A yeast biomass composition and uses thereof

Abstract

A yeast biomass composition is formed by culturing a strain of yeast in a substrate such as whey permeate to product a yeast broth, dewatering the broth to produce yeast biomass, and hydrolysing the yeast biomass. The yeast biomass composition finds utility as a feed additive for broiler chickens to reduce the adherence of pathogenic bacteria like Salmonella and E.coli to the gut wall of the chickens, thereby reducing intestinal stress and improving life daily weight gain in the chickens.

Description

[0001] TITLE

[0002] A yeast biomass composition and uses thereof

[0003] Technical Field

[0004] The present invention relates to a yeast biomass composition, methods for the production and uses thereof.

[0005] Technical Background

[0006] The term ‘microbial biomass’ describes a supplemental protein source obtained from processes in which bacteria, yeasts, other fungi or algae are cultivated in large quantities (Vieira, Andrietta & Andrietta 2013). Protein is a major component of yeast biomass, and yeast proteins are known to have an amino acid composition close to the ideal protein level (Jach, Serefko, Ziaja, & Kieliszek, 2022). Yeast is currently a common ingredient in the animal feed industry. The average yield of these products about 30 - 40 % crude protein [https: / / www.ingredients101 .com / molasses.htm].

[0007] Despite the extensive literature in the field of yeast biomass, few products have been successful commercially. One successful product is a yeast biomass produced from a molasses feedstock using a saccharomyces yeast (BIO-MOS feed supplement, Alltech Inc., Nicholasville, KT, USA). The protein yield using this saccharomyces yeast and molasses feedstock is about 40%. The process also employs yeast extract which is an expensive ingredient. There are many limitations to protein ingredients with yields of 40 %. Typically this is not concentrated.

[0008] However, a more concentrated protein product would cut costs and provide a more sustainable product in additional to have better functionality as a nutritional product. In addition, it would be beneficial to have a process for generating a yeast biomass by fermentation that does not require use of yeast extract. Salmonella spp. is one of the most common foodborne pathogens causing foodborne illness called “salmonellosis” in various parts of the world. The illness is initiated by the ingestion of contaminated foods or consumption of beverages. Salmonella spp. are involved as the main cause of a wide range of diseases. The contamination of foods with Salmonella is generally associated with meat production. Food-producing animals and poultry including broilers, play a major role in the spreading of this pathogen. It can be transmitted at the pre-harvest stage (on farms) to various other post-harvest steps along the food value chains, all the way to consumers. Consequently, it poses a significant public health concern as well as a cause of economic losses for producers.

[0009] US 5,486,368 describes a method of making a yeast biomass comprising culturing yeast on clarified whey permeate (5% Total Solids), separating the fermentation broth into yeast cream and centrate, and then subjecting the yeast cream to autolysis or hydrolysis. The concentrated centrate is then combined with the hydrolysed yeast cream to provide a partially hydrolysed yeast biomass, which is then dried and bagged. Various strains of yeast are mentioned, including a number of strains of Kluyveromyces for human consumption and Candida for animal consumption. The dry product contains up to 45% protein.

[0010] It is an objective of the invention to overcome at least one of the above-referenced problems.

[0011] Summary of the Invention

[0012] The objective is met by the provision of a hydrolysed yeast biomass composition, typically made by culturing a strain of yeast (e.g. Kluyveromyces ) in a substrate (e.g. whey permeate or delactosed whey permeate) to produce a yeast broth, dewatering the broth to provide a yeast biomass, and hydrolysing the yeast biomass. The yeast is typically adapted to grow in higher levels of the substrate, for example at least 10%, 12% or 14% whey permeate, which increases the throughput and efficiency of the process significantly. The protein yield in the yeast biomass obtained using an adapted strain of Kluyveromyces marxianis has been found to reach 50-55% (w / v), far greater than what has been achieved using saccharomyces yeast. In addition, the Applicant has surprisingly discovered that feeding an animal with an animal feed that is supplemented with the hydrolysed yeast biomass produced on the substrate and using the process described below causes a reduction in the adherence of pathogenic bacteria such as Salmonella and E-coli to the gut wall of the animal, thus lowering the intestinal stress of the animals while also improving daily live weight gain.

[0013] In a first aspect, the invention provides a process of making a yeast biomass composition comprising: culturing a yeast strain in a culture medium comprising a substrate and a nitrogen source, fermentation salts and other fermentation nutrients to produce a yeast broth; dewatering the yeast broth to provide a yeast biomass; and hydrolysing the yeast biomass to provide the yeast biomass composition.

[0014] The yeast biomass composition is generally a single cell composition.

[0015] In any embodiment, the yeast strain comprises a Kluyveromyces yeast.

[0016] In any embodiment, the substrate is a by-product of dairy processing, for example a whey permeate (including delactosed whey permeate) or another carbon source.

[0017] In any embodiment, the yeast strain is adapted to grow on a substrate (e.g. a whey permeate) having at least 10%, 11 %, 12%, 13% or 14% protein.

[0018] In any embodiment, the substrate comprises 2-3% protein on a dry matter basis. In any embodiment, the substrate comprises at least 7%, 8%, 10%, 11 %, 12%, 13% or 14% protein (w / v).

[0019] In any embodiment, the substrate is not treated to reduce the solids content, e.g. by a filtration method.

[0020] In any embodiment, the hydrolysing comprises thermal hydrolysis, chemical hydrolysis, enzymatic hydrolysis, or a combination thereof (for example, thermal and chemical hydrolysis, thermal and enzymatic hydrolysis, chemical and enzymatic hydrolysis, or thermal hydrolysis, chemical hydrolysis and enzymatic hydrolysis).

[0021] In any embodiment, the enzymatic hydrolysis employs one or more proteases and I or one or more sugar hydrolysing enzymes.

[0022] In any embodiment, the enzymatic hydrolysis employs one or more sugar hydrolysing enzymes (e.g. a cellulase, invertase, hydrolase).

[0023] In any embodiment, the enzymatic hydrolysis employs one or more proteases and I or one or more sugar hydrolysing enzymes.

[0024] In any embodiment, the thermal hydrolysis comprises heating the yeast biomass to 55° to 75°C for 240 to 600 minutes. The time v temperature combination may be modified by employing a higher temperature and lower time or vice versa.

[0025] In any embodiment, the chemical hydrolysis comprises treating the yeast biomass with acid, for example one or more organic acids, for example selected from one or more of Hydrochloric, Sulphuric, or Phosphoric acid.

[0026] In any embodiment, the culture medium comprises a source of nitrogen, phosphate and potassium. In any embodiment, the source of nitrogen includes ammonia water.

[0027] In any embodiment, the culture medium comprises one or more fermentation nutrients, trace elements or both.

[0028] In any embodiment, the culture medium comprises a single strain of Kluyveromyces yeast.

[0029] In any embodiment, the liquid whey permeate is pasteurised prior to formulation of the culture medium.

[0030] In any embodiment, the liquid whey permeate is diluted with water prior to pasteurisation.

[0031] In any embodiment, the hydrolysed yeast biomass is dried to a moisture level of less than 10%, 8%, or 6% moisture (v / w). Drying may be performed on a conventional dryer, for example a rotary drum dryer or a fluidised bed dryer. The inlet air temperatures combined with the drying conditions are generally sufficient to inactivate any enzymes present in the biomass.

[0032] In any embodiment, the yeast biomass is dewatered in at least two stages, an initial dewatering stage (e.g., decanting or centrifugation) and a second stage of drying (e.g., drum drying, fluid bed drying, spray drying, evaporation, lyophilisation) performed on the hydrolysate..

[0033] In any embodiment, the process is a fed-batch process. In any embodiment, the process is a continuous process.

[0034] In any embodiment, the substrate is added to the culture medium periodically during the culturing step. In any embodiment, sources of N, P and K are added to the culture medium periodically during the culturing step. In any embodiment, sources of fermentation nutrients and trace elements are added to the culture medium periodically during the culturing step.

[0035] In any embodiment, the culturing step is performed in a reactor vessel, wherein fermentation broth is optionally periodically removed from the reactor vessel.

[0036] In any embodiment, the culture medium is aerated and heated during the culturing step. These are conventional steps during fermentation processes.

[0037] In any embodiment, the pH of the culture broth is maintained at about pH 4-8 (e.g. about pH 5) during the culturing step. Culture conditions suitable for culturing Kluyveromyces strains are described in Fonseca et al. (FEMS Yeast Research, Volume 7, Issue 3, May 2007, Pages 422-435).

[0038] In any embodiment, the dried yeast biomass is milled.

[0039] In any embodiment, the culture medium is substantially free of yeast extract.

