CA1050911A - Process for the conversion of starch and protein-containing cellulosic substances into nutriments richer in proteins - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Processes for the conversion of starch and protein containing cellulosic substances such as cereals, bran from cereal and flour mills and broken pieces and crumbs of essentially fat-free baked goods from bakeries into nutriments richer in proteins by dispers-ing in water the cellulosic substances containing starches and pro-teins in comminuted form, subjecting the resulting slurry of the cellulosic substance in water to hydrolysis to convert the starches therein to glucose or other fermentable sugars, separating the glucose-containing liquid from the remaining solids by ultrafiltra-tion, aerobically cultivating in the said glucose-containing liquid a yeast such as Candida utilis or a bacterium that ferments and utilizes glucose for growth and subsequently recovering from the fermented liquid the protein-containing yeast or bacterium that was grown therein for use as a protein concentrate in human nutrition or as a supplement for animal feeds.

Description

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1 The present invention pertains to processes for the utiliza-tion of cellulosic starch and protein-containing substances such as wheat and cereals of all kinds as well as bran from cereal and flour mills and broken pieces and crumbs of baked goods from bakeries by converting the starches therein into protein-contain-ing nutriments or concentrates that can be used in human nutrition and as supplements for animal feeds and that are richer in proteins and accordingly more valuable than the original cellulosic substan-ces from which they were produced. The processes of the present invention are accordingly adapted for converting cereals such as wheat, rice, barley, rye, sorghum and grains and plants of all kinds into more valuable protein-containing concentrates or nutriments.
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In the production of cereal products, large quantities of waste cellulosic substances containing starches and proteins are produced which are used almost exclusively for animal feed.s that nonetheless have inferior nutritional valueO Considerable quanti-ties of such cellulosic starch and protein-containing waste prod-ucts are also produced, for example, in the production of baked goods such as biscuits and crackers in bakeries.
Processes are known for the conversion of starches and starch-like substances into protein-containing plants,such.as yeast cells or solids. One such process, which is described in United States Patent No. 3,105,799, consists in symbiotically cultivating in a substrate consisting essentially of potatoes or similar vegetable material containing starch with two microorganisms, namely, a : .
microorganism which hydrolyzes s*arch, such as Endomycopsi.s fibul-igera, and a sugar-fermenting yeast such as Torulopsis utilis, and separating and using the yeast that were grown therein during the fermentation as a nutriment~ Such cereals and waste cellulosic ~0 substances, however, also contain a substantial proportion of highly ~)51D~

1 valuable proteins which had heretofore been overlooked or ignored.
Processes have also been described for the recovery of pro-teins by ultrafiltration but such processes have been heretofore used chiefly in the dairy industry, for example, for recovery of proteins from whey, for example, in an apparatus such as is de-scribed in German published application No. 2,220,308.
The principal object of the present invention is to provide a ; process for the production of valuable pro-teins or protein-contain-ing concentrates or nutriments from the aforementioned cellulosic : 10 starch and protein-containing substances that can be used in human as well as animal nutrition. Other ob~ects and advantages of the invention, some of which are referred to hereinafter, will be ob-vious to those skilled in the art to which it perta.ins.
The foregoing object is achieved in accordance with the pro-cesses of the present invention, in which processes the cellulosic starch and protein-containing substance is first subjected to hydrolysis to convert the starches thereinto into glucose or other fermentable sugars. The resulting slurry is then subjected to ultrafiltration to separate the sugar or glucose-containing liquid from the protein-containing solids that remain therein. These :
protein-containing solids are also valuable nutriments. Therea:Eter the sugar or glucose-containing liquid is inoculated with a yeast such as Candida utilis (also known as Torulopsis utilis), Saccharo-myces cerevisiae or Hansenula polymorpha, or a species of Lacto-bacillus or Aerobacter bacteria that has been approved by the ap-propriate governmental authority for the production of nutriments for use in human nutrition, and the yea~tor bacterium is aerobic-ally cultivated therein. The yeast or bacterium that has been grown therein is then recovered by conventional methods. Both the yeasts : 30 and bacteria that are thus grown, as well as the proteln----3~ ..

