Description:FIELD OF THE INVENTION
[0001] The present disclosure broadly relates to the field of fermentation technology. More particularly, the present invention relates to a two-stage co-fermentation process for providing high-protein feedstuffs. The multi-approach fermentation process disclosed herein results into a significant quality improvement of protein-rich feedstuffs.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Animals play a vital role in the livelihoods of people around the world in various ways. The importance of animals in human livelihood can be seen in agriculture and food production where animals are a primary source meat, milk, eggs, and other animal products. Livestock such as cattle, goats, sheep, and poultry are essential for protein and nutrient-rich food production. They also contribute to the livelihoods of farmers and herders who rely on animal husbandry for income. Accordingly, it is important to provide the feedstuff to these animals which is specifically formulated to provide essential nutrients, including carbohydrates, proteins, fats, vitamins, and minerals that are necessary for the growth, health, and production of the animals.
[0004] Reportedly, high-protein feedstuffs have a high acid binding capacity and high pH, which paly a negative role for efficient (protein) digestion, especially in young animals. High-protein feedstuffs also have, in general, high levels of anti-nutritional factors (i.e., trypsin inhibitor, glucosinolates, phytate-phosphor, indigestible sugars), which negatively influences the efficiency of livestock production.
[0005] Also, several antibiotics are used while preparing the feedstuffs as animals, feeds, or production units are found to be infected with undesirable microbes (e.g., Salmonella, Campylobacter) which, in turn, result in high costs and loss of production. Accordingly, there is a need to minimize the use of antibiotics but at the same time maximize the yield from production units against minimizing production costs.
[0006] Generally, liquid fermentation is frequently used to prepare liquid feeding as the duration of liquid fermentation is relatively short and takes in general only 24 to 48 hours. However, the technique becomes unusable where dry feed is required. In order to provide the dry feed, solid-state fermentation came into picture which is relatively a simple technology. However, there are different challenges when Solid-state fermentation is used. For instance, it does not offer the option to mix at different time of starter cultures, enzymes, or other additives during the fermentation process. In solid-state fermentation concepts, all materials must be mixed at one time and putted into the fermenter vehicle.
[0007] Therefore, there is a dire need to develop a co-fermentation process for preparing the feedstuff which is efficient, simple and capable of overcoming the shortcomings of solid and liquid fermentation when performed separately. The results may be obtained by combining their properties of both the techniques to provide improved and efficient process.

OBJECTS OF THE INVENTION
[0008] An objective of the present invention is to provide a multi-approach fermentation process for significant quality improvement of protein-rich feedstuffs.
[0009] An objective of the present invention is to provide a co-fermentation process for preparing the feedstuff which is efficient, simple and easy to perform.
[0010] Another objective of the present invention is to provide a process for producing solid-state fermented high-protein and high nutritional value feedstuffs.
[0011] Another objective of the present invention is to provide a fermented high-protein livestock feed.

SUMMARY OF THE INVENTION
[0012] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0013] An aspect of the present disclosure is to provide a multi-approach fermentation process for significant quality improvement of protein-rich feedstuffs. The inventive uniqueness is laid down in the combination of liquid and solid fermentation methods where solid-state fermentation is treated with a multi-starter culture approach to produce fermented protein-rich feedstuffs.
[0014] In an embodiment, the present invention discloses a two-stage co-fermentation method for providing fermented high-protein feedstuffs, the method comprising the steps of:
a. selecting and activating freeze-dried bacterial cultures 1 and 2;
b. providing a mixture of high-protein feed material, cereal or cereal by-product, or a combination thereof, for liquid fermentation;
c. inoculating the activated starter of culture 1 of step (a) and starter of culture of 2 step (a) to the mixture of step (b);
d. mixing the product obtained from step (c) with distilled water wherein the moisture content during liquid fermentation is in the range of 99% and 70%, preferably between 95% and 85%.;
e. fermenting the product of step (d) under anaerobic and liquid conditions for the time ranging from 8 to 36 hrs., preferably, 12 to 24 hours; at the temperature ranging from 20 to 60 degrees Celsius, preferably 30 to 50, most preferably 35 to 40.
f. introducing solid-state fermentation material comprising high-protein feedstuff and cereal or cereal by-product or a combination thereof, to the liquid fermented product obtained at step (e);
g. mixing the seeded liquid ferment obtained at step (e) to the solid-state fermentation material introduced at step (f);
h. optionally providing a liquid enzyme in step (f);
i. fermenting the product of step (g) under anaerobic, semi-moisture / solid conditions for the time ranging from 48 to 120 hours; at the temperature ranging from 20 to 40 degrees Celsius, preferably 25 to 35, most preferably 28 to 32-degree Celcius.
j. drying the mixture obtained from step (j) at the temperature ranging from 30 ºC, more preferably above 40 ºC but not above 60 ºC.
k. obtaining fermented high-protein feedstuffs;
l. optionally grinding and packaging the fermented high-protein feedstuffs.
[0015] In still another embodiment, the media used for culturing the bacteria comprises a combination of nutrients (sugars, protein source(s), minerals) and moisture for an optimal multiplication and activation of the freeze-dried bacteria.
[0016] In yet another embodiment, the present invention provides a fermented high-protein livestock feed, which is characterized by its improved protein-content, high protein digestibility and availability, and low microbial spoilage level, and enhanced bioavailability of nutrients.
[0017] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