[0040] In any embodiment, the strain of Kluyveromyces yeast is a strain of Kluyveromyces marxianus or Kluyveromyces fragilis. Exemplary strains of Kluyveromyces marxianus include the strains described in Table 1 of Perpetuini et al. (Frontiers in Microbiology, Vol. 10, 2019), Kluyveromyces marxianus ATCC 26548, and Kluyveromyces marxianus A1 to A5 (GenBank Accession Numbers MT791345, MT793595, MT793593, MT793596 and MT793594, respectively). Other strains of yeast suitable for use with the process and composition of the disclosure include strains of yeast belong to the genus Saccharomyces, Hansenula, Torulaspora, Candida, or Kluyveromyces. Strains of Saccharomyces include S. cerevisiae, S. pastorineus, S. exiguus and S. bayanus, In any embodiment, the strain of yeast is an adapted strain (e.g. adapted to grow in / on a test culture medium supplemented with a higher concentration of a specific substrate compared to a non-adapted strain). Adapting a strain thus increases the tolerance of the strain to the specific substrate. Methods of adapting strains of microorganisms to grow on substrates is described in WO2017 / 072748 (especially page 10, lines 9-29, and pages 12-13).

[0041] In any embodiment, the strain of yeast is adapted to grown in / on a substrate containing at least 8%, 10%, 12%, 13% or 14% protein (w / v).

[0042] In any embodiment, the strain of yeast is adapted to grown in / on a whey permeate containing at least 8%, 10%, 12%, 13% or 14% protein (w / v).

[0043] In any embodiment, the adapted strain of yeast is a strain of Kluyveromyces, e.g. Kluyveromyces marxianis.

[0044] In another aspect, there is provided a hydrolysed yeast biomass obtainable by the process of the invention.

[0045] In another aspect, there is provided a yeast biomass composition obtainable by culturing a strain of Kluyveromyces yeast in liquid whey permeate to form a yeast broth, dewatering the broth to provide yeast biomass, and then optionally hydrolysing the yeast biomass.

[0046] In another aspect, there is provided a hydrolysed yeast biomass, in which the yeast comprises or consists of a strain of Kluyveromyces, ideally a strain of Kluyveromyces marxianis.

[0047] In any embodiment, the hydrolysed yeast biomass has a solubility in water of at least 90%, 95% or 99%. In any embodiment, the hydrolysed yeast biomass has a bulk density of 400 to 500 g / l, 440 to 480 g / l, 450 to 470 g / l, or about 460 g / l.

[0048] In any embodiment, the yeast biomass comprises at least 45%, 46%, 48%, 50%, 53%, 54% or 55% protein. In any embodiment, the yeast biomass comprises at 46- 60%, 48-57% or 50-55% protein. % protein is determined by the Kjeldahl method [Codex Alimentarius as the standard for quantifying milk protein (FAO, 2017)].

[0049] In any embodiment, the hydrolysed yeast biomass has a content of organic nitrogen of at least 7.0%, 7.5% or 8.0% (as determined by the Kjeldahl method).

[0050] In any embodiment, the hydrolysed yeast biomass comprises at least 25%, 28%, 30% or 31 % amino acids (w / w).

[0051] In any embodiment, the hydrolysed yeast biomass comprises 28 to 35%, 30 to 32% or about 31 .35% amino acids (w / w).

[0052] In any embodiment, the hydrolysed yeast biomass comprises threonine, valine, methionine, isoleucine, leucine, phenylalanine, histidine and lysine.

[0053] In any embodiment, the hydrolysed yeast biomass comprises at least 1.5% threonine, at least 1.5% valine, at least 0.4% methionine, at least 1.3% isoleucine, at least 2.1 % leucine, at least 1 .3% phenylalanine, at least 0.5% histidine and / or at least 2.3% lysine.

[0054] In any embodiment, the hydrolysed yeast biomass comprises substantially all of the amino acids aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine (e.g. at least 14, 15 or 16 of the amino acids).

[0055] In any embodiment, the hydrolysed yeast biomass comprises substantially all of (w / w): Aspartic 3.13%

[0056] Threonine (essential) 1 .63%

[0057] Serine 1.50%

[0058] Glutamic 6.02%

[0059] Proline 1.06%

[0060] Glycine 1 .42%

[0061] Alanine 2.89%

[0062] Cystine 0.26%

[0063] Valine (essential) 1.83%

[0064] Methionine(essential) 0.51 %

[0065] Isoleucine (essential) 1.53%

[0066] Leucine (essential) 2.37%

[0067] Tyrosine 0.95%

[0068] Phenylalanine(essential) 1.46%

[0069] Histidine (essential) 0.68%

[0070] Lysine (essential) 2.55%

[0071] Arginine 1.57%

[0072] In any embodiment, the hydrolysed yeast biomass comprises vitamins B1 , B2, B3, B6, B7 and B12. In any embodiment, the hydrolysed yeast biomass comprises at least 15 mg / kg vitamin B1.

[0073] In any embodiment, the hydrolysed yeast biomass comprises at least 30 mg / kg vitamin B2.

[0074] In any embodiment, the hydrolysed yeast biomass comprises at least 150 mg / kg vitamin B3.

[0075] In any embodiment, the hydrolysed yeast biomass comprises at least 15 mg / kg vitamin B6.

[0076] In any embodiment, the hydrolysed yeast biomass comprises at least 1750 pg / kg vitamin B7. In any embodiment, the hydrolysed yeast biomass comprises at least 25 pg / kg vitamin B7.

[0077] In another aspect, there is provided a use of the hydrolysed yeast biomass of the invention as a nutritional supplement. The supplement may be provided as a powder, beverage, food product, or unit dose product (e.g., a tablet, capsule, sachet of powder, gel)

[0078] In another aspect, there is provided a use of the hydrolysed yeast biomass of the invention as an animal feed supplement (additive).

[0079] In another aspect, there is provided an animal feed comprising an animal feed component and a hydrolysed yeast biomass of the invention as an additive. In any embodiment, the animal feed comprises 90.0% to 99.999% animal feed component and 0.001 %-10% hydrolysed yeast biomass (w / w). In any embodiment, the animal feed comprises 95-99% animal feed component and 1-5% yeast biomass composition as a supplement (w / w). In any embodiment, the animal feed comprises 97-99% animal feed component and 1-3% yeast biomass composition as a supplement (w / w). In any embodiment, the animal feed comprises 99.91 to 99.99% animal feed component and 0.01 to 0.09% yeast biomass composition as a supplement (w / w). The animal feed component may be any conventional animal feed, for example one or more of corn, cane molasses, diary proteins (whey), soybeans, sorghum, and grains such as oats and barley. The animal feed may be for avians (e.g. chicken, duck or game birds), beef cattle, dairy cattle, calves, sheep, swiner (e.g. pigs and hogs) and horses. The pig feed may be a weaner, finisher / fattener, lactating sow or dry sow type feed product. The chicken may be a layer or broiler.

[0080] In another aspect, there is provided a method of reducing a load of pathogenic bacteria (e.g. Salmonella or E.coli) in the gut of an animal comprising feeding a hydrolysed yeast biomass or animal feed of the invention to the animal. In another aspect, there is provided a method of reducing adherence of a pathogenic bacterium (e.g. Salmonella or E.coli) to a gut wall of a mammal comprising orally administering a hydrolysed yeast biomass to the animal.

[0081] In another aspect, there is provided a method of improving daily live weight gain of an animal comprising feeding a hydrolysed yeast biomass or animal feed of the invention to the animal.

[0082] In any embodiment, the animal feed comprises 1-1000, 10-1000, 10 to 500, 50 to 200, or about 80 to 120 mg hydrolysed yeast supplement of the invention per Kg of animal feed. In any embodiment, the animal feed comprises 0.0005 to 2%, , 0.001 to 0.01 %, or about 0.05 % hydrolysed yeast supplement (by weight).

[0083] Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

[0084] Brief Description of the Figures

[0085] Figure 1 shows an Overview of the experimental strategy to determine the effect of the yeast biomass on inflammatory pathways in innate immune cells.

[0086] Figure 2 shows the effect of the yeast biomass on basal cytokine secretion in J774A.1 Macrophages.

[0087] Figure 3 shows the effect of the yeast biomass on LPS Stimulated cytokine secretion in J774A.1 Macrophages.

[0088] Figure 4 shows the effect of the yeast biomass on basal cytokine secretion in JAWSII dendritic cells.

[0089] Figure 5 shows the effect of the yeast biomass on LPS stimulated cytokine secretion in JAWSII Dendritic cells. Figure 6 shows the evolution of the average faecal scores from 0 to 46 days postweaning by treatment.

[0090] Figure 7 shows the E. coli counts in faecal samples at days 7 and 14 post-weaning by treatment.

[0091] Figure 8 shows the results of the meta-analysis by Xu et al., 2021 comparing the effect of different antibiotic substitutes (89 trials) on ADG. The dot shows the average effect of each type of additive on ADG with 95% confidence interval. The dotted line shows the no effect point. The red line indicates the effect of the new biomass yeast product of the invention tested in this trial.

[0092] Figure 9 shows the effect of the test products on the ileal microbial gas production after 12 hours of simulation. The error bars indicate SE between the replicate simulation vessels and asterisks the statistical difference to the unamended control (grey column) with t-test.