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1 containing solids that have been recovered in the ultrafiltration step, are valuable sources of protein and may be used not only for the production of animal feeds and fodder but also in human nutrition.
The invention is described further in connection with the e~-amples and description which follow, which were selected solely for the purposes of illustration and consequently are not to be con-strued as restrictive of the invention or its scope.

The general procedure for carrying out the process that is described in this example is a preferred method and was used in all of the subsequent examples that are referred to hereinafter.
Ten hundred (1000) grams of comminuted defatted zwiebach crumbs was stirred into 3 liters of water in an autoclave to which 30 milliliters of concentrated hydrochlorio acid (32~i by weight of hydrogen chloride) was then added and the resulting slurry was then heated at a temperature of 120C for a period of approximately 2 hours, during which period it was stirred vigorously. At the end of ; this period, no starch could be detected in the mixture, indicating that it had been completely hydrolyzed. The resulting hydrolyzed mixture contained between 500 and 700 grams of glucose which was identified by chromatography. This mixture was then diluted with tap water to a volume of approximately 20 liters and subjected to ultrafiltration to separate and recover the protein-containing ~solids therefrom. Suitable ultrafiltration apparatus that lS com-' mercially available was used for this purpose. The filtrate con- ~
tained between 500 and 700 grams of fermentable sugar and had a -concentration of between 25 and 30 grams per liter.
Three (3) liters of this sugar solution was then placed into a fermentor having a capacity of 7 liters and 15 milliliters of .
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1 concen~rated phosphoric acid (85% by weight of orthophosphoric acid) and nutrient salts that are required for growth were added thereto.
Ammonium hydroxide was then added to the solution in such an amount as to bring the hydrogen-ion concentration of the solution to a value corresponding to a pH of 4.0 and the mixture was sterilized by heating it for 20 minutes at a temperature of 121C, after which it was cooled to a temperature between 28 and 32C and inoculat~d with a culture of Candida utilis yeast in an amount equivalent to be-tween 2 and 5 grams (dry weight) of the yeast. The fermentation was then continued at a temperature of approximately 32C while air at a space velocity between 0.5 and 1.0 volume per volume per minute was bubbled through the fermenting culture for a period of approxim-ately 25~ hours, or until all of the sugar had been completely con-sumed by the yeast for its growth and additional yeast cells had been produced.
After the fermentation had been completed, the entire mass of solid living yeast cells was separated in a centrifuge, washed with water, and formed into a slurry having a solids content between ap-proximately 16 and 20~ on a dry-weight basis. The yeast slurry was -then dried to a solids content of approximately 93~ in a drum drier. The drying could alternatively have been effected by spray-drying.
The yield of solid yeast cellular matter on a dry-weight basis was 50 grams, which is between 45 and 48% relative to the weight of the sugar that was fermented. The yeast thus produced by cultiva-tion of Candida utilis contained 7% by weight of water, approximately 50% by weight of protein (determined by the Kjeldahl method), ap-proximately 30% by weight of nonnitrogenous compounds, 5.1% by weight of fat, and 7.6~ by weight of ash. It had a nucleic acicl content of 9.3% by weight. The amino acids of the proteins and the percentages . .

