Dr. Sandro Andreoli*
Dr. Paola Lovrovich ( statistical analysis)
*Doctor in Medicine and Surgery; Specialist in Diseases of the Degistive System
Endoscope specialist at the S.Camillo Hospital, Treviso
Gastroenterology consultant at the Città della Salute, Torreano di Martignacco (Ud)
Gastroenterology consultant at the Salus Alpe Adria Pagnacco (Ud)
Gastroenterology consultant at the Clinica S. Eufemia Grado (Go).
Private Studio in Via Duino 1, 33100 Udine

SUMMARY:

Irritable Bowel Syndrome is provoked by a multitude of causes (physiological, psychological, environmental and behavioral) therefore no effective specific therapy exists, but rather multiple therapies that are aimed at the causes which are subjectively most important. This study, based on the gathering of symtomatological data before and after the treatment, demonstrates that the prolonged use of Kluyveromyces marxianus fragilis B0399 significantly improves the clinical picture and above all improves the quality of life of these patients.

INTRODUCTION:

Since 1998, the use of the lactic yeast Kluyveromyces marxianus fragilis B0399 is applied as a support aimed at significantly limiting the symptomology of the irritable colon, give significant proof of effectiveness, so much so as to encourage further study.
The objective of this study was to verify if the cyclical use of the lactic yeast Kluyveromyces marxianus fragilis B0399 improves the symptomology in patients affected by Irritable Bowel Syndrome (IBS). The utilization of subjects affected by IBS is recognized internationally (as illustrated in “The EFSA Journal (2008)853,1-15) as experimentation aimed at proving the effectiveness in reducing “intestinal discomfort”.

Numerous conferences have been dedicated to IBS with the objective of establishing a definition and the symptomology .
International groups of experts, brought together for the theme of Functional Gastrointestinal Disorders, have elaborated diagnostic criteria, known by the name of Roma I, II and III and have defined IBS as being “a group of functional intestinal disorders, in which abdominal pain is associated with defecation or the modification of the routine of the intestine or the subjective perception of altered defecation.” IBS is also defined as being a dysfunction and can include disorders known as: irritable colon, spastic colitis or simply colitis.

Abdominal pain or discomfort (disturbing sensation) recurring for at least three days a month in the last 3 months, associated with at least two of the following symptoms:

• Improvement with evacuation
• Appearance associated with variations of the frequency of evacuations.
• Appearance associated with variations of the consistency of the feces.

Abdominal pain can manifest itself as a cramp, predominantly during the day, alleviated by defecation and exacerbated by stress and the intake of food. This symptomology often conditions the quality of life and daily routine. The altered frequency of evacuations is expressed with a prevalence of constipation as opposed to diarrhea, especially among women. Men, instead, have a prevalence of diarrhea.

IBS is not a disease but a dysfunction, caused by an exaggerated synergy between the Central Nervous System and the Enteric Nervous System. It doesn’t have a well-defined etiologic factor; it is considered a “bio-psychosocial” disorder, caused by Physiological, psychological, environmental and behavioral factors.

IBS frequently has cyclical occurrences, with periods of well-being alternating with painful attacks provoked by emotional tension, fatigue, stress, diet. These painful crises create further tension in the patient which can cause a worsening of the symptoms. Genetic factors combined with psychosocial factors determine an increase of intestinal inflammatory cells, with an increase and activation of the mast cells, which cause colic hypersensitivity and unmobility, with bloating, alteration of transit time, meteorism, hypertonia of the sigmoid flexure, appearance of fecal mucous. Colonoscopy is often macroscopically negative but the histology highlights the microscopic colitis.

According to epidemiological data, IBS is present in 30% of the Italian population with a ratio woman/man between 2:1 and 4:1. This tendency is also found in our clinical experience with a ratio of 7 out of ten. This might indicate a greater sensitivity and knowledge on the part of the women.

Diagnosis

The diagnosis of IBS is based on an accurate evaluation of clinical symptoms and on the exclusion of other pathologies. A method had recently been set up consistent measuring fecal Calprotectina. In this way, the organic disease (colitis) is distinguished from the alteration of the functional state and is therefore considered to be the best means to confirm the diagnosis of IBS, excluding other pathologies such as intestinal inflammatory disease (IBS), poor absorption, tumors, infections (Histolytic entamoeba, Yersinia, Campylobacter jejuni, Giardia, etc), diets too rich in sorbitol, fructose, fiber, lactose, the use of laxatives or drugs rich in magnesium.

MATERIALS AND METHODS

The active ingredient

The active ingredient is the lactic yeast Kluyveromyces marxianus fragilis B0399, produced by Turval Laboratories of Udine.
The product in capsule is already in commerce in pharmacies and has been notarized by the Ministry of Health.

Selection of the group to be examined.

Fifty people who came under our observation during the past two years were selected; fifteen were men with an average age of 33 (20-62) and 35 women with an average age of 31(22-55). None of the subjects presented other pathologies, gastroenterological or otherwise. None were using any type of drug. They had symptoms which indicated Irritable Bowel Syndrome. To exclude ulterior pathologies, haematic-chemical exams such as bood count, Ves, Hepatic profile, amilasy test,total IgE, sideremia, search of Hb in huaman feces (three sample),fecal parasitological exam, feces culture exam (in patients with diarrhea), fecal Calprotectina dosage.
Patients over 50 years old and those who had a family history with many cases of colic polyps underwent a total colonoscopy (11). Three had colic polyps which were removed endoscopically (slight and mediumdysplasia ).

The 50 patients whose absence of pathological alterations worthy of mention was verified were asked to answered a series of question asked by the physician that summarize the diagnostic criteria of Roma (I, II and III). This questionnaire, defined as pre-administration, refers back to the study conducted at the University Hospital of Manchester (Francis at all, 1996) recommended in “Design of Treatment Trials for Functional Gastrointestinal Disorders” (Irvine at all., 2006), considered the reference guide of the EFSA.

The people who resulted in being suitable and were part of the program were adequately instructed on the lifestyle to follow, foods and dietary mistakes to avoid (coffee, carbonated drinks, alcohol, hot spices, legumes, cocoa, milk and dairy products, oily dry fruit, etc.)

Daily dosage
The experimentation in exam consisted in the administration of one capsuleevery twelve hours for a month and a maintenance period with one capsulebefore breakfast for two months, as normally recommended by the producing company and by doctors (pediatricians, gastroenterologists, gynecologists, family practitioners, etc.) as a dietary supplement.
Based on the information provided by the producing company, one capsule contains not less than 10/20 million live cells of lactic yeast

Analysis of the questionnaires
At the end of the period of administration of the product, the patients were visited again and a post-administration questionnaire was collected for a verification of the symptoms.
From the analysis of the answers obtained in the questionnaire before the administration and the one after administration, the evidence of an improvement in IBS, or lack thereof, was examined.

Diagnostic criteria.
Before the experimentation, diagnostic criteria for the determining of cases of IBS and their characteristics was selected that go by the name of Roma (I,II and III). This criteria helped to determine the suitability of the people chosen for the experimentation.

Tab.2.1 Answers to which it is necessary that all the answers satisfy the diagnostic criteria before considering the patient suitable for the experimentation.

Tab.2.2 Answers to which it is necessary that at least two answers satisfy the diagnostic criteria before considering the patient suitable for the experimentation.

Tab.2.3 Answers which define the subtype IBS. Each outline of answers corresponds to a particular subtype.

Question 12 (pre-administration) and XII (post-administration), relative to the use of the information obtained in the test, verified as being suitable only those who answered “0”.

Tab. 2.4 Outline of the questions comparable between pre-administration and post-administration testing.