[0093] Figure 10 shows the effect of the test products on acetic acid production after 12 hours of ileal simulation. The error bars indicate SE between the replicate simulation vessels and asterisks the statistical difference to the unamended control (grey column) with t-test.

[0094] Figure 11 shows the effect of the test products on lactic acid production after 12 hours of ileal simulation. The error bars indicate SE between the replicate simulation vessels and asterisks the statistical difference to the unamended control (grey column) with t-test.

[0095] Figure 12 shows the effect of the test products on total ileal bacterial numbers after 12 hours of simulation. The error bars indicate SE between the replicate simulation vessels and asterisks the statistical difference to the unamended control (grey column) with t-test. Figure 13. shows the effect of the test products on the caecal microbial gas production after 12 hours of simulation. The error bars indicate SE between the replicate simulation vessels and asterisks the statistical difference to the unamended control (grey column) with t-test.

[0096] Figure 14 shows the effect of the test products on the total caecal VFA production after 12 hours of simulation. The error bars indicate SE between the replicate simulation vessels and asterisks the statistical difference to the unamended control (grey column) with t-test. Note: lactic acid is excluded in the total VFA results of K. marxianus 40 mg / mL.

[0097] Figure 15 shows the effect of the test products on the caecal butyric acid production after 12 hours of simulation. The error bars indicate SE between the replicate simulation vessels and asterisks the statistical difference to the unamended control (grey column) with t-test.

[0098] Figure 16 shows the effect of the test products on the caecal propionic acid production after 12 hours of simulation. The error bars indicate SE between the replicate simulation vessels and asterisks the statistical difference to the unamended control (grey column) with t-test.

[0099] Figure 17 shows the effect of the test products on the caecal BCFA production after 12 hours of simulation. The error bars indicate SE between the replicate simulation vessels and asterisks the statistical difference to the unamended control (grey column) with t-test.

[0100] Figure 18 shows the effect of the test products on total caecal bacterial numbers after 12 hours of simulation. The error bars indicate SE between the replicate simulation vessels and asterisks the statistical difference to the unamended control (grey column) with t-test. Figure 19 shows the effect of the test products on the adherence of Escherichia coli on piglet intestinal mucus. The identities of test products are shown below the patterns of columns. The adherence inhibition % is set as 100 in the absence of any test products. Error bars indicate SE between 4 replicate reaction vessels. Statistical difference between the negative control and the test products is indicated by the asterisks. The statistical analysis is done with Student’s t-test.

[0101] Figure 20 shows the effect of the test products on the adherence of Salmonella enterica on piglet intestinal mucus. The identities of test products are shown below the patterns of columns. The adherence inhibition % is set as 100 in the absence of any test products. Error bars indicate SE between 4 replicate reaction vessels.

[0102] Figure 21 shows the Final survival rate (%) of fish based on different feed formulations.

[0103] Figure 22 shows the Individual absolute growth (g / fish, initial weight-final weight) per feed type.

[0104] Figure 23 shows the Individual growth rate (% / day / fish) per feed type.

[0105] Figure 24 shows the Individual Body Condition index per feed type.

[0106] Figure 25 shows the Total Biomass gained (g / tank) per feed type.

[0107] Figure 26 shows the Absolute Feed Intake (g / tank) per feed type.

[0108] Figure 27 shows the Daily Feed intake (g / day / tank) per feed type.

[0109] Figure 28 shows the Feed Conversion ratio per feed type.

[0110] Figure 29 shows the Feed Consumption time (s, as a measure of palatability) per feed type. Figure 30 shows the time to consume feed per feed type.

[0111] Detailed Description of the Invention

[0112] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

[0113] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0114] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0115] As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method / process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open- ended and does not exclude additional, unrecited integers or method / process steps.

[0116] As used herein, the term “hydrolysed yeast biomass composition” refers to a yeast biomass which is produced by culturing a strain of yeast in a culturing broth comprising a yeast substrate as a primary source of carbon (and typically supplemented with a source of nitrogen, potassium and phosphorus (N / P / K)), dewatering the fermentation broth to provide a yeast biomass, and hydrolysing the yeast biomass to provide the hydrolysed yeast biomass composition which is then optionally dried, milled and bagged. The composition is suitable for use as a food ingredient or a food supplement, for example an animal feed supplement / additive. The composition typically has greater than 40% protein on a dry matter basis, for example more than 50% or 55% protein. Typically, the method for making the composition broadly comprises the steps of: providing liquid whey permeate or delactosed whey permeate or other carbon source (Substrate); pre treating the diluted Substrate; adding the pretreated Substrate to a reactor vessel in a controlled manner during the culturing period along with a yeast inoculum, a source of N / P / K, and optionally one or more fermentation nutrients and trace elements; aerating and controlling both the temperature and the ph of the mixture in the reactor vessel during a culturing period; removing the fermentation broth from the reactor vessel after the culturing period; centrifuging the broth to isolate the yeast biomass; hydrolysing the yeast biomass to produce a hydrolysed yeast biomass composition; drying the hydrolysed yeast biomass composition; optionally, milling the dried hydrolysed yeast biomass composition to a suitable particle size; and optionally, packaging the milled biomass.

[0117] “Substrate” refers to a substrate that the strain of yeast can grow on to produce yeast biomass. Examples of a yeast substrate suitable for use in making the composition of the disclosure include by-products of the dairy industry, for example a whey product such as whey permeate and delactosed whey permeate, both of which contain carbon sources for the yeast. Residues of molasse or cornstarch may also be employed. “Whey permeate”: Whey is the liquid remaining after milk has been curdled and strained. It is a by-product of the manufacture of cheese, casein and whey protein. It can exist as sweet whey or acid whey. The whey may be obtained from bovine milk or milk from other mammals such as goats or sheep. Preferably, the milk is bovine milk. Whey permeate is produced by removing protein and other solid components from whey. It is generally produced by treating liquid whey to ultrafiltration or diafiltration. Whey permeate typically contains at least 40 g lactose per litre. Typically, whey permeate contains 1900 to 7600 ppm chloride. The term “whey permeate” as used herein also includes concentrated whey permeate (CWP), a product derived from whey permeate evaporation and delactosed whey permeate (DWP or DLP). Typically, concentrated whey permeate contains 200-240 g lactose per litre. An exemplary composition of CWP is provided in Table 3 below. In any embodiment, the whey permeate used as a feedstock (prior to dilution) contains at least 30g, 40g, 50g, 60g, 70g, 80g, 100g, 150g, 175g, 200g, 220g or 240g per little of whey permeate (w / v). In any embodiment, the whey permeate used as a feedstock (prior to dilution) contains at least 1000, 1500, 1800, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500 or 7000 ppm chloride.

[0118] “Delactosed whey permeate” or “DWP” or “DLP” is a by-product of processing of whey permeate to remove lactose. However, it has a higher amount of lactose post filtration compared with whey permeate, and higher amounts of salts and minerals, especially chlorides and phosphates. Typically, DLP contains at least 200g, 220g, or 240g lactose per litre. Typically, DLP contains 15,000 to 62,000 ppm chloride. An exemplary composition of delactosed whey permeate is provided in Table 1 below.

[0119] As used herein, the term “source of N / P / K” refers to a source of nitrogen, phosphorus and potassium. This may be provided from one component that contains sufficient amounts of each element, or from three sources (one for each element). The N:P weight ratio may be 1 : to 2:1. The N:K weight ratio may be from 1 :1 to 1 :3. The P:K weight ratio may be from 1 :1 to 1 :3. Sources of nitrogen include inorganic sources such as ammonium sulfate, nitrates, urea, and ammonia, and organic sources such as soybean, peanut, and cottonseed meal, protein hydrolysates, corn pulp and steep, meat extract, peptone, and fish meal.

[0120] As used herein, the term “fermentation nutrients” should be understood to include without limitation riboflavin (vitamin B2), niacin (vitamin B3), vitamin B6, biotin (vitamin B7), folate and folic acid.

