All feeds: TURVAL B0399

Probiotic additive for all feeds
with active typified lactic yeast
( Kluyveromyces BO399 )

Turval B0399: informative sheet for technicians

STRAIN DEPOSITED IN ACCORDANCE TO THE TREATY OF BUDAPEST and DIRECTIVE 70/524 CEE SHEET N° 2903/00

TURVAL B0399

Probiotic additive for all feeds with active typified lactic yeast

(Kluyveromyces B0399)

INTRODUCTION

Turval B0399 is a new product composed of an exclusive balanced mix of typified and selected lactic yeasts (Kluyveromyces B0399), supported by a wide range of cellular metabolites, fermenting products and hydrolyzed vegetable proteins.The lactic fermenting yeasts (or lactic yeasts) belong to the Kluyveromyces genus and have been used in the food and pharmaceutical industries for quite some time now, giving very interesting results.(Chart 1) Chart 1. List of yeast sources of industrial enzymes (Larpent, 1991)

Microorganisms	Enzymes
Saccharomyces cerevisiae	Invertasi
Kluyveromyces lactis	Beta-glicosilasi
Saccharomyces alluvius	Amilasi
Saccharomyces diastaticus	Glicoamilasi
Kluyveromyces Fragilis (B0399)	Beta-galattosidasi
Candida cylindraceae	Lipasi
Pichia guillermondii	Alfa-galattosidasi
Cryptococcus albidus	Beta-1,4 xilanoidrolasi

Up until now, the practical use of these yeasts in the animal diet (K. Fragilis or marxianus, in particular) was limited due to the difficulty in stabilizing these microorganisms (and the relative final products) using the standard industrial processes.A lengthy process of research and experimentation resulted in the refinement of a particular biotechnological method to select and typify the lactic yeast and moreover to produce the yeast on the industrial scale which stabilizes the product and exalts the modulation activity of the intestinal flora.The result of this innovative and laborious procedure is the probiotic additive Turval B0399 with active typified lactic yeast Kluyveromyces marxianus/fragilis B0399 .

FUNCTIONAL AND STRUCTURAL CHARACTERISTICS OF TYPIFIED LACTIC YEASTS

Kluyveromyces B0399The principal characteristics of Kluyveromyces fragilis B0399 (which sets it apart from brewer’s yeast, for example) are:· The capacity to grow and develop with extreme facility on a whey medium or other dairy by-products;· The ability to enzymatically degrade lactose (due to the presence of the enzyme beta-galactosidase or lactase in the citosol) in its constituents galactose and glucosesee (fig.1 and chart 2); In regards to the structural components of these active yeasts, it should be noted that the cellular wall contains a considerable quantity of polymers of mannose ( glucomannani, galactomannani e peptidomannani) and of N- acetil-glucosammina organized in complex structures (chitin), as well as the endocellular presence of the natural vitamins C, E and B complex (especially B1 and B2

Fig. 1: Mechanism of action of the enzyme beta-galactosidase

GAL-O-G + H2O = GAL + G

lattosio + H2O = galattosio + glucosio

Chart 2. Glicosidase identified in the yeasts (Bartnett 1981, Suomalainen and Oura 1971)

Enzima	Yeast	Substratum	Localization
Alfa -amilasi	Candida tropicalis	Amido, destrine	Extracellulare
Treleasi	Saccharomyces cerevisiae	Trealosio	Citosol, m.citoplasmatica
Glicoamilasi	Saccharomyces cerevisiae	Amido, destrine	Extracellulare
Inulinasi	Kluyveromyces fragilis (B0399)	Inulina	Extracellulare
Maltasi	Saccharomyces cerevisiae	Maltosio, saccarosio	Citosol
Isomaltasi	Saccharomyces cerevisiae	Isomaltosio	Citosol
Invertasi	Saccharomyces cerevisiae	Saccarosio, raffinosio	M. citoplasmatica
Beta-galattosidasi Lattasi	*Kluyveromyces fragilis B0399*	Cellobiosio Lattosio	Citosol
Alfa-galattosidasi	Saccharomyces cerevisiae	Raffinosio	M. citoplasmatica