STATISTICAL ANALYSIS:

Statistical analysis of the Chi-square Yates/Pearson
For the analysis of the data obtained from the answers of the questionnaires both pre and post-administration the analytic procedure of the Chi-squareof Yates and the Chi-squarePearson (with Vassar Stats: Statistical Computation Web Site) was selected, with the aim of verifying if their differences are due to chance or not. If the differences are not due to chance, they would be considered “statistically significant.”

Chi-square: Initially, whatever the existing differences between the two series of data to compare, the “zero hypothesis” is advanced. The zero hypothesis (or null hypothesis) simply affirms that the difference observed – of whatever entity – is due to chance. This hypothesis can either be accepted or rejected on the basis of the results of the statistical test.
If on the basis of this hypothesis the calculated value of χ² is greater than a certain critical value, we’ll have to conclude that the frequencies observed differ significantly from the frequencies expected and we will have to refuse the H0 at the corresponding level of significance. Otherwise, we will have to accept it, or at least not refute it. This procedure is called chi-squareof the hypothesis.

Grouping of the data

To proceed in determining the difference between a “healthy” state of a person and one affected by IBS, the answers to the questions were grouped, according the description shown below.

First question: following the diagnostic criteria described in paragraph 2.5, the “healthy” people reply with the options 0-1-2 while those affected by IBS reply with options 3-4-5-6.
Fourth question: following the diagnostic criteria described in paragraph 2.5, the “healthy” people reply with the options 0-1 while those affected by IBS reply with options 2-3-4.

Seventh, eighth, ninth, tenth and eleventh question: following the diagnostic criteria described in paragraph 2.5, the “healthy” people reply with the options 0-1 while those affected by IBS reply with options 2-3-4.

Furthermore, the division of these two cases identifies in answers 0-1 the sporadic nature of the event and in answers 2-3-4 indicate association with pain and elevated frequency.

5-Initial questionnaire (pre-administration):

6. Since this discomfort or pain has started, has defecation been less frequent?

9. In the past 3 months, how often were your feces hard and lumpy?

10.  In the past 3 months, how often were your feces loose, soft or watery?

12.  Knowing that your answers (anonymous) will be used for statistical purposes, are you contrary?

7-RESULTS:

7.1-comparison between the pre-administration questionnaire filled out at the doctor’s office and the one mailed from home by the patient.

The comparison between the initial forms collected by the doctor and those collected from the patients at both time 0 and at the end (after three months of the administration of Kluyveromyces marxianus fraglis B0399) showed an almost total correlation between the forms collected by the doctor and those collected from the patients: the discordance is minimal (2% of the answers).

The interpretation of the answers to the questionnaire is shown in the following paragraphs:

7.2-FREQUENCIES OBTAINED IN THE ANSWERS AND ANALYSIS OF THE DATA:

First question:

• a) All the subjects suffer from abdominal discomfort or pain.. (YES =50)
• b) Analysis of the frequency of episodes of discomfort/pain. In the past three months have you experienced abdominal discomfort or pain?

Tab 7.2.1

Tab. 7.2.1/stat Results of the statistical analysis of the data obtained in the first question. Comparison of the answers given before the administration and those given after (as described in paragraph 3, page 5)

Fig.7.2.1 Graph of first question :

Second question: Is the discomfort present only during menstruation?

Tab. 7.2.2

Fig.7.2.2 Graph of second question:

Third question: Have you been suffering from this discomfort for 6 months or more?

Tab. 7.2.3

Fourth question: How often does this discomfort or pain subside or disappear after defecation?

Tab. 7.2.4

Tab. 7.2.4/stat Results of the statistical analysis on data obtained from the fourth question. Comparison of the answers given before administration and those given after (as described in paragraph 3, page 5)

Fig.7.2.4 Graph of fourth question

Fifth and sixth questions:
Analysis of the frequency of defecation in relation to abdominal discomfort or pain.

Tab 7.2.5/6

Seventh question: frequency of soft feces.
Tab. 7.2.7

Tab 7.2.7/stat Results of the statistical analysis on the data obtained in the seventh question. Comparison of the answers given before administration and those given after (as described in paragraph 3, page 5)

Fig. 7.2.7 Graph of the seventh question:

Eighth question: frequency of hard feces.

Tab 7.2.8

Tab 7.2.8/stat Results of the statistical analysis on the data obtained in the eighth question. Comparison of the answers given before administration and those given after (as described in paragraph 3, page 5)

Fig.7.2.8 Graph of the eighth question.

Ninth question: frequency of hard and lumpy feces

Tab.7.2.9

Tab 7.2.9/stat Results of the statistical analysis on the data obtained in the ninth question. Comparison of the answers given before administration and those given after (as described in paragraph 3, page 5)

Fig.7.2.9 Graph of the ninth question

Tenth question : frequency of loose, soft or watery feces.

Tab 7.2.10

Tab 7.2.10/stat Results of the statistical analysis on the data obtained in the tenth question. Comparison of the answers given before administration and those given after (as described in paragraph 3, page 5)

Fig.7.2.10-Graph of the tenth question:

Eleventh question : How much does pain or discomfort influence your social life?
Tab.7.2.11

Tab 7.2.11/stat Results of the statistical analysis on the data obtained in the eleventh question. Comparison of the answers given before administration and those given after (as described in paragraph 3, page 5)

Fig.7.2.11 Graph of the eleventh question :

Twelfth question: request to utilize data obtained for experimentation
Tab.7.2.12

Chart of the results of question X (post-administration): During the treatment did you notice new symptoms that you didn’t have in the past?

Tab.7.2.14- Chart of the results of question XI (post-administration): If yes, which ones?

COMMENTS:

As previously stated, Irritable Bowel Syndrome (IBS) is a gastrointestinal dysfunction caused by multiple “bio-psycho-social” factors which determine symptoms which may even be contrasting (constipation and diarrhea, soft feces alternating with hard feces) and which are often irritating, embarrassing, unpredictable, uncontrollable and painful.
Non biological markers of reference exist, therefore the diagnosis is made by exclusion of pathologies and by consulting the established symptomatic criteria.
This study recruited 50 patients (15 males and 35 females) who fell into the symptomology critera of IBS.

Through an initial questionnaire and one in the end, we wanted to verify if, after treatment with Kluyveromyces for three months, the symptomology changed.
The first question (pre-administration, question 1) determined the admission to the study. In the test-pre-administration (question 1) 50 patients gave an answer greater than 2, revealing to be affected by IBS; 29 of them (58%) gave 5 as an answer, which corresponds to a pain for more than one day a week. At the end of treatment, 37 patients (74%) had a score of less than or equal to 2, which would exclude them from the diagnostic criteria for IBS. The remaining 13 patients (26%) still had discomfort or pain for 2/3 days a month (8 patients) or for 1 day a week (5 patients).

These results are important, as shown by the statistical analysis which compares the answers that indicate a healthy person (0-1-2) and those which indicate the presence of IBS 3-4-5-6), revealing a definite, significant difference between the answers given before the treatment and those given after ( Chi-square Yates = 55,6 with p < 0,001; Chi-square Pearson = 58,73 with p < 0,001). The answers to the first question underline how treatment with Kluyveromyces marxianus fragilis B0399 drastically reduces the symptomology with the exclusion of most of the patients from the diagnostic criteria for IBS.

The second question (Is this discomfort present just during menstruation?) (pre-administration question 2 and post-administration question II) remained unchanged. The answers of all the women revealed an independence of menstruation from the symptomology of IBS both before and after treatment.