[0121] The yeast biomass composition is made by hydrolysing yeast biomass, by thermal, chemical, or enzymatic hydrolysis, of any combination thereof. Hydrolysis of the yeast biomass disrupts the cell wall of the yeast releasing bioactive compounds.. An animal feed supplemented with hydrolysed yeast biomass reduced the adherence of Salmonella and E.coli to the digestive tract of chickens fed with the animal feed compared with chickens fed with the same animal feed without the hydrolysed yeast extract. In any embodiment, the yeast biomass is hydrolysed by chemical, thermal and enzymatic hydrolysis, ideally simultaneously. For example the yeast biomass may mixed with a suitable acid and protease preparation and or one or more sugar hydrolysing enzymes, and then incubated at elevated temperature for a suitable period of time until the correct degree of hydrolysis is achieved. Suitably, the yeast biomass composition is hydrolysed to achieve a degree of hydrolysis (%DH) of 10-95%, 10-90%, 10-30%, 30-50%, 50-70%, 70- 100%, 10-20%, 10-30%, 10-40%, 10-50%, 50-90%, 60-90%, 70-90%, 20-90%, or 18-85%. Degree of hydrolysis (%DH) is defined by observing increase in water soluble proteins, and is determined using protein analysis assay described in Neilsen et al (Journal of Food Science, DO - 10.1111 / j.1365-

[0122] 2621.2001. tb04614.x). In any embodiment, the yeast biomass composition comprises denatured protein. The hydrolysis step may be performed in acidic conditions. The pH of the yeast biomass may be adjusted with acid to about 2-3, 3- 4, 4-5, 5-6, 2-6, 2-5, 2-4, 3-6, 3-5, 3-4. The hydrolysis step may be performed at a temperature of 30-50, 30-45, 35-45, 30-40, 25-40, 25-35 or 25-30 °C. The duration of the hydrolysis may be adjusted according to the temperature of hydrolysis and the enzymes employed; for example, at 65-75°C and using two proteases, the duration of the hydrolysis may be 240-600 minutes. “Protease” refers to an enzyme that can break the peptide bonds of proteins converting them into smaller polypeptides, peptides and amino acids; they do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. The protease may be an acid, neutral, and alkaline proteases. If the hydrolysis step of the process of the disclosure is carried out in acidic conditions, an acid protease is preferred. The protease may be selected from a serine protease, a cycteine protease, a threonine protease, an aspartic protease, a glutamic protease, a metalloprotease, or an asparagine peptide lyase. Proteases may be obtained commercially from Novozymes of Bagsvaerd, Denmark and Merck of St. Louis, USA.

[0123] “Sugar hydrolysing enzyme” refers to an enzyme that catalyses the hydrolysis of sugar naturally present in yeast cells, for example a complex sugar such as a starch or polysaccharide, or simple sugars such as disaccharides. Examples of such enzymes include sucrases, cellulases, invertases, and hydrolases.

[0124] “Intestinal stress” refers to a condition suffered by intensively farmed animals such as broiler chickens, turkeys and pigs where stress caused by environmental conditions such as, for example, heat or overcrowding disrupts the gut integrity of the animal, including opening tight junctions in gut epithelial cells and eliciting an inflammatory response. In particular, the condition disrupts the microbiome of the animal (dysbiosis) which provides the conditions for opportunistic pathogens like Salmonella and E.coli to infect the animal. This has been shown to reduce the animals food intake in the animal with consequent reduced weight gain (Ducatelle R., Animal Nutrition Vol. 13, June 2023).

[0125] Exemplification

[0126] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

[0127] Example 1 - Production of adapted strain of Kluyveromyces marxianis

[0128] The adaption process described in WO2017 / 072748 was performed on strains of Kluyveromyves marxianis to isolate an adapted strain capable of growing on 14% whey permeate (w / v).

[0129] Production of K. Marxianus using mineral and ammonia salts on substrates of mainly liquid whey permeate. An aerated fermentation vessel is inoculated with seed material which is maintained at pre-determined operating conditions. Yeast biomass is propagated between 35°c and 40°c, pH is maintained between 4-8, aeration is maintained between 1-2wm.

[0130] Enzymatic hydrolysis is carried out down-stream from the fermenter. The yeast slurry produced in the fermenter is concentrated to form a cream. The cream may treated with hydrochloric or sulphuric acid. The cream is heat treated between 60 and 70°c. Protease enzymes are added at a rate of 0.0005% and 0.4% of yeast biomass. The cream is incubated for a duration of 240-600minutes. Enzyme rate and incubation time is determined by enzyme type used in hydrolysis. After hydrolysis, the biomass is dried on a rotary dryer and bagged.

[0131] Immunomodulation of the yeast biomass

[0132] The effect of the Yeast biomass on inflammatory pathways in innate immune cells was investigated. Sample preparation was based on Food for Health Ireland (FHI) laboratory standardized protocols. The immunomodulatory effect was tested in macrophages and dendritic cells and the experimental procedure is shown in Figure 1.

[0133] Figures 2 and 3 show the effect of the yeast biomass on cytokine secretion in basal (unstimulated) J774A.1 macrophages and on LPS stimulated macrophages, respectively. As can seen in Figure 2, it was found that the yeast biomass caused a rise in the basal levels of IL-6 and TNF-a concentrations, suggesting immune boosting properties. In contrast, the yeast biomass caused a suppression of IL-1 , which may also indicate a benefit in gut health.

[0134] In LPS stimulated macrophages (Figure 3), the yeast biomass resulted in a dose dependent downregulation of secreted IL-10 thus indicating potential gut benefit effects. Furthermore, a dose dependent enhancement of IL-6, IL-10 and TNFa also suggests immune boosting properties.

[0135] When tested in JAWSII dendritic cells, in was found that the yeast biomass caused a dose dependent increase in IL-10, IL-6 and TNFa suggesting immune boosting properties (Figure 4). Lastly, when tested in LPS stimulated JAWSII dendritic cells, a dose dependent decrease in IL-10 and TNFa was seen (Figure 5), suggesting anti-inflammatory potential in dendritic cells. TNFa is an important mediator of inflammatory disease.

[0136] Effect of the yeast biomass supplementation in pigs

[0137] Weaning pigs in a commercial farm is often characterised by a series of stressors (mixing, separation from the sow, new environment, new diet) that result in a reduction in feed intake which may lead to reduced substrate availability for microbial fermentation, reducing bacterial diversity and providing an opportunity for the proliferation of pathogenic bacteria, such as Escherichia coii, leading to postweaning diarrhoea. Incidences of diarrhoea may affect productive performance by reducing average daily gain (ADG) and average daily feed intake (ADFI) which will have a negative impact on bodyweight (BW) and feed conversion efficiency (FCE). The preventive use of antibiotics in feed or the use of therapeutic doses of zinc oxide will not be available to producers after January and June 2022 (EU regulations 2019 / 04-2019 / 06). Therefore, there is a need to examine the role of alternatives to protect the recently weaned pig against some of the stresses associated with weaning.

[0138] Objective The objective of this study was to evaluate the effects of supplementing the diet of newly weaned pigs with the yeast biomass product of the invention using 3 different doses.

[0139] Hypothesis

[0140] The addition of the novel yeast biomass product to the diet of recently weaned pigs will result in improved animal performance, improved bacterial populations and lower incidence of scour.

[0141] Animals and Facilities

[0142] A total of 278 animals were allocated to 24 pens of 11 / 12 pigs each as intact litters. The pens were fully slatted and equipped with one feeder and one nipple drinker. Water and pelleted feed were provided ad libitum. Pigs were fed diet 1 for 2 weeks and diet 2 for the rest of the trial. The rooms are automatically ventilated, and pens were enriched with rubber toys.

[0143] Experimental design and measurements

[0144] Piglets were weaned at 28 days of age with an average weight of 8.8 ± 0.98 kg. At weaning, litters were homogenized to a maximum of 12 pigs per pen and assigned to one of the 4 treatments in a randomized design considering weight and parity.

[0145] Treatments used were:

[0146] - A- Control diet (No yeast added).

[0147] - B- Yeast at equivalent to 0.5kg / ton;

[0148] - C- Yeast at equivalent to 1 kg / ton;

[0149] - D-Yeast at equivalent to 2kg / ton;

[0150] Group weight and feed intake were recorded on day 0, 15, 25, 35 and 46 of experiment (data not shown) and average daily gain, average daily feed intake and feed conversion efficiency were calculated for days 15, 25, 35 and 46.

[0151] Faecal scores were recorded on the day 1 , 4, 6, 10, 14, 19, 25, 35 and 46 of experiment (data not shown) with methodology adapted from Vigors et al. (2020): 1 = hard firm faeces;

[0152] 2 = slightly soft faeces;

[0153] 3 = soft, partially formed faeces;

[0154] 4 = loose, semi-liquid faeces;

[0155] 5 = watery, mucous-like faeces.

[0156] Faecal samples for Escherichia coli counts were collected from each pen on the day 7 and 14 of experiment.

[0157] Microbiology analysis

[0158] Each sample was weighed and buffered peptone water (Oxoid Ltd, Basingstoke, UK) was added to make a 1 : 10 suspension. The suspensions were placed in a stomacher for 2 minutes. The suspensions were serially diluted (104- 107) and 100uL aliquots were plated onto MacConkey no. 3 agar (Lab M Ltd, Heywood, UK). Plates were incubated for 24 hours at 37°C. Quantification of Escherichia coli in each sample was determined by enumeration of red colonies on the plate with between 30 and 300 colonies.

[0159] Statistical analyses

[0160] All data were analysed using SAS v9.4 (SAS Institute Inc., Cary, NC, USA). Each pen was considered as the experimental unit for all data analyses. Models for BW, ADG, ADFI, FCE, FS and microbiology were analysed using general linear models. Bacterial counts were log transformed for normality. Initial BW was used as a covariable for BW, ADG, ADFI and FCE.