FIGURES OF THE INVENTION
[0018] FIG. 1 illustrates a flow chart of two stage co-fermentation process for providing high-protein feedstuffs.
[0019] FIG. 2 depicts a detailed flowchart process for solid state fermentation

DETAILED DESCRIPTION OF THE INVENTION
[0020] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0021] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0022] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
[0023] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
[0024] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it is individually recited herein.
[0025] All processes described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0026] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0027] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0028] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0029] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description that follows, and the embodiments described herein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0030] It should also be appreciated that the present invention can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0031] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0032] The present disclosure focuses on a multi-approach fermentation process for significant quality improvement of protein-rich feedstuffs. The inventive uniqueness of the invention is laid down in the combination of several fermentation methods including liquid and solid stage fermentation displaying multi-stage fermentation steps including to obtain fermented protein-rich feedstuffs. Also, the chosen fermenter type and size allow the use of industrial production method, several degrees of automatization (depending on labour costs and labour availability in different regions of the world) and use of smart-sensors and software allowing the production of a constant high-quality fermented product. The present invention of multi-approach fermentation allows a shortening of fermentation time in solid-state process between 24 and 96 hours, whereas in other solid-state fermentation technologies fermentation times of minimum 1 week up to 6 weeks are common.
[0033] In an embodiment, the present invention discloses a two-stage co-fermentation method for providing fermented high-protein feedstuffs, the method comprising the steps of:
a. selecting and activating freeze-dried bacterial cultures 1 and 2;
b. providing a mixture of high-protein feed material comprising high-protein feedstuff and cereal or cereal by-product or a combination thereof, for liquid fermentation;
c. inoculating the activated starter of culture 1 of step (a) and starter of culture of 2 step (a) to the mixture of step (b);
d. mixing the product obtained from step (c) with distilled water, wherein the moisture content during liquid fermentation is in the range of 99% and 70%, preferably between 95% and 85%;
e. fermenting the product of step (d) under anaerobic and liquid conditions for the time ranging from 8 to 36 hours; preferably 12 to 24 hrs. at the temperature ranging from 20 to 60 degrees Celsius, preferably 30 to 50, most preferably 35 to 40;
f. introducing solid-state fermentation material comprising high-protein feedstuff and cereal or cereal by-product, or a combination thereof, to the liquid fermented product obtained at step (e);
g. mixing the seeded liquid ferment obtained at step (e) to the solid-state fermentation material introduced at step (f);
h. optionally providing a liquid enzyme in step (f);
i. fermenting the product of step (g) under anaerobic, semi-moisture / solid conditions for the time ranging from 48 to 120 hours; at the temperature ranging from 20 to 40 degrees Celsius, preferably 25 to 35, most preferably 28 to 32;
j. drying the mixture obtained from step (j) at the temperature ranging from 30 ºC, more preferably above 40 ºC but not above 60 ºC.
k. obtaining fermented high-protein feedstuffs;
l. optionally grinding and packaging the fermented high-protein feedstuffs.
[0034] In still another embodiment, the media used for culturing the bacteria is based on a proven combination of nutrients (sugars, protein source(s), minerals) and moisture for an optimal multiplication and activation of the freeze-dried bacteria.
[0035] In yet another embodiment, the activated and liquid starter culture 2 is added to liquid fermentation at the same time as starter culture 1 and/or between 1 and 12 hours after starter culture 1 is added, preferably between 2 and 8 hours after starter culture 1 is added and/or to the dry mixture of feedstuffs.
[0036] In still another embodiment, said culture 1 comprises enzyme-producing bacteria selected from B.Subtilis, B. Coagulens, B. Licheniformis or a combination thereof.
[0037] In yet another embodiment, said culture 2 comprises lactic acid-producing bacteria selected from Lactobacillus, Enterococcus, Lactococcus, and Pediococus including L. Plantarum, L. Rhamnosus, E. Faecium, P. Acidilactici, L. Lactis, P. Acidilactici, P. Pentosaceus or a combination thereof.