The third question (Have you been suffering from this discomfort or pain for 6 months or more?)
(pre-administration question 3)showed that all the patients suffered from abdominal discomfort or pain for more than 6 months (chronic symptomology) and was not asked again in the final control because it’s a normal course for the symptomology of IBS. metteva in evidenza che tutti i Pazienti soffrivano dolore odisagio addominale da più di sei

The fourth question (How often does this discomfort subside or disappear after defecation?)
(pre-administration question 4 and post-administration question III) regarded the association between pain or discomfort and defecation. The statistical analysis showed that there is a significant difference (with p< 0,05) between the answers in the questionnaire pre-administration and the one post-administration ( Chi-square Yates = 4,58 with p = 0,032347; Chi-square Pearson = 5,54 with p = 0,018587).

Initially, 28 patients (56%) reported an improvement after defecation. At the end, 20 patients (40%) reported an improvement in pain after defecation. This is due to the improvement of the overall symptomology and therefore less evidence of an association between pain – defecation.

The fifth and sixth questions (analysis of the frequency of defecation in relation to abdominal discomfort or pain.)
(pre-administration questions 5 and 6) compared the relation of pain with a change of intestinal cavity (more frequent or less frequent defecation). 36 patients (72%) reported having diarrhea and 23 (46%) constipation. Apparently, some patients have a mixed form of IBS: both diarrhea and constipation (9 patients=18%).
In the questionnaire post-administration (question IV), all the patients reported an improvement with 64% (32 patients) reporting a normalized intestinal cavity.

The seventh question (frequency of soft feces)
(pre-administration question 7 and post-administration question V) analyzed the relationship between abdominal pain or discomfort and soft feces: In the first questionnaire, 14 patients reported having soft feces all the time or most of the time, while the other 22 had soft feces often or sometimes. After the treatment with Kluyveromyces, 18 patients (36%) didn’t have soft feces and 12 patients had them just sometimes. The statistical analysis of the data describes how the answers of the patients vary in the pre-administration questionnaire and the one post-administration, with a change from the answers that indicate an elevated frequency of soft feces (2-3-4) to ones that describe sporadic episodes (0-1) ( Chi-square Yates = 9,72 con p = 0,001823; Chi-square Pearson = 11,18 con p = 0,000827)

The eighth question (pre-administration question 8 and post-administration question VI) looked into the relationship between pain and hard feces. This symptom was present in 23 patients (46%), 13 of which had the symptom just sometimes and 8 others often or always.
After the treament (question V) the hard feces disappeared in 10 patients and were present in 8 patients just sometimes. Even though in this case the statistical analysis doesn’t show a significant difference between the pre-administration answers and those post-administration, the graph representing the data shows a rather evident change in frequency, with a diminishing presence of hard feces.

The ninth question (frequency of hard and lumpy feces)
(pre-administration question 9 and post-administration question VII) looked into the correlation between pain and hard and lumpy feces. In 9 patients (18%) the feces were often hard and in 8 they were hard most of the time or always. After the treatment (question VII), 12 patients reported not ever having hard feces and 8 only sometimes. The answers of the pre-administration test and post-administration test are significantly different, with a change in the answers that indicate an elevated frequency of soft feces (2-3-4) to ones that describe sporadic episodes (0-1) ( Chi-square Yates = 10,9 con p = 0,000962; Chi-square Pearson = 12,9 con p = 0,000329), indicating that the treatment with Kluyveromyces diminishes the frequency of hard and lumpy feces.

In the tenth question (frequency of loose, soft or watery feces)
(pre-administration question 10 and post-administration question VIII) the relationship between pain and loose and watery feces was investigated. 12 patients reported having this symptom sometimes while 22 patients reported having it most of the time or often. After treatment with Kluyveromyces marxianus fragilis B0399, 26 patients did not have watery feces any more and only 10 patients had them just sometimes (question VIII). The verification of the statistical analysis confirms a considerably significant change in the answers from those that indicate an elevated frequency (2-3-4) of soft feces to those that describe sporadic episodes (0-1) (Chi-square Yates = 21,64 with p < 0,001; Chi-square Pearson = 23,97 with p < 0,001), demonstrating that the appearance of loose or watery feces was diminished.

The eleventh question (How much does abdominal pain or discomfort influence social life?)
(pre-administration question 11 and post-administration question IX ) looked into the relationship between IBS and quality of life. The first questionnaire showed that 34 patients (68%) were conditioned often or every day by IBS and 14 patients were conditioned sometimes. After the treatment (question IX) 40 patients (80%) reported being conditioned in their social life only sometimes. The verification of the statistical analysis confirms a considerably significant change in the answers, from those that indicate a considerable discomfort (2-3-4) to ones that describe a light or discomfort or none at all (0-1) (Chi-square Yates = 34,52 with p < 0,001; Chi-square Pearson = 36,95 with p < 0,001),

Beyond all the symptomatic improvement demonstrated by the above questions, this information leads to considering the importance that treatment with Kluyveromyces marxianus fragilis B0399 can have in mutating not only physical factors but psychological ones as well in patients with IBS.

In the post-administration questionnaire a question was added that inquires about eventual side effects (X). Ten patients (20%) answered in the affirmative, specifying the particular symptom in the following question (XI): 7 patients reported having halitosis, 2 headache, meteorism, burping, heartburn, 1 epigastric pain and myalgia. No one had allergies, intolerance or other symptoms.

The last question (corresponding to XII) had the purpose of informing the patients of the collection and use of data (though anonymous) and getting their permission (100% of the patients accepted).

CONCLUSIONS:

Acknowledging the fact that IBS has multiple causes (physiological, psychological, environmental and behavioral) and therefore there is no single effective therapy but multiple therapies which are aimed at the most important subjective causes, this study, based on the symptomological data gathered before and after the treatment, demonstrates that the prolonged administration of Kluyveromyces marxianus fragilis B0399 significantlyimproves the clinical condition and above all improves the quality of life of the patients.
A correct diet, together with physical exercise and stress control, further improve the situation, giving the doctor the job of informing the patient, educating him and giving him confidence and then prescribing the most effective drug.

BIBLIOGRAFHY:

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Giorgio Mustacchi (Oncological Centre, Local Health N1-Trieste; Trieste University) ; Antonio Miclavez ; Alessandro Turello;
Franca Bearzi;Flaviano Collavini; Paolo Valles.

This study proposed to evaluate the colonization of the gastrointestinal tract after the utilization of Il Kluyveromyces marxianus fragilis B0399. The product was administered to 17 healthy volunteers for a period of 14 day.The intake of Kluyveromyces fragilis marxianus B0399 B0399 on the part of the healthy subjects at the dosage of 20 million CFU/diem for 2 weeks proves to be more than sufficient for intestinal  colonization to take place without any side effects.

It is clearly demonstrates its ability of resistance to the gastric barrier and suggests that it can be effectively taken even at much lower dosages than those examined.

Also noteworthy, since it is a probiotic composed of yeasts, Kluyveromyces fragilis marxianus B0399 could be taken together with antibiotics.

SUMMARY

Kluyveromyces marxianus fragilis B0399 is a probiotic yeast used as a dietary supplement.  This study proposed to evaluate the colonization of the gastrointestinal tract after the utilization of Il Kluyveromyces marxianus fragilis B0399.

The product was administered to 17 healthy volunteers for a period of 14 day. The feces of the subjects were analyzed to evaluate the presence of Kluyveromyces marxianus fragilis B0399at T0 (time zero), before the administration of the product (absence verification) and then at the end, after 14 days (T14 – tempo 14.)

The intake of Kluyveromyces fragilis marxianus B0399 on the part of the healthy subjects at the dosage of 20 million CFU/diem for 2 weeks proves to be more than sufficient for intestinal colonization to take place without any side effects.

The elevated magnitude of the colonization found clearly demonstrates its ability of resistance to the gastric barrier and suggests that Kluyveromyces fragilis marxianus B0399 can be effectively taken even at much lower dosages than those examined (for example: 5–10 million CFU/diem).