[0161] Multiple means comparisons were done using Tukey-Kramer’s correction in all cases. Alpha level for determination of significance was 0.05 and trends were identified as alpha of 0.10. Results for fixed effects are reported as least square means ± standard (SE) error mean.

[0162] Results and discussion

[0163] Table 1 shows the results for average body weight of the pigs by treatment. The inclusion of the novel yeast resulted in a higher BW in each instance with this difference reaching significance (P < 0.10) at days 35 and 46, with dose 0.5 kg / ton tending to be higher than control (p = 0.069 for day 35 and 0.054 for day 46). The D46 increase is attributable to a higher ADG of (60.2 g / d) which is, in turn, driven by a higher ADFI (53.6 g / d) and FCE of 1.27 (see tables 2 and 3).

[0164] Table 1. Average body weight (kg)

[0165] Values are least square means ± standard error mean.A B cWithin each row, values that do not share a common superscript tend to be different (p<0.10).

[0166] Table 2 shows the results for average daily gain of the pigs by treatment. Inclusion of the novel yeast resulted in a higher ADG in each instance becoming significant (p < 0.10) at day 35 and 46 with dose 0.5 kg / ton having higher ADG than controls (p = 0.068 and 0.054 respectively).

[0167]

[0168] Table 2. Average daily gain (g / d).

[0169] Values are least square means ± standard error mean.A B cWithin each row, values that do not share a common superscript tend to be different (p<0.10).

[0170] Table 3 shows the results for average daily feed intake of the pigs by treatment. Pigs on dose 1 kg / ton showed a higher ADFI than those on control diet at day 46 (p = 0.039).

[0171] Table 3. Average daily feed intake (g / d)

[0172] Values are least square means ± standard error mean.a b cWithin each row, values that do not share a common superscript were to be different (p<0.05).

[0173] Table 4 shows the results for feed conversion efficiency of the pigs by treatment. Dose 0.5kg / ton showed a significantly higher FCE (p < 0.05) compared to dose 1kg / ton on day 25 (p = 0.034).

[0174] Table 4. Feed conversion efficiency Values are least square means ± standard error mean.a b cWithin each row, values that do not share a common superscript are significant different (p<0.05).

[0175] Table 5 summaries the results of the feed conversion ratio, body-weight daily gain and % improvement of the different treatments.

[0176] Treatment Feed Bodyweight % Improvement Conversion Average Daily Ratio Gain (g / d) D46

[0177] A - Control 1.31 506.6

[0178] B (0.05%) 1.27 566.8 +11.88

[0179] C 1.33 556.3 +9.81

[0180] D 1.28 559.5 +10.44

[0181] Table 5. Summary of feed conversion efficiency

[0182] The evolution of the faecal score for the different treatments is shown in Figure 6. Dose 2kg / ton showed a significantly (p < 0.001) higher FS compared to all other treatments at day 6 post-weaning. No other differences were found between treatments.

[0183] The E coli counts for the different treatments 7 and 14 days post-weaning is shown in Figure 7. No significant difference was seen between treatments. However, dose 2kg / ton showed numerically higher counts on both days compared to all other treatments.

[0184] Discussion

[0185] With the imminent ban of in-feed antibiotics and ZnO in 2022, there is a need to examine the role of alternatives to protect the early weaned pig against the stresses associated with weaning. In this trial we assessed the effects on growth performance and scour of supplementing the diet of newly weaned pigs with the biomass yeast product of the invention development using 3 different doses. The piglets were followed after weaning for 7 weeks and the new yeast product improved 12% the ADG and 8% the ADFI of the piglets at the lowest dose 0.5kg / ton reducing the FCE numerically from 1.31 to 1.27.

[0186] In a recent meta-analysis, Xu et al. (2021) compared the effects of different types of antibiotic substitutes including plant extracts, probiotics, micronutrients, organic acids, bacteriophages, and oligosaccharides, among others, in 89 trials at international level.

[0187] Figure 8 shows the comparison of the effect of the different additives using standardised mean differences (Cohen’s d). When translated into actual growth, the average effect of these additives was around 30g (considering a growth of 500g / day) or 6% of improvement on ADG. In another meta-analysis carried out by Sales 2013 including 26 trails comparing zinc oxide with control diets, they found a similar effect (+31 g / g) of zinc oxide on ADG.

[0188] Thus, the improvements of the new yeast product tested in this trial were superior to the average effect expected for antibiotic substitutes or zinc oxide. As can be seen in figure 8, the effect of the new yeast product was similar to the average effect of probiotics and oligosaccharides (prebiotics) and better than the average effect of organic acids.

[0189] Conclusion

[0190] Based on the results described above, the novel yeast product tested in this trial improved productive performance. In particular, at the lower doses studied (0.5g / ton) the new yeast product improved daily gain by 12% and intake by 8%.

[0191] The impacts of the inclusion of the novel yeast on bacterial populations and incidence of scour were marginal at lower dosage levels. However, it is apparent that the highest dose studied may promote growth of E.coli and induce softer faeces a week after weaning. The recommended dose for its use in weaner pigs would be 0.5 kg / ton.

[0192] Effect of yeast biomass product on ileal and caecal fermentation of broiler chickens

[0193] -ex vivo fermentation model Background

[0194] Since the ban of antimicrobial growth promoters (AGP) in the European Union, feed manufacturers have been actively looking for alternatives for the control intestinal microbiota in production animals. Even though the exact mode of action is still not fully understood, the current hypothesis is that AGPs have not only controlled the growth of true pathogens, but also reduced the level of commensal bacteria in the small intestine of the animals. Bacteria in the small intestine utilize the same nutrients that are essential for the productivity of the host. Therefore, reduced bacterial load in this section of the gastrointestinal tract is believed to improve feed conversion efficiency and body weight gain of production animals.

[0195] Fibrous carbohydrates as well as oligo- and polysaccharides may improve intestinal health of a host by providing substrates for the bacteria in the distal intestine. While simple sugars never reach distal intestinal sections, the more complex ones escape small intestine and support the growth of beneficial bacteria in the caecum and colon of the host. Such carbohydrates are referred to as prebiotics. In the present study, we evaluated the effect of Kluveromyces marxianus yeast strain in intact and hydrolysed form on several ileal and caecal fermentation parameters in the broiler chicken ex vivo simulation model of Alimetrics Research.

[0196] Hydrolysis of Kluveromyces marxianus yeast

[0197] The Kluveromyces marxianus yeast product was hydrolysed with heat treatment according to the method of the invention. Briefly, the yeast was suspended in deionised water (1 :2) and the slurry was incubated at 60°C for 18h with constant shaking. Next, the hydrolysate was freeze-dried and the homogenised dry material was applied in the simulation. Therefore, both intact and hydrolysed yeast had the same dry matter content in the study.

[0198] Treatments (both ileal and caecal simulation)

[0199] 1 . Negative CTRL with no amendment 2. Negative CTRL with no amendment

[0200] 3. Positive CTRL (Yea-Sacc); 2.5 mg / mL

[0201] 4. Positive CTRL (Yea-Sacc); 10 mg / mL

[0202] 5. Positive CTRL (Yea-Sacc); 40 mg / mL

[0203] 6. K. marxianus (intact); 2.5 mg / mL

[0204] 7. K. marxianus (intact); 10 mg / mL

[0205] 8. K. marxianus (intact); 40 mg / mL

[0206] 9. K. marxianus (hydrolysed); 2.5 mg / mL

[0207] 10. . marxianus (hydrolysed); 10 mg / mL

[0208] 11. . marxianus (hydrolysed); 40 mg / mL

[0209] In both ex vivo models, four replicate vessels were simulated for each of the treatments. Thus, the total number of vessels was 88 in the fermentations (2 gut compartments x 11 treatments x 4 replicates).

[0210] General simulation protocol - ileum and caecum

[0211] For the chicken ileal ex vivo model, the authentic growth substrates for the fermentations were prepared from the small intestinal digesta recovered from live animals grown in Alimetrics Research broiler facility. For the chicken caecum ex vivo model, the respective substrates were prepared from a mixture of digesta from distal ileum and caecum. The final growth medium was maintained anaerobic and amended with the test products.

[0212] Next, the fresh inocula for the fermentations were recovered from five broiler chickens (age 30 days) grown at a commercial farm three hours before the initiation of the fermentation simulations. The inoculum digesta samples of the birds were pooled and kept under conditions ideal for the viability of the bacteria. Finally, inoculum from fresh pooled ileal and caecal digesta (respectively) was introduced into the simulation vessels in an anaerobic glove box. It is noteworthy that the inoculation was performed in a random order to avoid any potential systematic shifts. Simulation vessels were sealed with thick butyl rubber stoppers, transferred to 37°C and continuously mixed in a gyratory shaker at 100 r.p.m. Incubation was continued for 12 hours prior to sampling of the vessels for various analyses as described below.

[0213] Parameters measured from the fermentation simulation

[0214] Gas production

[0215] During the fermentation, gas production was measured at 3, 6, 9 and 12 hours in order to study the general metabolic activity of the intestinal microbes and the effect of the test products.