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1 by weight in which they were present in the yeast were as follows:
Aspartic acid 10.4%, threonine 5.8%, acrine 6.0~, glutamic acid 12.4%, proline 3.5%, glycine 5.1%, alanine 6.7%, valine 5.3%, methionine 2.0%, isoleucine 4.7%, leucine 7.9%, tyrosine 5.3%, phen-ylalanine 7.1%, lysine 8.0%, histidine 2.5%, arginine 5.8% and tryptophen 0.8%.
~ The yeast solids thus produced can be used as a protein-rich concentrate or supplement for addition to poultry, cattle and hog feeds in amounts between 10 and 30% by weight. It is also suitable for use as a yeast nutrient or supplement in human nutrition.
EXA~PLE 2 .
Ten hundred (1000) grams of finely ground defatted æwiebach crumbs were stirred into 20 liters of water and the slurry was then made homogeneous by vigorous stirring. A small amount of alpha-amylase enzyme was then added to the suspension and the mixture was allowed to stand for such a period and at such a temperature that the starch therein was completely hydrolyzed. The resulting hydro-lyzate was then subjected to ultrafiltration as in Example 1 and the filtrate was inoculated with a culture of Candida utilis yeast 20 as in Example 1. The fermentation and recovery of the yeast solids from the ~ermentation brew were completed as described hereinbefore in connection with Example 1, Instead of using zwiebach as in Example 1, 1000 grams of fine-ly ground defatted cake crumbs which on a weight basis consisted of 4.9% of water, 16.0% sucrose, 1.5% of sodium chloride, and 77.6%
of starch, were substituted therefor, and in all other respects the procedure described in Example 1 was repeated.
The yeast solids that were recovered did not di.ffer to any substantial extent from the product that was described in Example 1 , . .

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1 and the amount that was obtained was equivalent to 55 grams on a dry-weight basis.
EXAMPLE 'l Ten hundred (1000) grams of defatted wheat bran were sub-stituted for the zwiebach crumbs that were used in Example 1 and the procedure therein described was repeated with essentially the same results.

, EXAMPLE 5 One hundred (100) kilograms of wheat kernels are ground together with 400 liters of water and 6 liters of concentrated hydrochloric acid are added thereto and the slurry is heated at a temperature between 110 and 125C in a sealed autoclave for a per-iod between 1.5 and 2.5 hours, thereby hydrolizing the starches therein to soluble sugars.
The same results are obtainable when dry whole wheat flour is used in preparing the slurry and when at least 6 and at most 12 liters of concentrated hydrochloric acid is used for the hydrolysis.
The course of the hydrolysis is followed by periodically test-ing the mixture of unhydrolyzed starch with a solution of iodine prepared by dissolving iodine crystals in an aqueous solution of ' potassium iodide.
After the wheat has thus been hydrolyzed, the slurry is di-luted with water until it has a concentration of approximately 60 grams of soluble sugars per liter. The mixture is then filtered through an ultrafilter consisting of a plurality of ultrafiltration membranes at a temperature between 30 and 60C. The residue is washed thoroughly with water until most of the soluble sugars are extracted therefrom. The aqueous extracts are collected and com-bined with the ultrafiltrate which then will contain between 35 ~ and 40 grams of soluble sugars per liter.

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1 The ultrafiltrate is then inoculated with a suitable yeast culture nutrient salts and other substances that are required in accordance with conventional fermentation methods, and is aerobically fermented in conventional manner. At the conclusion of the fermenta-tion, the beer contains between 17 and 20 grams per liter of yeast based on its dry weight. This yeast is recc,vered by centrifugation and dried. It has a protein content of approximately 50~ by weight and a gross dry weight of between 25 and 32 kilograms.
The li~uid remaining after removing the yeast cells from the beer by centrifugation contains the major portion of the proteins that were originally present in the starch and protein-containing cellulosic substance together with some carbohydrates. The solids that are obtained when this liquid is evaporated have a protein content between approximately 30 and 50% by weight. E'rom 100 kilo-grams of wheat kernels, between 25 and 50 kilograms of these pro-tain-containing solids or concentrates can be obtained in accordance with this process.

-Five (5) kilograms of whole wheat flour containing approximate-ly 12~ protein, between 60 and 70% starch, approximately 15% moisture, approximately 1% ash, and 0.5% fibrous substances (all percentages being in percentages by weight) is stirred into 15 liters of water.
; Then 3.5 grams of an enzyme -- alpha-amylase, type HT-1000 Miles Laboratories was used -- is added to the resulting slurry and the slurry is allowed to stand while it is maintained at a temperature of 80C until substantially all starches therein are hydrolyzed to ` dextrin. Subsequently, the dextrin therein is converted to glucose or other soluble sugars by adding a second enzyme thereto -- amylo-slucosidase, Diazyme L-100 Miles Laboratories was used -- and al-lowing the slurry to stand for several more hours.