AN INNOVATIVE PRODUCT

Within the ambit of the vast category of probiotic products, Turval B0399 stands out for its unique characteristics, which sets it apart from common brewer’s yeasts and the major part of lactic yeasts. As the in vitro tests conducted at the University of Udine (Italy) have demonstrated, Turval B0399 produces (from the fermentation of the organic substrata) lactic acid and acetic acid much more efficiently than many of the Saccharomyces cerevisiae (Brewer’s yeast) employed in zootechnical diets. (Chart 3) Chart 3. Comparization of fermentation profil of Turval B0399 vs Brewer’s yeast ( pH6,8, 39° C, 24 ore). (Univ. Udine 1999).

	TURVAL B0399	Brewer’s yeast
Acido lattico (mg/l)	2152	214
Ac. lattico + ac. acetico (mg/l)	3483	330

Compared to other products with a lactobacilli basis, Turval B0399 stands out for its elevated stability at room temperature (at least 24 months) and for the possibility, thanks to its particular resistance, of combining it with antibiotics such as the Ampicillin, Erythromycin, Tetracyclines and Amoxicillin (to which the major part of lactic bacteria is sensitive) and others.

INGREDIENTS

The additive TURVAL B0399 is composed of a balanced mix of natural products, such as: tipifyed active lactic yeast Kluyveromyces m/f B0399, wheat bran pre-treated and sterilized, whey powder, lactose, hydrolysed casein

ANALYSIS of additive
Parameter	Percentage value % on additive as is
Humidity	10.9%
Total fats	4.8%
Proteins	20.8
Raw fiber	6,7%
Ashes	4,92%


Analysis of heavy metals
Parameter	Value on additive as is (mg/kg)
Cd	0,08
Hg	0,05
Cr tot	1,00
Cu	0,50
Pb	0,25

PHYSICAL STATE, DIMENSION OF PARTICLES

The additive is under the form of powder which is easily miscible. Regarding the powder quality, an absence of particles under 36 micron is observed. Granulometric analysis
Granulomtry (mm) Percentage fraction

Granulomtry (mm)	Percentage fraction
> 170	58 %
> 100	26 %
> 36	16 %
< 36	0 %

CHARACTERISTICS OF THE SELECTED STRAIN

The active ingredient is a yeast,, Kluyveromyces marxianus (B0399) (Hansen) van der Walt, synonymous of Kluyveromyces fragilis; Genus Kluyveromyces, ascosporigenous, of which morphological characteristics and reproduction system are described. Isolation, purification, preservation, identification and classification were executed according to the dictates of “Bergey’s Manual of Systematic Bacteriology” and according to Lodder e Kreger van Rij, at theUniversitt di Perugia, Italy, Department of Botanical Biology , Applied Microbiology Sector. The mother strain (B0399) is deposited at BCCM – Belgian Coordinated Collection of Microrganism – Colture Collection Mycotheque de l’Université Catholique de Louvain. Biological activity: · Colony Forming Units of Kluyveromyces fragilis (B0399) : >5 x 106CFU/gr · production of b-galactosidase ( lactase )· demolition of lactose in glucose and galactose Morphological characteristics: The morphology of the cell and the mode of reproduction varies depending on whether the strain was cultivated in liquid culture or on “morphology agar”.· Kluyveromyces marxianus ( Hansen ) van der Walt var. Marxianus ( synonymous of Kluyveromyces fragilis ) registration number B0399 at BCCM. · Genus: Kluyveromyces ( Ascosporigenous ).· Vegetative cells: spheroidal, ovoidal, ellipsoidal or cylindrical ( 2,0 - 6,0 ) x ( 3,0 - 10,0 )mm· Reproduction : through single budding ( monopolar ) or in pairs ( bipolar ) and occasionally ( in conditions of anaerobiosis ) in short chains.· Asci : jugated or not jugated, with one or more ascospores ( 4 max.) , evanescent.· Ascospores: crescent-shaped, kidney-shaped, ellipsoidal or spheroidal with a tendency to stick together after liberation.· Culture in agar : colonies of a creamy type, cream colored, with wavy or lobe-shaped border .· Base nitrogenous percentages (mol % G+C): 43.91 ± 0,10 which include values characteristic to the species (see doc 1 attached and also vol 4/5). Physiological characteristics :q vegetative phase : cells predominantly spheroid but also ellipsoidalq reproduction by monopolar budding. q aschi : jugated, of the faded kind, with one or two ascospores up to a maximum of four. q ascospores : kidney-shaped and ellipsoidal with chitinose membrane.