[0216] Short-chain fatty acids

[0217] At the end of the 12-hour fermentation simulation, the vessels were analysed for short-chain fatty acids (SCFAs) by gas chromatography. The SCFAs quantified were acetic, propionic, butyric, branched-chain fatty acids (iso-butyric, 2-methyl-butyric and iso-valeric), valeric and lactic acid. Total SCFAs indicate the overall fermentation activity and the relative abundance of individual acids indicates the activity of different bacterial fermentation routes.

[0218] Total bacterial numbers

[0219] At the end of the fermentation, simulation vessels were analysed for the total bacterial numbers. The analysis was carried out by quantitative real-time PCR technique. It is worth noting that the individual DNA samples were stored in freezer, and therefore, it is possible to conduct a more detailed analysis of the microbial community at later point.

[0220] Statistical analyses

[0221] Statistical analysis consisted of two-tailed t-tests for all measured parameters. The tests were performed against the control treatment with no test product amendment (Negative CTRL) just as Dunnett’s post hoc test would have been used, but t-test was chosen to let the individual treatments be independent of the other treatments tested simultaneously. Significance according to Student’s t-test:

[0222] - p-value < 0.05 *

[0223] - p-value < 0.01 **

[0224] - p-value < 0.001 ***

[0225] Effect of test products on ileal microbial gas production

[0226] Gas production analysis is a general measure of microbial activity in biological systems. Under the conditions and the redox potential relevant for the small intestine, the gas is almost pure carbon dioxide. It is noteworthy, however, that the stoichiometry of different metabolic pathways and substrates may and often do differ with regards to moles of gas produced from a mole of sugar. For example, some microbes produce more gas from xylose than from glucose. The cumulative gas production results (0 to 12 hours) of ileal simulation treatments are presented in Figure 9.

[0227] - Yea-Sacc showed a significant dose-dependent stimulation of cumulative gas production during the 12-hour simulation. Gas production boost can be derived from the metabolic activity of the yeast itself or, alternatively, components of the yeast mass can serve as a substrate for ileal bacteria.

[0228] - K. marxianus stimulated the ileal gas production only with the highest 40 mg / mL dose.

[0229] - No difference in the gas production patterns was observed between the intact and hydrolysed K. marxianus product.

[0230] Effect of test products on ileal acid production

[0231] Acetic and lactic acid production

[0232] Short-chain fatty acids (SCFAs) are formed during microbial fermentation in anaerobic biological systems such as small-intestine. Total acid concentration indicates the overall fermentation activity and the relative abundance of individual acids indicates the respective activity of different fermentation pathways. In the small intestine, lactic acid bacterial fermentation which produces lactic and acetic acids at different ratios can be expected to dominate. However, in the case of difficult pathogen attack, other acids may grow in concentration.

[0233] Lactic acid is the strongest of the common acids produced by bacteria in the small intestine. In the upper gastrointestinal tract, the residual concentration of lactic acid is typically high since lactic acid bacteria (LAB) are normally the most prevalent microorganisms present in this gut compartment. LAB are classified as homofermentative or heterofermentative based on their end-products of sugar fermentation. Homofermentative LAB ferment glucose with lactic acid as the primary product, while heterofermentative LAB ferment glucose with lactic acid, acetic acid, ethanol and CO2 as end-products. Figure 10 and 11 show the acetic and lactic acid production at 12-hour sampling point.

[0234] - Lactic acid was the most abundant individual SCFA produced by ileal microorganisms, indicating that the fermentation was generally driven more by homofermentative than heterofermentative lactic acid bacteria.

[0235] - Yea-Sacc showed a significant dose-dependent stimulation of acetic acid production, while lactic acid formation was reduced with all three doses tested. This result suggests that Yea-Sacc can modify the ileal fermentation strongly towards heterofermentative metabolism or produces acetic acid in its own metabolism.

[0236] - Both intact and hydrolysed K. marxianus stimulated dose-dependently acetic acid formation; however, the observed increase was smaller as compared to the corresponding Yea-Sacc doses.

[0237] - Unlike Yea-Sacc, K. marxianus significantly stimulated lactic acid formation in the ileal environment. With the highest 40 mg / mL dose the final concentration was approximately 3-fold compared to the Negative CTRL. Again, no difference between the intact and hydrolysed product was observed.

[0238] - The results therefore suggest that small-intestinal bacteria are able to utilise some components of the K. marxianus product as a substrate for growth. Effect of test products on ileal bacterial numbers

[0239] In monogastric animals, bacteria compete directly with the host for specific nutrients in the small-intestine. Hence, increase in these bacterial populations may reduce nutrient capture by the host thus impairing body weight gain and feed conversion ratio. On the other hand, imbalance and reduction of the resident commensal microbiota (mostly lactic acid bacteria in the small-intestine) facilitates the proliferation and colonisation of opportunist pathogens in ecological niches previously unavailable to them. The effect of test treatments on the bacterial growth at 12 hours of ileal fermentation are presented in Figure 12.

[0240] - As expected based on the SCFA production results, K. marxianus at the highest dose stimulated significantly (2-fold) the ileal bacterial growth during the 12-hour simulation. Taken the SCFA and microbial results together, it is likely that the microbes that were increased were mainly homofermentative lactic acid producers.

[0241] - Again, the stimulatory effect on microbial growth was rather similar with intact and hydrolysed K. marxianus, when tested at the highest 40 mg / mL dose.

[0242] - Interestingly, Yea-Sacc did not influence the ileal bacteria growth. Therefore, the observed acetic acid formation was likely caused by the viable yeast itself.

[0243] Effect of test products on caecal microbial gas production

[0244] The cumulative gas production results (0 to 12 hours) of caecal simulation treatments are presented in Figure 13.

[0245] - Similarly to ileal simulation, Yea-Sacc at the highest dose showed also the most considerable stimulation of caecal gas production during the 12-hour fermentation.

[0246] - Both intact and hydrolysed K. marxianus stimulated caecal microbial gas production. However, the results showed that no difference between the 10 mg / mL and 40 mg / mL was observed. This suggest that adding the product concentration from 10 to 40 mg / mL did not provide additional boost to the gas production of caecal bacteria. It is worth noting that, unlike in ileum, the magnitude of stimulation was very similar to one another with the two lower doses of both Yea-Sacc and K. marxianus.

[0247] - Again, the stimulatory effect on microbial gas production was rather similar with intact and hydrolysed K. marxianus, suggesting that hydrolysis did not affect the yeast product properties or mode-of-action in intestinal environment.

[0248] Effect of test products on caecal acid production

[0249] Total VFA production

[0250] Volatile fatty acids (VFAs) function generally as systemic sources of energy in lower intestine such as broiler chicken cecum. Total acid concentration indicates the overall fermentation activity and the relative abundance of individual acids indicates the respective activity of different fermentation pathways. The reason why increased VFA formation (e.g. butyrate, acetate and propionate) in the lower intestine is considered beneficial is the fact that a large part of the acids produced during carbohydrate fermentation are utilised by the host, providing additional energy for the animal. Moreover, nutritional disorders and enteritis problems often reflect to the profile of lower intestinal VFAs, which can therefore be used as health indicators. Therefore, for long term productivity it is important that VFA concentration in caecum is maintained or boosted. Figure 14 shows the total VFA production at the 12-hour sampling point.

[0251] - Yea-Sacc stimulated the total VFA production in the caecum environment with a statistical significance in a dose-dependent manner. This result is in line with the observed increase of gas production with Yea-Sacc.

[0252] - Also, K. marxianus (both intact and hydrolysed) stimulated the caecal VFA production already at the lowest dose tested. Similarly to gas production, the highest 40 mg / mL dose did not increase VFA concentration from the intermediate 10 mg / mL dose.

[0253] - The results showed that K. marxianus at 10 mg / mL dose stimulated VFA formation in a significantly greater extent than the corresponding dose of Yea- Sacc, suggesting that caecal bacteria are more efficiently utilising K. marxianus product as a growth substrate and producing beneficial fatty acids as metabolic end-products; those again serve as an additional energy source for the host.

[0254] - It is noteworthy that lactic acid was highly accumulated (~40 mM concentration) with the highest dose of K. marxianus (both intact and hydrolysed), while it was not detected with any other treatments (data not shown). As lactic acid is typically efficiently converted to other, perhaps more beneficial VFAs by caecal lactate-utilising bacteria, it is evident that the 40 mg / mL dose was a clear overdose in the simulation model resulting in caecal acidosis.

[0255] Butyric acid production

[0256] Among the VFAs, butyrate has gained particular interest, as it is the preferred energy source for the enterocytes and is known to regulate cellular differentiation and proliferation within the intestinal mucosa, thereby improving the condition of the intestinal tissue. The epithelium provides a highly selective barrier that prevents the passage of toxic and pro-inflammatory molecules from the external milieu into the submucosa and systemic circulation. Hence, the contribution of butyrate and other VFAs to epithelial development is essential in the maintenance of normal intestinal barrier functions. The effect of test treatments on the caecal butyrate formation at 12 hours of fermentation are presented in Figure 15.