[0038] In still another embodiment, said feed material comprises at least one high-protein feedstuff derived from Guar meal, rapeseed and its by-products, sunflower seed and its by-products, soybean seed and its by-products, legume seeds like peas, chickpeas, field beans and other protein-rich feedstuffs like cottonseed meal, peanut meal, and others and a combination thereof
[0039] In yet another embodiment, said feed material further comprises at least one cereal or cereal by-product selected from wheat, wheat bran, maize, rice, sorghum, rye or a combination thereof,
[0040] In still another embodiment, at least 1 feedstuff is selected with high presence of endogenous enzymes including phytase.
[0041] In yet another embodiment, said proportion of the high-protein feedstuff is at least 80% by weight of total dry matter.
[0042] In still another embodiment, the moisture content during liquid fermentation is in the range of 99% and 70%, preferably between 95% and 85%.
[0043] In yet another embodiment, the moisture content during solid fermentation is in the range of 60% and 25%, preferably between 45% and 35%,
[0044] In still another embodiment, said solid-state process is completed in the time ranging from 24 to 96 hours.
[0045] In another embodiment, the present invention discloses a fermented high-protein livestock feed, obtainable by the method as claimed in claim 1, characterized by its improved protein-content, high protein digestibility and availability, and low microbial spoilage level, and enhanced bioavailability of nutrients.
[0046] In yet another embodiment, said feed is a dry feed.
[0047] Hence, under this background, the present inventors have made intensive efforts to solve the above problems, and as a result they developed an improved method for producing solid-state fermented high-protein feedstuffs which displays high nutritional value advantageous for the animals, and in particular having a high level of lactic acid, high level of bioactive components, low acid binding capacity and low pH.
EXAMPLES
[0048] The present invention is further explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.
Example 1
Starter Culture 1.
[0049] In the present invention, microbes which are mainly, but not exclusively, microbes with enzyme producing bacteria of the genus Bacillus and Lactobacillus were used. For example, B.Subtilis, B. Coagulens, B. Licheniformis, and L. Plantarum, or a mixture of all or some of them.
[0050] The freeze-dried bacteria strain(s) were activated before they were used in fermentation processes. The freeze-dried bacteria were multiplied and activated by offering them a medium including moisture and necessary nutrient along with specific pH-value, temperature, and duration. The culture medium was selected based on a proven combination of nutrients (sugars, protein source(s), minerals) and moisture for an optimal multiplication and activation of the freeze-dried bacteria. Final pH of the activated and liquid inoculum was between 3.5 and 5.5, preferably between 4.0 and 5.0. In the present invention, the activated and liquid starter culture 1 was added in the liquid fermenters.
Starter Culture 2
[0051] For a starter culture 2, microbes were selected from lactic acid producing bacteria of the genus Lactobacillus, Enterococcus, Lactococcus, and Pediococus. For example, but not exclusively, L. Plantarum, L. Rhamnosus, E. Faecium, P. Acidilactici, L. Lactis, P. Acidilactici, P. Pentosaceus, or a mixture of all or some of them.
[0052] The freeze-dried bacteria strain(s) were activated before they were used in fermentation processes by offering them a medium including moisture and adding necessary nutrients along with a set of initial pH-value, temperature, and duration the bacteria start to grow and multiply. The duration and temperature chosen resulted in high numbers of bacteria which were in optimal stage of life cycle, i.e., in logarithm or stationary phase. Final pH of the activated and liquid inoculum was between 3.5 and 5.5, preferably between 4.0 and 5.0.
[0053] The activated and liquid starter culture 2 can be added to liquid fermentation step at the same time as starter culture 1 and/or can be added to liquid fermentation step at a different time to liquid fermentation step (i.e., between 1 and 12 hours after starter culture 1 was added, more specifically between 2 and 8 hours, and/or can be added to the dry mixture of feedstuffs (see Solid State Fermentation chapter).
Example 2: Liquid Fermentation
[0054] The first step of the process disclosed herein is a liquid fermentation in liquid fermenters. In this liquid fermentation phase, the goal was to produce specific bacteria strain or mixture of strains using several types of microbial enzymes such as proteases, amylases, phytases or other fermentation metabolites such as lactic acid, acetic acid, bacteriocins, to prepare well for the second stage, i.e., solid-state fermentation.
[0055] In the liquid fermentation phase, it can be chosen to add only starter culture 1, but it is also an option to add both starter culture 1 and starter culture 2. It is an option to add them both at same time, but it is also an option to firstly add starter culture 1, followed by a delay of between 1 and 12 hours, more specifically between 2 and 8 hours, before starter culture 2 is added into the liquid fermenters.
