac disease, an intestinal occlusion and sub-occlusion, previous abdominal surgery with the exception of hernia and appendectomy, taken antipsychotic drugs in the previous three months or steroids in the preceding month, an intolerance to lactose or immunodeficiency, scare compliance.
 
During the period of treatment the assumption of drugs which could alter the motor function or intestinal absorption were not allowed, including laxatives and anti-diarrhea, products capable of altering the intestinal bacterial flora (antibiotics and commercial products containing probiotcs.) 
Examination of the fecal samples
 
 
The feces of the people selected were analyzed for the verification of the presence of Candida albicans.
 
Before the beginning of the treatment with Kluyveromyces marxianus fragilis B0399, samples were collected at time T0 (T with 0) to determine the initial state of the fecal composition.
 
To determine the evolution in time and the influence of the treatment with Kluyveromyces marxianus fragilis B0399, the analysis of the fecal samples was repeated after 14 days of administration (time T14).
 
Initial criteria of evaluation
 
To determine the presence of Candida albicans in the fecal sample, a screening of the colonies (of yeast) positive at the Sabouraud agar was conducted through the identification of the colonies of Candida (Medium Chromoalbicans agar) and through their examination at the microscope ( contrast of phase 12,5 x 40).
 
Collection of samples:
The fecal material was collected from the individual subjects in sterile containers (BIO-BOX , container for feces samples, for.me.sa.) and delivered to the doctor in charge. All containers with fecal material were carefully preserved in refrigerators at a constant temperature of 4°C.
 
Homogenization of the samples for analyses
 
All original samples were prepared with the aim of obtaining the most homogeneous material possible to execute the analyses and the count of the yeasts quantitatively.
The laboratory prepared the suspension of known concentration beginning with the initial solid sample.
The dilution of the fecal material was done with sterile physiological solution.
The dates of the collection and arrival of the samples are indicated in Tab 1.
 

 

Tab 1 Summary chart of collection and arrival dates of samples, both at Time zero (T/0) and Time 14 (T/14)
 
Sample
 No.
Date of sample collection
Date of sample arrival
Sample
 #
Date of sample collection
Data of sample arrival
 
 
 
 
 
 
1 T/0
 
23/06/09
24/06/09
1 T/14
06/07/09
07/07/09
2 T/0
 
23/06/09
24/06/09
2 T/14
06/07/09
07/07/09
3 T/0
 
23/06/09
24/06/09
3 T/14
06/07/09
07/07/09
4 T/0
 
‘23/06/09
24/06/09
4 T/14
06/07/09
07/07/09
5 T/0
 
23/06/09
24/06/09
5 T/14
 06/07/09
07/07/09
6 T/0
 
23/06/09
24/06/09
6 T/14
06/07/09
07/07/09
7 T/0
 
23/06/09
24/06/09
7 T/14
06/07/09
07/07/09
8 T/0
 
23/06/09
24/06/09
8 T/14
06/07/09
07/07/09
9 T/0
 
23/06/09
24/06/09
9 T/14
06/07/09
07/07/09
10 T/0
 
23/06/09
24/06/09
10 T/14
06/07/09
07/07/09
11 T/0
 
23/06/09
24/06/09
11 T/14
06/07/09
07/07/09
12 T/0
 
23/06/09
24/06/09
12/ T/14
06/07/09
07/07/09
13 T/0
 
23/06/09
24/06/09
13 T/14
06/07/09
07/07/09
14 T/0
 
23/06/09
24/06/09
14 T/14
06/07/09
07/07/09
15 T/0
 
23/06/09
24/06/09
15 T/14
06/07/09
07/07/09
18 T/0
 
23/06/09
24/06/09
18 T/14
S. not collected
S. not collected
19 T/0
 
23/06/09
24/06/09
19 T/14
06/07/09
07/07/09
2 VT/0
06/07/09
07/07/09
2VT/14
20/07/09
21/07/09
7VT/0
06/07/09
07/07/09
7VT/14
20/07/09
21/07/09
8VT/0
06/07/09
07/07/09
8VT/14
20/07/09
21/07/09
10VT/0
06/07/09
07/07/09
10VT/14
20/07/09
21/07/09
13VT/0
06/07/09
07/07/09
13VT/14
20/07/09
21/07/09
14VT/0
06/07/09
07/07/09
14VT/14
20/07/09
21/07/09
 

 

 
 
The principal characteristics of the subjects and the dates of collection are indicated in Tab2.
Tab 2 : Characteristics of the subjects and collection times
Subjects
 