After examining various official trials in the animal field, it becomes questionable or even inopportune to excessively increase the daily dosage.

Also noteworthy, since it is a probiotic composed of yeasts, Kluyveromyces fragilis marxianus B0399 could be taken together with antibiotics. These characteristics, together with the absence of side effects, demonstrate a profile not only of effectiveness but of convenience and safety in usage, worthy of further scientific investigation.

1 – Researchers and facilities involved in the experimentation

Giorgio Mustacchi (Centro Oncologico Azienda per i Servizi sanitari N° 1 – triestina, Università degli Studi di Trieste): ): Head of the experimentation and statistic calculation.

Antonio Miclavez, CSM s.r.l Trieste, promoter of the study

• Alessandro Turello, Laboratori Turval s.r.l. : owner of the active ingredient (attachments A1,A2)
• Franca Bearzi, Laboratorio analisi della Casa di Cura Pineta del Carso (Aurisina- Trieste), preparation of the sample in suspension. (attachment B)
• Flaviano Collavini, CATAS S.p.a, Laboratorio ambiente e agroalimentare (laboratorio microbiologico), CCIAA di Udine ( Allegato C)
• Paolo Valles, microbiologist, Analysis at the microscope (Allegato D)

INTRODUCTION:

The intestinal bacterial flora has a key role in both nutrition and health maintenance and, in its complexity and function, plays a unique role in our organism. Several thousand different bacterial species are present in the human intestine, some considered beneficial (ex. bifidobacteria, lactobacilli, lactic yeasts) and others benign (ex. Methanogens and sucrose species of Clostridum and Bacterium) capable of contrasting the excessive multiplication of species dangerous to human health (proteolytic species of Bacteroium, Clostridium difficile, C perfrigens and the pathogen species of Enterobacteria).
Probiotics are microorganisms with the ability to bypass the gastric barrier and favor the modulation of the intestinal bacterial flora capable of surviving and maintaining viability for long periods of conservation and be harmless for man.
By introducing foods with the addition of probiotics, the useful bacteria can survive the journey through the gastroenteric tract and reach the colon. They colonize it, altering favorably the microbial equilibrium.
Probiotics have shown to be useful in the treatment of a number of intestinal problems, such as childhood diarrhea, traveler’s diarrhea and certain chronic inflammatory diseases of the intestine.

Objective of the study

The object of this study was to verify and evaluate the ability of Kluyveromyces marxianus fragilis B0399 to bypass the gastric barrier and to colonize in the intestinal tract when administered at the normal dosages suggested by the distributing company.

Some notes on the active ingredient utilized in the trial.

Kluyveromyces marxianus fragilis B0399 is a lactic yeast with characteristics which are different from those of lactobacilli and of bifidobacteria, and of 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 eucaryotic type of cell (Lachance M.A 1) endowed with an elevated lactasic enzymatic activity (β-galactosidase). It ferments lactose with the production of lactic acid. The enzymatic activity, in anaerobic conditions typical of the intestine, is of a homofermenting type, in that it transforms all the resulting glucose is lactic acid, without the production of gas (C02) (ex.Saccharomyces) (Vananuvat-Kinsella2 ,Wasserman-Hopkins-Porges3). This contributes in modulation the intestinal environment, rducing the pH. Furthermore, it has shown to be particularly resistant to the action of antibiotics (Voughan 4 ).

Kluyveromyces marxianus fragilis B0399 appears to be capable of resisting gastric shock. This ability was also tested in vitro (Susmel and Stefanon5 ) by measuring the fermenting capacity before and after gastrointestinal digestion. The resistance to digestion proved to be elevated.
The ability of bypassing the gastric barrier was confirmed also by trials in vivo on monogastric animals like piglets, whose digestive apparatus is very similar to the human apparatus (Lovrovich P. 6 ) and on horses, by testing the presence of Kluyveromyces B0399 in the feces and the modification of the pH in the colon ( Lowell R. S. 7. Susmel-Stefanon 8,Bosi P. 9.)

The beneficial aspects of Kluyveromyces B0399 have also been indicated by the various studies regarding problems of the colon as well (Andreoli S.10, Irvine EJ 11, Bottona-Parisi-Zilli 12).

MATERIALS AND METHODS

As stated by the producing company (Turval Laboratories srl of Udine) the strain utilized is Kluyveromyces fragiilis marxianus B0399, deposited in BCCM-Belgium Coordinated Collections of Microorganisms, Culture Collection Mycoteque de l’Université Catholique de Lovain (Belgium) with the mark B0399, (Attachmentsi A, A2).

The experimentation was set up using guidelines from a project with the same objectives “Colonization and Immunomodulation by Lactobacillus reuteri ATCC 55730 in the Human Gastrointestinal Tract” (Valeur 13 ).

3.3.4 – Product object of the study and dosage

Products in capsule found on the market and already notarized by the Ministry of Health were used: Biosympa, which is normally found in pharmacies.
As certified by the producing company, Turval Laboratories Italia s.r.l, the product Biosympa is produced with 40 million (40*106 ) ufc/g (Attachments A, A1, A2).

The product “Biosympa” containing the active ingredient Kluyveromyces marxianus fragilis B0399, was administered at the dosage of two capsules per day (20*106 cfu per caps.), for a total daily dosage of 20*106 ufc di Kluyveromyces B0399, for 14 days.

3.1 – Enrolled subjects

17 healthy volunteering subjects of age were enrolled, (median 53, range 35-72 years old), 3 males and 14 females.
The evaluation of their state of health was done through a medical consultation to gather all t case-history information considered to be necessary. In particular, subjects were asked which drugs, if any, they had used during the six months prior to experimentation, if they’d had any recent diseases/pathologies, if they had any symptoms at present and what was the typology of their everyday diet.

Criteria for exclusion
Subjects were excluded who: in the three months prior to the experimentation had taken antibiotics or probiotics, had a severe chronic disease and/or other affection of the colon which results in the fragility of the mucosa,celiac, had intestinal obstruction orsub-obstruction, had previous abdominal surgery with the exception of hernia or appendectomy, had taken antipsychotic drugs in the previous 3 months or steroids in the previous month, were lactose intolerant or immune deficient, poor compliance.
During the period of treatment the assumption of the following were not allowed: drugs which could alter the intestinal motor function or absorption intestinal, including laxatives and anti-diarrhea medications, products able to alter the intestinal bacterial flora (antibiotics and products containing probiotics).

Examination of fecal samples

The feces of the people participating were analyzed for the verification of the presence of Kluyveromyces marxianus fragilis B0399.
Before the beginning of the treatment with Kluyveromyces marxianus fragilis B0399, samples were taken at time T0 (time zero) with the aim of determining 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 fecal samples was repeated after 14 days of administration ( at time T14).

Initial criteria of evaluation

The test described here was conducted using the criteria recognized at the European level (Valeur 13 , EFSA 14 , , Francis C. Y 15).
With the aim of verifying the presence of Kluyveromyces marxianus fragilis B0399 in the feces, and not having any data regarding lactic yeasts, an increase in value in the feces superior to 103 ufc/g in accordance with the value taken in the reference study about lactobacillus reuteri (Valeur 13), would be considered significant.

Collection of sample:

The fecal matter was collected from each subject in sterile containers (BIO-BOX raccoglitore per ricevere il campione di feci, for.me.sa.) and delivered to the appointed doctor. All containers with fecal matter were carefully preserved in refrigeration at a constant temperature of 4°C.

Homogenization of the sample for the analyses

All the original samples were prepared with the aim of obtaining specimen as homogeneous as possible in order to execute the analysis and the count of the yeasts on a quantitative basis.
The laboratory prepared the suspension at known strength starting with the initial solid sample. The dilution of the fecal matter was done with sterile physiological solution.
The dates of collection and arrival of the samples are indicated in Tab 1.