[0056] The mixture of feedstuffs chosen for the liquid fermentation step, were kept identical to the mixture of feedstuffs to be fermented in the solid-state fermentation step. This was done to create best circumstances for the bacteria to optimize their metabolism in solid-state fermentation phase. Mixture of at least 2 feedstuffs was selected with water, whereas at least 1 feedstuff with proven high presence of endogenous enzymes like phytase was selected.
[0057] The fermentation was performed under anaerobic circumstances or semi-anaerobic circumstances, at the temperatures between 30 and 39 ºC, most preferably between 36 and 38 ºC, but should not exceed 40 ºC and the fermentation time was kept between 12 and 24 hours, and more specifically between 16 and 24 hours, but was not exceeded after 36 hours.
[0058] The moisture level of the fermentation was maintained between 99% and 70% moisture, specifically between 95% and 85% moisture, specifically between 90% and 80% moisture, but is not limited thereto.
Example 3: Solid-State Fermentation
[0059] The second step of the process was a solid-state fermentation which was a lactic-acid fermentation, and performed by using inoculum able to produce high quantities of lactic acid and a low to moderate quantities of acetic acid. The fermentation was done under anaerobic circumstances or semi-anaerobic circumstances. The fermentation was performed at temperatures above 20 ºC, most preferably above 30 ºC, but was not exceeded after 40 ºC. The fermentation time was kept between 24 hours and 120 hours, more specifically between 48 and 96 hours for the best results (FIG. 2).
[0060] It was observed that the application of liquid ferment (instead of water) as moisture source for the solid-state fermentation improves the speed and quality of the solid-state fermentation process. It was assured that the total moisture amount during the solid fermentation was maintained between 60% and 25%, more specifically between 45% and 35%, 70% and 100% (w/w) using the liquid ferment produced during the liquid fermentation step. The remaining 0% to 30% (w/w) of moisture was maintained by combining hot (between 60 and 80 ºC) and cold water (lower than 15 ºC).
[0061] Eventually, an enzyme or mixture of enzymes selected from phytase, xylanase, cellulase, protease, B-glucanase or mixture of two or more was added into said water, and subsequently mixed into the solid-state fermentation mixture. In case, where moisture was provided solely via liquid ferment produced during the liquid fermentation step, the enzyme or mixture of enzymes was added to the liquid ferment just before adding to the solid-state fermentation mixture.
[0062] Regarding the feedstuff, most preferred feedstuff was a mixture of at least 2 feedstuffs where 1 feedstuff was selected with proven high presence of endogenous enzymes like phytase and the other feedstuff was selected with high-protein level. The feedstuffs were selected from Guar meal varieties, rapeseed and its by-products, sunflower seed and its by-products, soybean seed and its by-products, legume seeds like peas, chick peas, field beans and other protein-rich feedstuffs like cottonseed meal, peanut meal, and others. At the start of the solid-state fermentation step, starter culture 1, or starter culture 2, or both starter cultures were added to the mixture depending on the desired results.
Example 4: Big Boxes
[0063] The mixed product is packed into the “fermenter” i.e., a plastic big box with a cover of the size ranging from 100 Kg to 2000 kg, preferably between 500 Kg to 1000 kg. The fermentation mixes and moisture product were packed into the big box, and the top-layer was compacted by a load which is pushed on top-layer mechanically or by manpower to create an anaerobic or semi-anaerobic environment (FIG. 1).
Example 5: Smart Sensors
[0064] During the whole process, both, liquid fermentation process and solid-state fermentation process were supervised by smart sensors by measuring the temperature and pH which were further reported to the control software.
Example 6: Moderate Drying and Packaging
[0065] Drying of solid-state fermented feedstuff was carried out in a moderate way with temperature preferably above 30 ºC, more preferably above 40 ºC but not above 60 ºC. The feedstuff was further grounded or pulverized depending on the product characteristics after fermentation steps.
Example 7: Trial SSF 2022
[0066] The fact that inclusion of 10% wheatbran resulted in equal Crude Protein-% after fermentation, means that fermentation resulted in higher Crude Protein. It is to be noted that wheatbran has a remarkably lower Crude Protein % compared to Rapeseed Expeller : 10% versus 30% Crude Protein respectively. In theory, it would be expectable that Crude Protein of the 90/10 RSC/Wheatbran mixture is about 2% lower compared to non-fermented RSC The addition of Wheatbran has as goal the addition of endogenous enzymes present in wheatbran (i.e., phytase high content).
Non-fermented Rapeseed Expeller Fermented Rapeseed Expeller (90%) + Wheatbran (10%)
Moisture (%) 8.7 8.8
pH 5.9 4.1
Glucosinolates (umol/g) 25.6 0.2
Crude Protein (%) 30.7 30.6