RESISTANCE TO PELLETIZATION

It has been demostrated that TURVAL B0399 is fir for pelletization as the cell wall is rich in chitin and provides a useful protective bvarrier thus making the cell similar to a spore.· In cold conditions (t°C< 45°C): no mortality.· In hot conditions (t°C < 70°C): mortality < 20%

DURATION AND CONSERVATION

Expiry time: 2 yearsConservation: store in a dry and room-temperature place (3-30°C).The product is thermostabilized.
USE AND DOSAGEIn cattle· Milch cows: 8/10 gr. per head per day; · Cattle for fattening: 8/10 gr. per head per day;· Calves: 8/10 gr. per head per day.We suggest to use the additive Turval B0399 in combination with yeast Saccaromyces Cerevisae: in this case the abow dosage can be reduced up to 30%.In swine For piglets up to 30kg:· Weaning phase ( up to 8 kg. ): 1.20-1.50 kg per tonn of fodder· First phase of post-weaning ( from 8 to 15 kg. ca ): 1.20-1.50 kg per tonn of fodder.· Second phase of post-weaning (from 15 to 30 kg.ca.): 1.20-1.50 kg per tonn of fodder.The dosage can be reduced to 0.80-1.20 kg per tonn if the piglets were treated since weaning or since the first phase of post-weaning.- For fatten piglets: 0.40 kg per tonn of fodder for piglets which have been previously treated with Turval B0399..· To obtain best results Turval B0399 should be administered continually. · Larger doses may be used only during periods of stress ( at birth, during weaning, during change of diet, during change of seasons, after transportation, etc).

USE AND DOSAGE

In cattle.

Cattle, Dairy cows: 0.28 - 0.35 oz (8-10 g) per head per day;

Calves : 0.10 - 0.20 oz (3-6 g) per head per day;

In piglets up to 30kg:

Weaning phase, and post weaning phase: 1.2-1.5 lb per 1,000 lb complete feed;

If the piglets were fed the yeast since weaning or since the first phase of post-weaning, the suggested inclusion rate can be reduced to 0.80-1.20lb per 1,000 lb complete feed,

In swine over 30 kg: 0.40 lb per 1000 lb complete feed;

In Horses:

TURVAL B0399 is added to the daily ration of complete feed in the following quantities:

Foals: 0.25-0.35 oz (7-10g) per day;

Weaning colts: 0.35-0.53 oz (10-15g) per day;

One year old colts, adult horses, stallions and brood-mares: 0.53-0.70 oz (15-20 g) per day;

In Camels and Camelids (llamas and alpacas):

TURVAL B0399 is added to the daily ration in the following quantities:

Crias: 0.11-0.17 oz (3-5 g) per day;

Alpacas: 0.28-0.35 oz (8-10 g) per day;

Llamas: 0.88-1.10 oz (25-30 g) per day;

Calves: 0.71 oz (20 g) per day;

Female camels: 0.16 oz per 100 lb (10 g per 100 kg) live weight per day;

Bulls: 0.16 oz for 200 lb (10 g per 200 kg) live weight per day;

PACKAGES

25 Kg triple paper layers sacks

More information and detailed reports of trials are available.

INFORMATION SERVICE

For further information and samples please contact Turval laboratories.