Tab 1 Summary table of the dates of collection and arrival of the samples of both Time zero (T/0) and Time T/14.

The main characteristics of the subjects studied and the dates of collection are indicated in Tab2.

Tab 2 : Characteristics of the subjects and times of collection

Analyses conducted on the samples obtained at time T 0

Count of total yeasts and elimination of interference of candida.
1) Presence and quantification of microorganisms (yeasts) positive on Sabouraud agar.
2) To eliminate the interference of candida , a screening of the colonies (of yeast) positive on Sabouraud agar was conducted through the identification of the colonies of Candida (Medium Chromoalbicans agar) and by their examination at the microscope (Phase contrast : 12.5 x 40).

Analyses conducted on samples obtained at time T 14

Count of total yeasts, elimination of interference of candida and verification of the presence of lactic yeasts Kluyveromyces) ,
1) Presence and quantification of the microorganisms (yeasts) positive on Sabouraud agar.
2) To eliminate the interference of candida, a screening, like above, of the colonies (of yeast) positive on Sabouraud agar was conducted through the identification of the colonies of candida (Medium Chromoalbicans agar) and an exam at the microscope was executed on all the plates (Phase contrast : 12.5 x 40)
3) The identification of Kluyveromyces : the method used (supplied by Turval Laboratories) was the one found in the dossier approved by the EU (norm 377/2006 ) for the identification of lactic yeasts (Kluyveromyces through sowing on Medium MV1 Agar specific to lactic yeasts. (Lab CATAS CCIAA UD all. C)
4) Observation at the microscope was conducted (Phase contrast: 12.5 x 40) (Dr. Valles) attachment D.

STATISTICS ANALYSIS:

The evaluation of the presence of the yeasts in the feces was determined using the following parameters: (Tab 3):
Negligible: (< 100 ufc/g)
Positive: (lactic yeast by identification on MV1 agar –medium selected for Kluyveromyces sp.)
Positive**: (lactic yeast by identification at the microscope.)
Non determinable*: (presence of patina)

The count (CFU) refers to the weight of the feces as it.

The difference between frequencies (positive/negative for the threshold level pre-selected between T0 and T14) was analyzed with the chi-square test. The analysis of the quantitative differences between T0 and T14 was evaluated with the “Two-Sample T-test” a one-tailed test ( Alpha=0.05). The analyses were executed with the software NCSS 2007 (NCSS Statistical software, Kaysville, Utah, USA).

RESULTS:

The results in 16/17 subjects enrolled were able to be evaluated (94.12 %)

At Time T/0. considering the results obtained by the total count of the yeasts and subtracting from them the count of the colonies of Candida Albicans, the presence of total yeasts was negligible or absent in 13 out of 17 samples (76.47 %). The remaining 4 had a minimal presence of colonies (Tab 3).

At Time T14 16/16 samples (100 %) resulted positive in terms of presence of yeasts, with a statistically significant increase (chi-square Test: X=19.2, p < 0.00001) compared to T0.

Analogically, the quantitative evaluation between T0 and T14 shows a statistically significant difference in terms of presence of colonies (medium values 1373.33 versus 460873.30 (p = 0.035).

The average increase was about 3 logarithms (range from 1 to 6). The increase appears to be major in cases where at Time T0 there was no trace of yeasts compared to those in which a negligible or modest presence was detected (Tab 3).

At T/14 the yeasts or microorganisms positive on Sabouraud-agar through the identification test executed on MV1- agar on 7 randomly selected samples (3, 7, 9, 10, 12, 13, 19) all resulted in being lactic-fermenting yeasts (kluyveromyces marxianus/fragilis). (Tab 3).

From the microbiological comparative exam executed at the microscope on all the samples Sabouraud-agar positive, results show that the isolated colonies can be identified in 100% of the cases exclusively with a lactic yeast (kluyveromyces marxianus fragilis).

Tab 3: Evaluation of the presence of total yeasts in the feces (excluding candida) ‘
At Time 0 and 14 days and verification of lactic yeasts at Time 14

• Negligible: (< 100 UFC/g)
• Positive*: (lactic yeast by identification on MV1 agar –medium selective for Kluyveromyces sp.)
• Positive**: (lactic yeast by identification at the microscope)
• Non determinabile*: (presence of patina)
• Note : the count (CFU) refers to the weight of the feces as it.

Fig 5.1 Increase of Kluyveromyces marxianus fragilis BO399 (Ufc/g) in the fecal matter after 14 days of administration ( at time T14).
On the x-axis there is decimal interval of variance in Kluyveromyces BO399 between T/0 and T/14 (Ufc/g)
On the y-axis there are the number of subjects on 17.

Profile of toxicity

None of the subjects treated complained of any type of side effects.

This study demonstrated that the presence of yeasts ( different from candida) in the feces of healthy subjects is present in less than 25% of cases. It’s reasonable to think that these are yeasts different from Kluyveromyces fragilis marxianus B0399 (for instance, yeasts which are present in foods, like Sacccaromyces cervisiae and others which are similar) since none of the subjects in the study had taken the active ingredient in the three months preceding the testing.
In any case, the administration of Kluyveromyces fragilis marxianusB0399 at the pre-selected dosage of 20 M/die taken orally for two weeks is able to show very high levels of yeast in 100% of the subjects treated, and further analyses of confirmation proved it to be the lactic yeast Kluyveromyces fragilis marxianus B0399.
In our study we chose the threshold value of positiveness at T14, based on the reference literature (Valeur 13). This value, however, is in reference to lactobacilli (lacttobacilus reuteri) , usually saprophytes of the human intestine. The lactic yeast Kluyveromyces fragilis marxianus , as confirmed in the present study, is rarely present in a relevant amount, therefore it is reasonable to assume an even lower threshold of positiveness, of about 2 logarithms.
Apart from these considerations, the quantitative increase of the active ingredient present in the feces is considerable—it, in fact, varies from three to five logarithms.

The entity of the results is such that a colonization time of less than 14 days can be considered, and also the hypothesis of lower dosages, as seen in monogastric animal systems like piglets of 35kg, to whom, according to the European commission, 4.56 million CFU/die are sufficient. (Valles-Lugano16 , Commission Regulation (EC) 17, Bottona-Parisi-Zilli 12 )
It is also important to note the negligible interference of the gastric barrier which emerges

CONCLUSIONS:

The dosage of 20 million CFU/die taken by the healthy subjects for two weeks resulted in being fully sufficient for intestinal colonization , and with no side effects.
The elevated entity of the colonization found clearly demonstrates the capacity to resist the gastric barrier and can therefore be reasonable to think that Kluyveromyces fragilis marxianus B0399 could be effectively be taken at considerably lower dosages than those examined (for example 5- 10 million CFU/die)

To the contrary, it was observed that in the dietary trials conducted on monogastric animals of reference, such as piglets (Bosi 9), over-dosage (x 10 times) resulted in a worsening of the conversion index, (see dossier Kluyveromyces B0399 evaluated by the EFSA and approved by the European Commission (Bosi 9, Commission Regulation (EC) 17 ).
Therefore, it is probably ineffective or even inopportune to excessively increase the daily dosage (for example, more than a logarithm) without specific testing.

Also noteworthy, since it is a probiotic composed of yeasts, Kluyveromyces fragilis marxianus B0399 could be taken together with antibiotics. (Voughan 4)
These characteristics, together with the absence of side effects, demonstrate a profile not only of effectiveness but of convenience and safety in usage, worthy of further scientific investigation.