Example 7a: Trial SSF 2022 – march
Non-fermented Korma Guar Meal roasted Fermented Korma Guar Meal roasted (incl. addition of enzyme mixture)
Moisture (%) 8.5 6.8
pH 6.1 4.3
Crude Protein (%) 48.4 49.6
Protein Solubility (KOH) (%) 80.2 87.1
Lactic Acid (%) Non-detectable 4.3

Example 7b: Trial SSF 2022 – march
Non-fermented CHURI roasted Fermented CHURI roasted (incl. addition of enzyme mixture)
Moisture (%) 8.9 7.9
pH 5.7 4.4
Crude Protein (%) 32.8 34.6
Protein Solubility (KOH) (%) 74.9 85.2
Lactic Acid (%) Non-detectable 3.7

[0067] Briefly, two varieties of Guar Meal products: Korma, and, Churi were tested. As can been seen from the tables, the Crude Protein % is increased after fermentation (1.2 up to 1.8%), protein solubility (KOH) as indicator for protein digestibility is increased after fermenttaion (6.8 up to 10.3%), pH is decreased which is also a positive factor for an improved protein digestibility in first part of gastro-intestinal tract.
Example 7c: SSF 2022 – October
Non-fermented Korma Guar Meal roasted Fermented Korma Guar Meal
roasted
Moisture (%) 13.5 10.5
pH 5.6 4.4
Crude Protein (%) 47.2 49.2
Protein Solubility (KOH) (%) 64.4 76.0
Lactic Acid (%) Non-detectable 3.9

[0068] As can been seen from the tables, the Crude Protein % is increased after fermentation (2.0%), protein solubility (KOH) as indicator for protein digestibility is increased after fermenttaion (11.6%), pH is decreased which is also a positive factor for an improved protein digestibility in first part of gastro-intestinal tract.
Example 8: Trials Broilers
[0069] In Summer 2022, two big trials with broilers has been done (Department Poultry Science & Department of Animal Nutrition, India, CVSc, Rajendranagar farm). One trial was done with „raw Guar Meal” (RGM) and 4 treatments: non-fermented versus fermented, and both types with an inclusion rate of 10% or 15%. The second trial was done with „roasted Guar Meal” (TGM) and 4 treatments: non-fermented versus fermented, and both types with an inclusion rate of 10% or 15%.

Body Weight Gain (g) Feed Intake (g) Feed Conversion Rate
Control 2,199 a 3,293 1.498 a
RGM 10 2,169 a 3,329 1.539 ab
RGM 15 2,046 b 3,308 1.620 b
F-RGM 10 2,207 a 3,269 1.485 a
F-RGM 15 2,099 ab 3,260 1.564 ab

Number replicates 12 12 12
P-value * 0.77 **
SEM 19.18 18.48 0.01

Body Weight Gain (g) Feed Intake (g) Feed Conversion Rate
Control 2,271 a 3,398 1.495 ab
TGM 10 2,224 ab 3,376 1.519 bc
TGM 15 2,184 b 3,394 1.555 c
F-TGM 10 2,279 a 3,301 1.450 a
F-TGM 15 2,243 ab 3,280 1.465 ab