Sex
Zone
Age
Collection T/0
Prelievo T/14
 
# on label
Date
# on label
date
A. S.
F
Trieste
39
1 T/0
23.06.09
1 T/14
06.07.09
G. D.
M
Trieste
50
2 T/0
23.06.09
2 T/14
06.07.09
R. C.
F
Trieste
53
3 T/0
23.06.09
3 T/14
06.07.09
F. S.
F
Trieste
49
4 T/0
23.06.09
4 T/14
06.07.09
D. A.
F
Udine
38
5 T/0
23.06.09
5 T/14
06.07.09
D. A.
F
Trieste
38
6 T/0
23.06.09
6 T/14
06.07.09
S. L.
F
Trieste
57
7 T/0
23.06.09
7 T/14
06.07.09
M. N.
F
Trieste
66
8 T/0
23.06.09
8 T/14
06.07.09
C. M.
F
Udine
59
9 T/0
23.06.09
9 T/14
06.07.09
M. M.
M
Trieste
72
10 T/0
23.06.09
10 T/14
06.07.09
G. G.
F
Trieste
64
11 T/0
23.06.09
11 T/14
06.07.09
B. G.
F
Trieste
70
12 T/0
23.06.09
12 T/14
06.07.09
C. L.
F
Trieste
49
13 T/0
23.06.09
13 T/14
06.07.09
B. C.
F
Trieste
55
14 T/0
23.06.09
14 T/14
06.07.09
M. L.
F
Trieste
44
15 T/0
23.06.09
15 T/14
06.07.09
C. M.
F
Trieste
35
18 T/0
23.06.09
18 T/14
06.07.09
C. A.
M
Trieste
57
19 T/0
23.06.09
19 T/14
06.07.09
V.Z.
M
Venzone
44
2 VT/0
06.07.09
2VT/14
20.07.09
F.D.
M
Venzone
30
7VT/0
06.07.09
7VT/14
20.07.09
G.D.
M
Venzone
29
8VT/0
06.07.09
8VT/14
20.07.09
C.D.
F
Venzone
23
10VT/0
06.07.09
10VT/14
20.07.09
A.P.
M
Venzone
31
13VT/0
06.07.09
13VT/14
20.07.09
G.S.
M
Venzone
39
14VT/0
06.07.09
14VT/14
20.07.09
 

 

Analyses conducted on samples obtained at time T 0
Count of total yeasts at candida identification.
1) Presence and quantification of microorganisms (yeasts) positive at Sabouraud agar.
2. To evaluate the presence of candida, a screening of the colonies (of yeast) positive at Sabouraud agar was conducted through the identification of the colonies of Candida (Medium Chromoalbicans agar) and through their examination at the microscope (contrast of phase 12,5 x 40).
 
 
Analyses conducted on samples obtained at time T 14
 
Count of total yeasts at candida identification.
1) Presence and quantification of microorganisms (yeasts) positive at Sabouraud agar.
2) To evaluate the presence of candida, a screening of the colonies (of yeast) positive at Sabouraud agar was conducted ( like above) through the identification of the colonies of Candida (Medium Chromoalbicans agar) and an examination on all the plates were conducted at the microscope (contrast of phase 12,5 x 40).
 
 
 
 
 
4 – STATISTICAL ANALYSIS:
 
For the analysis of the information obtained from the answers in the pre-treatment and post-treatment questionnaires, the Chi-square test of Yates and the Chi-square test of Pearson (with Vassar Stats: Statistical Computation Web Site) was selected, with the aim of verifying if their differences were purely random or not. If the difference is not random, then it is considered “statistically significant.”
 
Chi-square: test Initially, the existing difference between the two series of data to compare is considered “hypothesis zero”.   Hypothesis zero simply asserts that the difference observed – whatever the entity- is purely random. This hypothesis can be either accepted or rejected on the basis of the result of the statistical test.
If on the basis of this hypothesis the calculated value of χ² is greater than a certain critical value, we will have to conclude that the frequencies observed significantly differ from the frequencies anticipated and we will have to refute HO at the corresponding level of significance. Otherwise, we will have to accept it, or at least not refute it. This procedure is called chi-square test of the hypothesis.
 
 
5 - RESULTS:
 
 
The results are evaluated in 22/23 of enrolled subjects, with the withdrawal of one patient on itinerary.
 
Tab 5.1a: Results obtained at time T0.
 