BIBLIOGRAPHY:

1. Lachance M.A. (1970) Kluyveromyces: systematics since Antoine van Leeuwenhoek 63: 95-104, 1993.

2. Vananuvat P. and Kinsella J.E. (1975) Production of yeast protein from crude lactose by Saccharomyces fragilis. Batch culture studies.J. Food Science 40: 336-41.

3. Wasserman A.E., Hopkins W.J., Porges N. (1958) Whey utilization – Growth conditions for Saccharomyces fragilis.Sewages Ind. Wastes 30: 913-20.

4. Vaughan Ann – University of Studies of Perugia Italy, Department of Biology and Agrodietary Biotechnologies. Resistance to antibiotics of the typified lactic yeast kluyveomyces marxianus fragilis B03999.

6. Lovrovich Paola Ricerca bibliografica.: Il maiale come modello adatto alla sperimentazione applicata del sistema digerente umano. Expertise – 2008; agg.2009

Lowell R. Smalley – (Kluyveromyces B0399) daily probiotic influence in chemistry in the large colon of the horse. H & S Lab Inc.. Omaha, Nebraska US TURVAL 6 agg.2009

Susmel P., Stefanon B., Del Savio R., Boccalon S.
Variation of large colon pH in horses after administration of lactic probiotic (kluyveromyces B0399) University of Udine – Dept. of Animal Science. , Trial#57, agg. 2009.

Bosi Paolo (DIPROVAL –UNIVERSITY OF BOLOGNA) –Effects of Turval B0399 in the diet of the weaned piglet, Tolerance test and effects on the intestine microorganism. Directives 70/524, 87/153, 94/40 EEC EU ( regolamento 377/2006 ) ; Trial# 22, agg. 2009.

10. Andreoli Sandro Gastroenterologo Risultati clinici del trattamento con lievito lattico Kluyveromyces B0399 nella Sindrome dell’Intestino Irritabile” Expertise. ; Trial# 132, agg.2009.

11. Irvine EJ, Whitehead WE, Chey WD, Matsueda K, Shaw M, Talley NJ, Veldhuyzen van Zanten SJ. (2006) Design of treatment trials for functional gastrointestinal disorders. Gastroenterology. 2006 Apr;130(5):1538-51.

12. Bottona E. , Parisi G., Zilli M. Gastroenterologia ed Endoscopia Digestiva ULSS 5-Arzignano(VI), Medicina Interna-Osp.S.M.del Prato ULSS2 Feltre(BL), Gastroenterologia ed Endoscopia Digestiva Osp.S.M.della Misericordia-Udine Valutazione del lievito lattico Kluyveromyces marxianus fragilis B03999.(cfr Linee guida dei probiotici . Min. della Salute Dic 2005 All 1)” ; Expertise, agg. 2009.

Valeur N. at all.( 2004) – Colonization and Immunomodulation by lactobacillus reuteri ATCC 55730 in the Human Gastrointestinal Tract. Applied and Enviromental microbiology, Feb 2004 p.1176-1181.

14. EFSA – Fondatezza scientifica di un’indicazione sulla salute relativa a LGG® MAX e riduzione del fastidio gastrointestinale ai sensi dell’articolo 13 paragrafo 5 del regolamento (CE) n. 1924/20061. Parere scientifico del gruppo di esperti scientifici sui prodotti dietetici, l’alimentazione e le allergie. The EFSA Journal (2008) 853,1-15;

15. Francis C. Y., Morris J. & Whorwell. P. J. (1997) The irritable bowel severity scoring system: a simple method of monitoring irritable bowel syndrome and its progress. Aliment Pharmacol Ther 1997; 11: 395±402.

16. Valles Paolo, Lugano Alessia. Laboratori Turval Italy Dosaggio giornaliero di lievitilattici Kluyveromyces B0399 nell’alimentazione umana.- , Expertise, agg. 2009.

17. COMMISSION REGULATION (EC) (2006)Use of the micro-organism preparation of Kluyveromyces marxianus-fragilis B0399 for piglets.- Official Journal of the European Union L 135/3 No 773/2006 of 22 May 2006;

Kluyveromyces marxianus fragilis B0399, in patients suffering from IBS. A double blind, monocentric study, controlled against a yogurt containing Streptococcus t, Lactobacillus b and Bifidobacterium lactis BB1, in parallel groups, in patients affected by IBS.The subjects assigned to the treated group presented a global satisfaction significantly superior compared to the control group.

experimenter: Prof. Enrico Roda (Bologna University – Italy)

Promoter: COOP Italia

Summary

This study evaluates the effects of a yogurt formulated with a new lactic yeast,
Kluyveromyces marxianus fragilis B0399 in patients suffering from IBS. A double-blind, monocentricstudy controlled against a yogurt containing Streptococcus t, Lattobacillus b and Bifidobacterium lactis BB12, in parallel groups, in patients affected by IBS. (Total patients- 92; active treated group- 46; control group- 46).

The study comprises a collection of data in three phases: pre-treatment (basal), treatment and “washout”, for a total duration of 8 weeks for every patient. In first analysis, a significant reduction of bloating and a regularization of the number of evacuations were observed.

Also, the “active” treated group had a statistically significant reduction in the characteristics of the evacuations, a composite score that includes both the urgency to evacuate in patients with diarrhea and the difficulty to evacuate in constipated patients (confirmed also by the comparison between the two treatments).

Furthermore, the subjective impression on the global effect of the treatment supports the objective clinical observation of a reduction in the difficulty correlated to the characteristics of the evacuation. The subjects assigned to the treated group presented a global satisfaction significantly superior compared to the control group, both at the end of the treatment as well as at the end of the study (P=0.002 e P =0.001) and therefore the patient was guaranteed a sense of subjective well-being by reducing personal discomfort.

Introduction

Irritable bowel syndrome is a common gastrointestinal functional disorder, characterized by coexisting abdominal pain, discomfort and alterations in the frequency and composition of the feces. IBS frequently reduces the quality of life with implications that reflect in the social sphere and in the personal activities of the patients, but a therapeutic target that can alter the natural history has not yet been found.

This clinical study wanted as its objective to demonstrate the effectiveness of a “new” yogurt in reducing the symptoms of the pathology; therefore, bloating and meteorism, abdominal pain or discomfort, the number of daily evacuations and the characteristics of the evacuations were examined. Because IBS is characterized by strong interpersonal differences in the perception of symptoms, and since the clinician is still not in possession of a test to objectively evaluate the symptoms of a patient, a daily clinical diary was used.

The results were evaluated as modifications of the symptomological scores in relation to the base course of the patient, so as to steer away from interpersonal differences of the perception of symptoms.

Materials and methods:

Layout of the study Monocentric double blind study, controlled against a yogurt containing Streptococcus t, Lattobacillus b and Bifidobacterium lactis BB12, in parallel groups, in patients suffering from Irritable Bowel Syndrome.

Phases of study. The study was divided into 3 periods, as follows:
1 Basal (pre-treatment)(2 weeks)
2 Treatment (4 weeks)
3 Wash- out (2 weeks)

In particular, it was divided by data collected in the pre-treatment observation (basal) phase, in the treatment phase and in the wash-out phase, for the total duration of 8 weeks for each patient.

Assignment of treatment. At the end of the basal period, the patients were consecutively randomly divided into 2 groups.
Treatments in study. The patients were randomly divided into two groups.
The treated group was supplemented with a yogurt, cod.AB262, containing Streptococcus t, Lattobacillus b, Kluyveromyces marxianus var fragilis B0399 (10⁷ CFU/dose) and Bifidobacterium lactis BB12(10⁹  CFU/dose).
The control group was supplemented with a yogurt containing Streptococcus t, Lattobacillus b and Bifidobacterium lactis BB12.