Number replicates 12 12 12
P-value * 0.15 **
SEM 11.39 15.84 0.01

[0070] The results of broiler trial: Feed Conversion Rate is significantly improved if toasted guar meal is fermented. The inclusion of 10% and 15% F-TGM has better FCR then TGM. And even numerically lower compared to Control Group. Also this is an indication for a better protein digestibility, because the diets were isoenergetic and isonitrogenic.
[0071] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.
, Claims:1. A two-stage co-fermentation method for providing fermented high-protein feedstuffs, the method comprising the steps of:
a. selecting and activating freeze-dried bacteria cultures 1 and 2;
b. providing a mixture of high-protein feed material comprising high-protein feedstuff, cereal or cereal by-product or a combination thereof for liquid fermentation;
c. inoculating the activated starter of culture 1 of step (a) and starter of culture of 2 (step a) to the mixture of step b;
d. mixing the product obtained from step c with distilled water wherein the moisture content during liquid fermentation is in the range of 99% and 70%, preferably between 95% and 85%.;
e. fermenting the product of step d under anaerobic and liquid conditions for the time ranging from 12 to 24 hours; at the temperature ranging from 20 to 60 degrees Celsius, preferably 30 to 50, most preferably 35 to 40.
f. introducing solid-state fermentation material comprising high-protein feedstuff, cereal or cereal by-product, or a combination thereof to the liquid fermented product obtained at step (e);
g. mixing the seeded liquid ferment obtained at step (e) to the solid-state fermentation material introduced at step (f);
h. optionally providing a liquid enzyme in step (f);
i. fermenting the product of step (g) under anaerobic, semi-moisture / solid conditions for the time ranging from 48 to 120 hours; at the temperature ranging from 20 to 40 degrees Celsius, preferably 25 to 35, most preferably 28 to 32.
j. Drying the mixture obtained from step (j) at the temperature ranging from 30 ºC, more preferably above 40 ºC but not above 60 ºC.
k. obtaining fermented high-protein feedstuffs;
l. optionally grinding and packaging the fermented high-protein feedstuffs.
2. The method as claimed in claim 1, wherein the activated and liquid starter culture 2 is added to liquid fermentation at the same time as starter culture 1 and/or between 1 and 12 hours after starter culture 1 is added, preferably between 2 and 8 hours after starter culture 1 is added and/or to the dry mixture of feedstuffs.
3. The method as claimed in claim 1, wherein said culture 1 comprises enzyme-producing bacteria selected from B.Subtilis, B. Coagulens, B. Licheniformis or a combination thereof.
4. The method as claimed in claim 1, wherein said culture 2 comprises lactic acid-producing bacteria selected from Lactobacillus, Enterococcus, Lactococcus, and Pediococus including L. Plantarum, L. Rhamnosus, E. Faecium, P. Acidilactici, L. Lactis, P. Acidilactici, P. Pentosaceus or a combination thereof.
5. The method as claimed in claim 1, wherein said feed material comprises at least one high-protein feedstuff derived from Guar meal, rapeseed and its by-products, sunflower seed and its by-products, soybean seed and its by-products, legume seeds like peas, chicken peas, field beans and other protein-rich feedstuffs like cottonseed meal, peanut meal, and others and a combination thereof
6. The method as claimed in claim 1, wherein said feed material further comprises at least one cereal or cereal by-product selected from wheat, wheat bran, maize, rice, sorghum or rye or a combination thereof,
7. The method as claimed in claim 1, wherein at least 1 feedstuff is selected with high presence of endogenous enzymes including phytase.
8. The method as claimed in claim 1, wherein said proportion of the high-protein feedstuff is at least 80% by weight dry matter.
9. The method as claimed in claim 1, wherein moisture content during liquid fermentation is in the range of 99% and 70%, preferably between 95% and 85%.
10. The method as claimed in claim 1, wherein moisture content during solid fermentation is in the range of 60% and 25%, preferably between 45% and 35%,
11. The method as claimed in claim 1, wherein said solid-state process is completed in the time ranging from 24 to 96 hours.
12. A fermented high-protein livestock feed, obtainable by the method as claimed in claim 1, characterized by its improved protein-content, high protein digestibility and availability, and low microbial spoilage level, and enhanced bioavailability of nutrients.
13. The fermented high-protein livestock feed according to claim 12, where said feed is a dry feed.