[0257] - Yea-Sacc showed a significant dose-dependent stimulation of caecal butyric acid production during 12-hours of fermentation.

[0258] - Interestingly, K. marxianus (both intact and hydrolysed) stimulated butyrate formation significantly already at the lowest, 2.5. mg / mL dose. At the intermediate 10 mg / mL dose the magnitude of butyrate production was equal to that obtained with Yea-Sacc at 40 mg / mL. This again suggests that beneficial caecal butyrate-producing microbes are able to efficiently utilise K. marxianus derived carbohydrates as a fermentation substrate. - The results with K. marxianus (both intact and hydrolysed) showed a quadratic dose-response effect on the butyric acid production, as the highest dose decreased considerably the butyrate production as compared to the intermediate dose. The reason for the dramatic reduction with the highest K. marxianus dose is very likely linked to the observed accumulation of lactic acid (see Appendix I). Lactate is the strongest of the common short-chain fatty acids produced by gastrointestinal bacteria and, therefore, it tends to reduce residual pH more than other acids. During normal caecal conditions, lactate is rapidly used as a substrate for lactate-utilising bacteria and metabolised to more reduced VFAs such as propionic and butyric acid. However, in cases where lactic acid is heavily accumulated in caecum due to increased activity of lactic acid bacteria, as seen here with K. marxianus amendment at 40 mg / mL, the pH-sensitive beneficial lactate-utilising and butyrate-producing bacteria are inhibited. This eventually leads to caecal acidosis and strong reduction of butyric acid production as observed with the highest K. marxianus dose.

[0259] Propionic acid production

[0260] Propionic acid is of great importance to the host as a gluconeogenic substrate. In addition, propionate plays a role in the maintenance of gut health and has antiinflammatory effects. Figure 16 shows the effect of test products on caecal propionic acid production at a 12-hour sampling point.

[0261] - All yeast treatments tested showed a significant dose-dependent increase in caecal propionic acid production. Hence, the results suggest that bacteria producing propionic acid as a metabolic end-product were not inhibited by the caecal lactic acid accumulation with the highest K. marxianus dose (as was the case with butyric acid).

[0262] Again, the stimulatory effect of propionate production appeared to be generally more pronounced with K. marxianus (both intact and hydrolysed) than with Yea-Sacc.

[0263] Branched-chain fatty acid (BCFA) production Branched-chain fatty acids (BCFAs) result from amino acid fermentation. Therefore, these harmless products are indicators of putrefaction (protein fermentation) which is producing many other metabolites such as amines, phenolic compounds and volatile sulphur compounds, some of which are harmful and even toxic to the host. Figure 17 shows the effect of test products on caecal BCFA production at a 12-hour sampling point.

[0264] - Results showed that the highest (40 mg / mL) doses of tested yeast products reduced the BCFA production in caecal simulation as compared to the two lower doses. In fact, K. maxianus at the highest dose suppressed the BCFA formation also from the Negative CTRL with a statistical significance.

[0265] - The observed results strongly suggest that introduction of the test products at very high concentrations suppresses microbial putrefaction in caecum. This reduction could be linked to the fact that bacteria preferentially ferment carbohydrates, when both proteins and carbohydrates co-exist in the intestinal habitat. Hence, the carbohydrates of yeast additives appeared to serve as preferential digestible substrates for caecal bacteria.

[0266] Effect of test products on caecal bacterial numbers

[0267] Caecum is a highly important site for the bacterial fermentation in broiler chicken intestinal tract. High bacterial density indicates breakdown of dietary components that are beyond the endogenous digestion system of the host. It is possible that with poorly digestible diets, a considerable proportion of total dietary energy comes from bacterial fermentation. The effect of test treatments on the bacterial growth at 12 hours of caecal fermentation are presented in Figure 18. The numbers of 16S rDNA gene copies were log transformed to achieve a normal distribution for the statistical analysis.

[0268] - The results showed that K. marxianus at the two highest doses (10 and 40 mg / mL) stimulated the caecal bacterial growth significantly during the 12-hour simulation (both intact and hydrolysed).

[0269] - On the other hand, no difference in bacterial counts between these two doses were observed, suggesting that increasing the concentration from 10 to 40 mg / mL did not provide any additional benefit to caecal microorganisms, most likely due to considerable lactic acid accumulation and subsequent inhibition of dominant lactate utilisers as discussed above.

[0270] - Also Yea- Sacc stimulated the bacterial growth, but the effect was somewhat smaller than with the corresponding K. marxianus dose. Therefore, this result in in line with the VFA results, in which the 10 mg / mL dose of K. marxianus had rather similar stimulatory effect as the 40 mg / mL dose of Yea-Sacc.

[0271] - Overall, the patterns of microbial results were very similar to those of total VFA production (see Figure 14).

[0272] Assessment of the efficiency of test products to prevent adherence of pathogenic Escherichia coli and Salmonella enterica on intestinal mucus in vitro

[0273] Introduction

[0274] Three test products were evaluated in their efficiency in preventing pathogen adherence on the piglet intestinal mucus layer in vitro. Inhibition of pathogen adherence to intestinal epithelium can be due to efficient co-flocculation of the pathogenic bacteria with the insoluble test product, which prevents bacterial binding and leads to washout of the agglomerates. The other possible mechanism is that a test product blocks the pathogen target receptors on intestinal epithelium thus preventing the adherence.

[0275] Outline of the work

[0276] Microtiter plates were coated with authentic mucus originating from distal ileum of piglets. Tryptic soy broth was used as growth medium for the enteropathogenic Salmonella enterica (serovarTyphimurium IR 715) and Escherichia coli F4+ bacteria. The bacteria were cultured without shaking at +37°C. Cultures were refreshed daily for three days prior to the test. In the final cultivation cycle, 3H-labelled thymidine was added in the culture medium to label the bacteria with a radioactive marker. The test products were added onto the mucus-coated microtiter plates at four doses, mannose was used as a positive control and negative control was treatment with no amendments. Four replicate test wells were generated for each treatment. Labelled bacteria were added onto the mucus-coated microtiter wells with or without the test products and the reaction mixtures were incubated for 1 h at +37°C. Unbound bacterial cells were removed by washing the wells twice with HEPES-Hanks buffer at room temperature. Scintillation cocktail was added onto the mucus-coated microtiter plate and the radioactivity of mucus bound bacteria was measured with a scintillation counter. The test products and doses are listed in Table 6. The test product C was a yeast slurry with 20 % dry weight content. The doses were calculated to represent the dry weight content of the sample in the reaction well.

[0277]

[0278] Table 6. Treatment list Statistical analysis

[0279] The data from mucus adherence assay was analysed with Student’s (2-sided) test. The treated samples were compared against the negative control (no amendments). Obtained p-values smaller than 0.05 are shown in the graphs as follows: p-value < 0.05 * p-value < 0.01 ** p-value < 0.001 ***

[0280] Results and discussion (Figures 19 and 20) The Escherichia coli adherence was significantly and substantially inhibited with the two highest doses of all test products (A, B, C) (Figure 19). The lowest doses of products B and C had the tendency to increase the pathogen adherence. This can be due to test product adhering both to mucus and the bacteria and therefore low product doses may increase the radioactive signal in the well. The adherence of Salmonella enterica was not affected by the test products in a statistically significant manner. However, the three highest doses of test product C were able to lower the salmonella load on mucus while the test products A and B increased the adherence slightly (Figure 20). In conclusion, the test product C has the potential to inhibit both E. coli and salmonella adherence on piglet mucus. The products A and B can inhibit the adherence of E. coli but possibly increase the adherence of salmonella

[0281] LSAQUA Feeding

[0282] This experiment was designed to test 7 feeds (A-G) for rainbow trout. The test was blind, the experimenters did not know the composition of the different feeds. The test was performed in triplicate, each feed was given to 3 separate tanks which each contained 10 fish. Tanks were divided randomly across the room using a random number generator. Water quality was assured by a continuous flow through in combination with a solid filter which was cleaned regularly. Feeding occurred at a rate of 5% body mass per day which was divided over 2 equal portions each day and adjusted weekly.

[0283] During the test, three tanks showed considerable mortality (50-100%). In our opinion, these mortalities are not related to the feeds, as this occurred independently with 3 different feeds (C-E-F) which had 100% survival in the other 2 replicate tanks. As the fish are acquired from an outdoor hatchery, small benign infections in one of the fish might evolve to become pathological due to the stress caused by transport and movement to the indoor facilities and could subsequently affect the whole tank. Sanitary measures were in place (separate dip nets, disinfection of equipment) and the infected tanks were not related (no neighboring tanks). So, in contrast with the preliminary report send earlier, we have decided to completely eliminate these tanks from the analysis as the remaining fish in these tanks also experienced different conditions (different densities, possibly also affected by the infection) and definitely distorted the tank related parameters. This change is reflected mainly in the results expressed per tank, not so much in the results expressed on an individual basis. Slight differences in trends in average absolute values compared to relative (%) values might be caused by slight differences in initial weights of the fish (e.g. comparing individual absolute growth and relative growth rate between A to D) when differences are so small that they are not significantly different.