Data of sample collection
Sample
Total yeasts
CFU/g of feces
Candida albicans
23/06/09
1 T/O
2X10 3cfu/g
absent
23/06/09
2 T/O
Absent
absent
23/06/09
3 T/O
4X10 3cfu/g
absent
23/06/09
4 T/O
1X10 3cfu/g
present
23/06/09
5 T/O
6X10 3 cfu/g
present
23/06/09
6 T/O
80cfu/g
present
23/06/09
7 T/O
Absent
absent
23/06/09
8 T/O
6,5X10 3cfu/g
absent
23/06/09
9 T/O
7X10 3cfu/g
absent
23/06/09
10 T/O
20 cfu/g
absent
23/06/09
11 T/O
8X10 2cfu/g
present
23/06/09
12 T/O
2X10 5cfu/g
present
23/06/09
13 T/O
4X10 2cfu/g
present
23/06/09
14 T/O
1X10 4cfu/g
present
23/06/09
15 T/O
1,6X10 2cfu/g
present
23/06/09
18 T/O
80cfu/g
absent
23/06/09
19 T/O
absent
absent
06/07/09
2 V T/O
2,4 X10 4cfu/g
absent
06/07/09
7 V T/O
absent
absent
06/07/09
8 V T/O
2,4X10 4cfu/g
absent
06/07/09
10 V T/O
2,4X10 4cfu/g
present
06/07/09
13 V T/O
2,4X10 2cfu/g
absent
06/07/09
14 V T/O
1,7X10 4cfu/g
present
 

 

Tab 5.1b: Results obtained at time T14
 
Date of sample collection
Sample
Lactic yeasts (kluyveromyces B0399)
CFU/g di feci
Candida albicans
06/07/09
1 T/14
7X104cfu/g
absent
06/07/09
2 T/14
5X10 4cfu/g
absent
06/07/09
3 T/14
5,7X10 4cfu/g
absent
06/07/09
4 T/14
7X10 4cfu/g
absent
06/07/09
5 T/14
2,5X10 4cfu/g
absent
06/07/09
6 T/14
2,5X10 4cfu/g
absent
06/07/09
7 T/14
1,8X10 5cfu/g
absent
06/07/09
8 T/14
3X10 4cfu/g
absent
06/07/09
9 T/14
Non determinabile
absent
06/07/09
10 T/14
3,2X10 5cfu/g
absent
06/07/09
11 T/14
2,5X10 4cfu/g
absent
06/07/09
12 T/14
4,4X10 5cfu/g
present
06/07/09
13 T/14
1,7X10 3cfu/g
present
06/07/09
14 T/14
2,5X10 4cfu/g
absent
06/07/09
15 T/14
3X10 4cfu/g
absent
06/07/09
18 T/O
withdrawn
withdrawn
06/07/09
19 T/14
1,5X10 5cfu/g
absent
20/07/09
2 V T/14
2,8X10 6cfu/g
absent
20/07/09
7 V T/14
4,6X10 3cfu/g
absent
20/07/09
8 V T/14
2,2X10 6cfu/g
absent
20/07/09
10 V T/14
4X10 6cfu/g
absent
20/07/09
13 V T/14
2,4X10 6cfu/g
absent
20/07/09
14 V T/14
3,9X10 5cfu/g
present
 
 

Tab 5.1: frequencies of presence and absence of Candida albicans before and after treatment with Kluyveromyces marxianus fragilis.

 

 

Pre-administration

Post-administration

Presenza di candida

10

3

Assenza di candida

12

19

 

Tab 5.3: risults of the statistical analysis of the  data shown  in Tab 2.

 

 

Chi-square

Phi

Yates

Pearson

-0,35

3,93

5,35

P

0,047432

0,020722

 

 

Fig 5.1: Representation of the cases of presence or absence of Candida albicans before and after treatment with Kluyveromyces marxianus fragilis BO399.
 
 
The statistical analysis shows that there is a significant difference (with p< 0,05) between the presence of Candida albicans found in the pre-treatment analysis and in those post-treatment ( Chi-square Yates = 3,93 con p = 0,04743; Chi-square Pearson = 5,35 con p = 0,020722).
 
 
Profile of toxicity
 
None of the subjects treated complained of any side effects.
 
 
7 - CONCLUSIONS:
 
The analysis in the fecal samples of Candida albicans shows a reduction of the presence of the fungus after treatment with Kluyveromyces marxianus fragilis BO399.
The statistical analysis shows that there is a significant difference (with p< 0,05) between the presence of Candida albicans found in the pre-treatment analysis and in those post-treatment ( Chi-square Yates = 3,93 with p = 0,04743; Chi-square Pearson = 5,35 with p = 0,020722). It was therefore demonstrated that Kluyveromyces marxianus B0399 has the capacity of influencing the development of colonies of C.albicans at the intestinal level.
We know that the intestine constitutes the natural receptacle of this type of fungus and therefore the importance of this observation in vivo is evident.
Since this probiotic may also be taken with antibiotics ( thanks to its proven resistance) the phenomenon of re-colonization of Candida albicans (spore-forming) after the treatment with fungicides and antibiotics would be inhibited since an impoverishment of the intestinal flora would be avoided.
 

Laboratori Turval Italia Srl – Head Office and Research Dpt: Scientific and Technological Park "L. Danieli"
via Jacopo Linussio, 51 – 33100 Udine (UD) – P. IVA 01883020305