The patients ate one small jar of yogurt a day, at any time during the day, for the entire period of the treatment (4 weeks).
Patients. From February, 2009, to June, 2009, 92 patients of both sexes were enrolled, under the supervision of the gastroenterology ward of the S’Orsola-Malpighi Hospital of Bologna. All the patients met the criteria of inclusion and exclusion; in particular, all suffered from Irritable Bowel Syndrome, according to the diagnostic criteria of Roma III.

Variables in study. Each patient was asked to fill out a daily clinical diary, in which to annotate the following daily symptoms:
3 bloating and/or meteriorism (scale of 0 to 10)
4 abdominal pain or discomfort (scale of 0 to 10)
5 number of daily evacuations (number)
6 characteristics of the evacuation, taking into consideration the following sub-characteristics: sense of difficulty, incomplete evacuation, urgency to evacuate (scale of 0 to 10)

Every two weeks the consistency of the feces was evaluated, according to the validated Bristol Stool Scale. At the end of the treated period and at the end of the wash-out period, the experimenter personally asked the patient his global impression of the efficacy of the treatment.

2.Results

From the data collected, a trend seems to emerge which confirms some of the hypotheses presented; in fact, both products in the study had a positive effect on the symptoms of the patients.

By further analyzing the data, a significant reduction of abdominal bloating and meteorism was noted, along with a regularization of the number of evacuations in both groups of the study.

In this study, the data of the effect on humans of a new probiotic strain is reported.
Kluyveromyces marxianus fragilis B0399, a fungus commonly found in fermented lactic-dairy products (cheese and kefir) has already been studied and introduced in clinical veterinary practice.

Studies in vitro, implemented with the aim of evaluating the possible implications of dietary technology, took into considerationKluyveromyces m. as a possible fungus with probiotic action (Kumura H, J. Dairy. Sci 2004). In these models, the capability of the fungus to both adhere to the enterocytes and to be able to grow in a culture even in the presence of bile (colic acid 0.1%) was demonstrated. In the rat (Yoshida Y, Biosci. Biotech. Biochem 2005) the dietary supplementation with Kluyveromyces m. increases the fecal concentration of sterols and the caecal concentration of SCFA (Short-Chain Fatty Acid). This result derives, in themurine model, from the suppression of the dietary absorption of cholesterol, from the action of the entero-hepatic circuit and on the synthesis of SCFA.

In first analysis, what emerges from the administration in humans is a profile of tolerance and effectiveness not inferior to other probiotics.
The primary end-point was, in fact, reached in that the treated group reported both a significant reduction of the bloating symptom and the regularization of the number of evacuations.

There was also another noteworthy result: the treated group reported a statistically significant reduction of the characteristics of evacuation, a composite score that comprises both the urgency to evacuate in patients suffering from diarrhea and the difficulty in evacuation in patients suffering from constipation. The data regarding the decrease of the characteristics of evacuation is also confirmed in the comparison between the two treatments.

Tab.2.1 – Reduction of the score vs. basal – Comparison between treatments

Results statistically significant for P<0.05.

Tab.2.2 –  Success relative to the characteristics of evacuations in the second week of wash-out.

Results statistically significant for P<0.05.

From a clinical standpoint, the theoretical hypotheses have been confirmed that a beneficial effect on humans would be observed as has already been reported in the veterinarian field.
It is not known today whether if in humans it’s the increased endoluminal synthesis of SCFA which influences the micro-inflammatory mucosa, or if it’s the colonization activity on the part of the fungus of the mucous bio-film,  or if therapeutic mechanisms not yet known come into play.

Finally, the subjective impression of the global effect of the treatment supports the objective clinical data of reduction of the difficulty correlated to the characteristics of evacuation. The subjects assigned to the treated group presented a global satisfaction superior to those in the control group, both at the end of treatment and at the end of the study (P=0.002 e P =0.001).

In reviewing literature, there are few studies which conclude with the demonstration of clear evidence of superiority of one preparation compared to another.

The use of a control group which was supplemented with a yogurt containing probiotic strains demonstrates that  Kluyveromyces marxianus fragilis B0399 is active and effective in controlling symptoms of IBS and, in particular, at least from the data in our possession, guarantees the patient a sense of subjective well-being, reducing personal discomfort.

Concluding considerations

The significant improvement of most of the symptoms on the part of both products is evident; Two new probiotics (Kluyveromyces marxianus fragilis B0399 and Bifidobacterium lactis BB12) are added to the list of the biologically active substances in gastroenterology; one ((Bifidobacterium lactis BB12) is already known for other strains of the same species and this is, in a certain sense, a confirmation; the other, (Kluyveromyces marxianus fragilis B0399) enters for the first time on the short list of correctly tested probiotics.

The second aspect, of notable scientific interest, concerns a novelty: the superior effectiveness of Kluyveromyces marxianus fragilis B0399 on symptoms tied to physical activity and visceral hypersensitivity, which represents one of the aspects that is one of the most difficult to control in this affection: the characteristics of the evacuations and the global assessment that stems from the evaluation of the state of well-being by both doctor and patient, jointly.

Therefore, this work confirms that Bifidobacteria is useful in this category of patients and suggests that the new addition, Kluyveromyces marxianus fragilis B0399, represents a new, more potent and specific resource. It’s possible that its effectiveness may prove to be greater in certain subgroups of patients.

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RESISTANCE TO ANTIBIOTICS OF THE FEED SUPPLEMENT TURVAL B0399 (probiotic with typified lactic yeast Kluyveomyces marxianus fragilisB03999).

Generalities
No type of incompatibility results.
In particular, the strain Kluyveromyces marxianus fragilis B0399 (TURVAL B0399) was found to be compatible with the most common antibiotics utilized in the zootechnical field, as the test results shown above, demonstrate.

Purpose of the tests :
To evaluate the sensitivity of Kluyveromyces marxianusfragilis (B0399) to the action of some of the antibiotics which are more commonly utilized for therapeutic purposes in the zootechnical field or those capable of inhibiting the intestinal lactic, bacterial flora (lactic ferments) compared to the antibiotics to which yeasts are notoriously sensitive.

Materials and Method used for the tests:
Method of diffusion in agar, Minimum Inhibiting Concentration ( MIC ).
This method, as with the others, cannot precisely represent a situation in vivo, but it can show the effect of a change of concentration of the antibiotic on a population of yeast in the developing phase. In regards to the procedure , the method NCCLS Kirby-Bauer was used, and the MIC method for the determination of the diameters of the halos of inhibition. Several changes had to be made regarding the culture terrain ( Mueller-Hinton Agar ) and the preparation of the bacterial suspension for the inoculum, due to the particular needs of the yeast ( Kluyveromyces marxianus fragilis B0399 ) for development.
For the test on the sensitivity to antibiotics , the antibiotic discs of the Oxoid company were used.

Results and comments:
The trials, dated Dec, 12 2000, July 8 2002 and Dec. 17, 2002, are conducted by Prof. Ann Vaughan at the University of Studies of Perugia Italy, Department of Biology and Agrodietary Biotechnologies .The results are reported below in Chart 1.

Chart 1 – Antibiogram of the strain Kluyveromyces fragilis (B0399)
(cfr. Department of Vegetation Biology of the University of Perugia, Italy.

Comments.
The results shown in antibiogram Chart 1. confirm that the Kluyveromyces marxianus fragilis B0399 presents an elevated degree of resistance to the antibiotics tested. Sensitivity is encountered only in the case of those antibiotics notorious for their effect on the sensitivity of many yeasts.

Mechanism that confers the resistance to several families of antimicrobials.
The resistance of Kluyveromyces fragilis to the antibiotic families considered in the tests is due to the different cellular organization which exists between the procariotic organisms (bacteria) and eucariotic organisms (yeasts and cells of superior organisms, man included).