[0284] Overall, conclusions drawn from the data expressed on an individual basis are stronger (N=20-30) as the statistical power per tank is limited (N=2-3), but the trends are very similar. It is clear that feed G performs poorer than any of the other feeds. More subtle differences seem to occur between the other feeds with feed B showing the best performance closely followed by F, and a slightly lesser performance of feed E. Between the 2 best performing feeds, it is striking that feed F has the best palatability. Despite being not significant due to the low tanks numbers (F is one of the feeds where we lost a tank), the time needed to consume the feeds was on average 45% lower. In contrast, feed G seems to become less and less palatable over time (Figure 31).

[0285] Experiment Information

[0286] Oncorhynchus mykiss juveniles (4.4 ± 1 .5 g) were obtained from the Tipsbosch trout hatchery, Hellendoorn, the Netherlands on October 22, 2020. The fish were transported to the laboratory facility of the Systemic Physiological and Ecotoxicological Research (SPHERE) group of the University of Antwerp. The fish were randomly distributed in 21 flow-through tanks (10 fish per tank, 3 tank replicate per diet) and were habituated to laboratory conditions for approximately 4 weeks prior to the start of experiment (November 17, 2020). The flow through tanks (40 cm with x 60 cm length x 43 cm height; filled with 50 L freshwater) have a flow rate of -300 ml per minute and are individually equipped with airlines and solids filters. A regular light dark cycle of 12 / 12 hours was maintained during the experiment. Dissolve oxygen levels were monitored daily by WTW ProfiLine 3310 meter and were maintained 80% saturation level throughout the experiment. Nitrite and ammonia levels were monitored twice a week using Tetra test kits and kept at concentrations below 0.3 mg L'1and 0.25 mg L'1, respectively. The fish were fed twice a day with experimental diets at 5% feeding rate (5% of the total biomass per day) for 30 days. Feed adjustment was done weekly using predicted biomass calculated from growth curves determined in the laboratory. Actual fish weigh sampling was also done every second week to assure that predicted biomass values were precise. Feed that was not eaten within approximately 10 minutes was recovered, dried, weighed, and discounted from the total amount of feed given to the fish. The time it takes to consume the feed given was recorded once every week as an additional measure of palatability.

[0287] On the last day of the experiment, all surviving fish were fasted for 24 hours, euthanized by an overdose of tricane-mesylate (MS-222), measured and weighed. Blood samples were immediately obtained from three randomly selected fish from each tank by puncturing the caudal vein using a heparinized syringe and needle. Collected blood in 1.5ml bullet tubes were quickly centrifuged (4,200 rpm for 5 minutes at 4°C). Plasma samples were isolated and stored in -80°C until the start of immunological analyses.

[0288] Data Analyses

[0289] Data analyses were performed in R version 4.0.3. One-way ANOVA models were run to analyze the effects of feed types on normally distributed (examined by visual observations of qq-plots, histograms and boxplots) and homoscedastic (checked by Levene’s Test) datasets, followed by Tukey’s post-hoc test to discern significant differences among feed treatments. Effects of feed types on datasets with questionable normality was analyzed by a nonparametric Kruskal-Wallis One-way ANOVA, followed by Wilcoxon signed-rank test with Bonferonni correction to determine significant differences among feed types. Statistical significance was accepted at p<0.05. All data in this report are expressed as average ± standard error.

[0290] Results are summarized in Table 7 and associated Figures 21 - Figure 29. Feed G performed significantly worse than feed A, B, D and F. The difference is not significant with

[0291]

[0292] The results for the feed consumption time per different treatment are shown below in Table 9 and represented graphically in Figure 30. Equivalents

[0293] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

CLAIMS1 . A hydrolysed yeast biomass, in which the yeast comprises a strain of Kluyveromyces marxianis, and in which the hydrolysed yeast biomass has a degree of hydrolysis (%DH) of 30% to 60% and comprises at least 45%, organic nitrogen.

2. A hydrolysed yeast biomass according to Claim 1 , in which the strain of Kluyveromyces marxianis is an adapted strain that is adapted to grow on whey permeate containing at least 12% protein (w / v).

3. A hydrolysed yeast biomass according to Claim 1 or 2, in which the yeast biomass contains at least 50% protein prior to hydrolysis.

4. A hydrolysed yeast biomass according to Claim 1 or 2, in which the yeast biomass contains 50% to 55% protein prior to hydrolysis.

5. A hydrolysed yeast biomass according to any preceding Claim, comprising hydrolysed whey protein.

6. A hydrolysed yeast biomass according to any preceding Claim, having a solubility in water of at least 99% (w / v).

7. A hydrolysed yeast biomass according to any preceding Claim, comprising at least 30% amino acids (w / w).

8. A hydrolysed yeast biomass according to any preceding Claim, comprising threonine, valine, methionine, isoleucine, leucine, phenylalanine, histidine and lysine.

9. A hydrolysed yeast biomass according to any preceding Claim, comprising at least 1 .5% threonine, at least 1 .5% valine, at least 0.4% methionine, atleast 1.3% isoleucine, at least 2.1 % leucine, at least 1.3% phenylalanine, at least 0.5% histidine and / or at least 2.3% lysine.

10. A hydrolysed yeast biomass according to any preceding Claim, comprising substantially all of the amino acids aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine.11 . A hydrolysed yeast biomass according to any preceding Claim, comprising vitamins B1 , B2, B3, B6, B7 and B12.

12. A hydrolysed yeast biomass according to any preceding Claim, formed by culturing the yeast in a broth comprising a yeast substrate and a source of nitrogen, potassium and phosphorus (N / P / K) to form a fermentation broth, dewatering the fermentation broth to provide a yeast biomass, and enzymatically hydrolysing the yeast biomass to provide the hydrolysed yeast biomass composition.

13. A hydrolysed yeast biomass according to Claim 12, in which the enzymatic hydrolysis employs a protease enzyme and a sugar hydrolysing enzyme.

14. A hydrolysed yeast biomass according to Claim 12 or 13, in which the yeast biomass contains 21 % to 25% yeast (w / v).

15. Use of the hydrolysed yeast biomass of any preceding Claim as an animal feed supplement.

16. Use of Claim 15, in which the animal feed supplement is selected from a pig feed supplement, a poultry feed supplement, and a fish feed supplement.

17. An animal feed comprising an animal feed component and a hydrolysed yeast biomass of any of Claims 1 to 14.

18. An animal feed according to Claim 17, selected from a pig feed, a poultry feed and a fish feed.

19. An animal feed according to Claim 17 or 18, comprising 0.025% to 0.075% hydrolysed yeast biomass (w / w).

20. A hydrolysed yeast biomass according to any of Claims 1 to 14, for use in a method of treating or preventing intestinal stress in an animal by reducing adherence of a pathogenic bacteria to a gut wall of the animal, in which the hydrolysed yeast biomass is fed to the animal.

21. A hydrolysed yeast biomass according to any of Claims 1 to 14, for use of Claim 20, in which the pathogenic bacteria is selected from Salmonella or Escherichia coli.

22. A hydrolysed yeast biomass according to any of Claims 1 to 14, for use of Claim 20, in which the animal is a species of swine or poultry.

23. A hydrolysed yeast biomass according to any of Claims 1 to 14, for use of Claim 20, in which the animal is a broiler chicken or pig.

24. A method of improving the daily live weight gain of an animal comprising feeding a hydrolysed yeast biomass of any of Claims 1 to 14 or an animal feed of any of claims 17 to 19 to the animal.

25. A method according to Claim 24, in which the animal is a chicken or a pig and the method comprises administering an animal feed of Claim 17 to the animal.

26. A process of making a hydrolysed yeast biomass comprising culturing a strain of Kluyveromyces marxianis yeast in a broth comprising a yeast substrate comprising at least 10% protein (w / v) and a source of nitrogen, potassium and phosphorus (N / P / K) to form a fermentation broth, dewateringthe fermentation broth to provide a yeast biomass, and enzymatically hydrolysing the yeast biomass to provide the hydrolysed yeast biomass composition.

27. A process according to Claim 26, in which the strain of Kluyveromyces marxianis yeast is an adapted strain that is adapted to grow in whey permeate comprising at least 12% protein (w / v).

28. A process according to Claim 26 or 27, in which the enzymatic hydrolysis employs a protease enzyme and a sugar hydrolysing enzyme.

29. A process according to any of Claims 26 to 28, in which the yeast biomass is treated with an acid prior to the hydrolysis step.

30. A process according to any of Claims 26 to 29, in which the yeast biomass is enzymatically hydrolysed to a degree of hydrolysis (%DH) of 30% to 60%.

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