Targets of the antibiotic activity are the procariotic cells: in particular, extremely peculiar structures of the cell itself — the peptidoglicanic wall and the enzymes involved in its synthesis. Structures which don’t have equals in any other cellular types.

Other target examples are the ribosomes and enzymes: the RNA polimerase, the topoisomerase ll, which even though are present in the eucariotic cells also, are different from a morphological standpoint as well as what regards the genetic sequence by which they are codified. These sequences are present on constitutive genes and are not acquired or led by plasmids or by transposable elements.

We would also like to point out that the eucariotic cells, among which yeast cells, are not involved in phagic infections (small viruses which have bacterial cells as a target and host). They are able to lead tracts of DNA from one procariotic cell to another. Often, what happens that in these “transported” genetic fragments also contain genes for antibiotic resistance which propagate within a population.

This problem, however, does not involve yeast cells, as was previously mentioned. Therefore, in conclusion, the resistance to antibiotics is natural and constitutional and not acquired.

Prof. A. Tulli – Director of the Clinic of Dermatology,
University of Studies “G. d’Annunzio” Chieti (Italy)

TRIAL 75 ~ PRELIMINARY STUDY ON EFFECTS OF KLUYVEROMYCES B0399 ON PATIENTS WITH ATOPIC DERMATITIS

INTRODUCTION

The study was conducted on the basis of recent considerations on patients affected by atopic dermatitis. In about 80% of these patients a reduction in the efficiency of cellular immunity was observed, along with an elevated increase of total IgE. This could explain why atopic and non-atopic patients have a different distribution of subclasses of T lymphocytes. Given that T lymphocytes are divided into two types – Th1 cells which produce IL2 and IFN gamma and Th2 cells which produce interleucine IL 4,5,6 and 10 – and given that IFN gamma inhibits the IgE which are induced by the IL4, it was found that patients with atopic dermatitis had very low levels of IFN gamma and therefore a tendency toward a high level of IgE. This high level of IgE induces a hypersensitivity of the disease with critical consequences of the pathogenesis itself.

METHOD OF STUDY

Ten (10) patients with atopic dermatitis and with high seric levels of IgE, between the ages of 6 and 10, were treated orally with capsules containing the active ingredient Kluyveromyces marxianus fragilis B0399 (typified lactic yeasts: KLUYVER B0399).
Dosage: 1 capsule a day for 30 days. During the treatment the normal specific therapies were also applied.
After 30 days the seric levels of the IgE of the patients were controlled and showed a normal range of total IgE .

CONCLUSION

The results obtained show that the administration of Kluyveromyces B0399 favors the therapeutic action. This oral therapy with exogenous microorganisms (lactic yeasts KLUYVER B0399) can be a valid aid and adjuvant in atopic dermatitis , with an improvement of the coenaesthesia of the patient.

Dr. S. Andreoli – medical director of the Gastroenterologic Service of the Hospital S. Maria della Misericordia of Udine Italy;

Introduction
Gastrointestinal disorders are often accompanied by alterations of the colic area, with ulterior worsening of the symptoms, especially in the lower part of the digestive tract. The now widespread use of antibiotics has resulted in an equally widespread diffusion of the so-called “syndromes of intestinal dismicrobism”, caused by the antibiotics’ modification and selection action on the common intestinal saprophytic flora which, as everyone knows, has a physiological function of major importance. The antibiotics which are predominantly responsible for these intestinal syndromes are those which are not absorbed (or only in part) by the gastric and intestinal mucosa when administered orally, thus reaching the lower intestine and colon unmodified, where the enteric bacteric flora resides.
Equally responsible for these syndromes can also be those antibiotics which, when administered directly into the intestine, are partially eliminated in active form with the feces (tetracycline, etc.). Even the irritable colon produces a change in the intestinal flora, with a production of fermentative processes which produce alterations of the digestive functions.This process leads to meteorism, diarrhea, etc.. The alteration of the intestinal flora also provokes the loss of vitaminic function. The lack of production of vitamin B leads to glossitis, cheilitis and trophic disturbances of the intestinal mucosa, while the deficiency of vitamin K causes disturbances in blood coagulation.

Lastly, when alterations of the ecologic intestinal equilibrium occur, external pathogenic germs (staphylococci), take root, and the “pathogenic opportunists” already present can become virulent, penetrating the intestinal wall causing septicaemia or visceral localizations (candidiasis). To avoid these syndromes of intestinal dismicrobism, the idea to use the exogenous contribution of a microbic flora which could correct the effects of the disappearance or decrease of the saprophytic flora with its enzimatic, vitaminic and antagonistic functions of pathogenic germs was elaborated. The attention was turned to probiotic Bioval plus with yeasts pertaining to the typified lactic yeast Kluyveromyces fragilis B0399 ( Turval B0399) balanced with Saccharomyces cerevisiae, which have a considerable natural resistance to antibiotics. They synthesize amylolytic enzymes and vitamin B and inhibit the development of the staphylococco and Candida.

Considering that 30/70 % of the gastroenterologic ambulatory visits regard colon irritability, with a frequency of morbidity in the general population of 15/25%, we wanted to verify the efficacy of BioVal plus in this pathology. In all the patients, organic pathology was excluded by means of proctoscope and/or opaque enema, or colonscope.

A search was executed for blood defects, parasites, as well as a control of total IgE, hemochrome, sideremia. In the elderly, a search for Cea, Alfa feto protein was also done.

Symtoms

The patients had the following symptoms for at least three months prior to the beginning of the trial:

• abdominal pain or distension which decreased after evacuation;
• variation of the womb’s frequency and/or consistency of the feces;
• alterations in defecation which becomes difficult, impelling and with the sensation of incomplete evacuation,
• myxorrhea

45 patients were treated, predominantly female, between the ages of 20 to 70 (average age: 38).

Dosage

• * Group A: 1 capsule every 8 hours for 20 days.
• * Group B: 1 capsule every 12 hours for 30 days.
• * Group C: 1 capsule a day for 60 days.

Results

No unwanted side effects, adverse reactions or similar undesirable manifestations were verified which could have caused suspension of the treatment.
It was not necessary to interrupt therapy by substituting it with cholinergics, prokinetics or other drugs.

In Group A a considerable improvement in abdominal distension was noted, with an accelerated rhythm in evacuation and an emission of tendentially soft feces .
In Group B an improvement abdominal distension and consistency of the feces was noted, with a regularization of the womb and regular defecation.
Even in Group C an improvement in symptoms, though not constant, was noted.

As an ulterior test, a treatment with BioVal plus was executed on 15 patients which were under antibiotic therapy for eradication of Helicobacter pylori.
Conclusions

In light of these results, treatment A is recommended for patients who are habitually constipated, especially in the first phase of treatment, which will then be followed by scheme B and C.

Treatment B constitutes the “golden standard” and can be alternated with scheme C. Treatment C can be used as maintenance in patients with irritable colons, after an initially more intense therapy or cyclically alternating with the latter. In the ulterior test, all patients confirmed a preventive effect on the various symptoms of intestinal dismicrobism, with a better tollerance of the considerably heavy antibiotic and anti-microbic load (claritromycine, thymidazol, amoxicillin, metronidazol).

In conclusion, the use of the synergic balanced mix of Saccaromyces cerevisiae and Kluyveromyces fragilis B0399 is recommended in all cases of intestinal dismicrobism induced by organic diseases and/or antibiotics, but also for putting intestinal flora back in equilibrium to improve bloating,abdominal swelling and irregularity of the intestine in the syndrome of irritable colon.

Dr. S. Andreoli – medical director of the Gastroenterologic Service of the Hospital S. Maria della Misericordia of Udine Italy;