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1 The resulting hydrolyzate is then diluted with water to a final volume of 65 liters and its hydrogen--ion concentration is adjusted to a value corresponding to a pH of ~.5.
The solid portion of the hydrolyzate which includes proteins that were originally present in the flour are separated by ultra-filtration from the liquid portion or ultrafiltrate which consists essentia]ly of a solution of sugars.
The concentration of total solids in the 65 liters of the diluted hydrolyzate, including the solid portion as well as the solids dissolved in the liquid portion, is 54 grams per liter, which corresponds to 3.27 kilograms or 65.4% by weight of the original whole wheat flour.
The solid portion of the hydrolyzate that is thus separated :is a protein~containing concentrate which, after washing twice with minial portions of water and drying, contains between approx-imately 40 and approximately 70% by weight of the gluten and other proteins that were originally present in the whole wheat flour, depending upon the degree of washing and hydrolysis. The less washing or hydrolysis, the higher the residual soluble salts and the higher the percentage of ash after dry:ing, so that the protein content is lower.
The ultrafiltrate or liquid portion of the hydrolyæate contains 39.2 grams of dissolved solids per liter, of which 77% by weight are sugars. This corresponds to a 72% conversion of the starches in the whole wheat flour to sugars. This ultrafiltrate on analysis was found to have a nitrogen content of only 0.11%, which is equivalent to 0.7% by weight of proteins which may consist of peptides or amino acids that passed through the ultrafiltrate mem-branes. Since this amount corresponds to only 1~ grams of the original 5 kilograms of the whole wheat flour, it may consequently be dis-_g_ ~ :

1 regarded.
The sugar-containing ultrafiltrate is then inoculated with a yeast culture and the yeast is grown there:Ln in accordance with conventional aerobic fermentation methods such as are referred to hereinbefore and the yeast that is thus produced is recoverëd ~from the brew in conventional manner. A 50% yield of dry yeast sub-stance, as related to the sugar in the filtrate, was obtained. A
quantity of 72 kg sugar was obtained per 100 kg of starches used, and 36 kg dry yeast substance was obtained per these 72 kg of sugar, i.e. 36% of yeast as related to the starch. Dry yeast substance should be understood to be 100~ yeast, i.e. completely dry yeast. -In the foregoing manner, the whole wheat flour is converted into two products, each containing a high percentage of protein, one being the solid concentrate containing gluten and other pro-teins that was direc-tly separated from the hydrolyzate by ultra-filtration, and the other being the yeast produced by fermentation ;~ of the sugars in the liquid portion of the hydrolyzate or ultra-filtrate.
In this example, the enzymatic hydrolysis can be completed within a relatively short period so that the solids remaining in the slurry that are separated :in the subsequent ultrafiltration step, consist, for example~ of approximately equal parts by weight of carbohydrates and proteins. By longer or more vigorous hydroly-sis, the carbohydrates remaining in the solids can be almost com-pletely converted to soluble sugars which remain dissolved in the li~uid portion of the hydrolyzate and can be separated from the solid portion of the hydrolyzate in the processes of the present invention in the ultrafiltrate.
The processses of the present invention are illustrated further in connection with the accompanying drawing wh:ich is a schematic ~, ~ . .. ..
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1 flow sheet of a preferred embodiment of the invention.
The starch and protein-containing cellulosic substances are crushed or milled in the pulverizer or grinder l that is represent-ed in the accompanying drawing to form a slurry with water which is then passed into one of the hydrolysis kettles 2 in which the starches that are present in the cellulosic substances are hydro-lyzed to sugars. The resulting hydrolyzate is then passed into and through a series of ultrafilter membranes 3 in which the protein-containing solids are separated from the liquid portion con-taining dissolved sugars. The separated solids, which include theproteins that were originally present in the cellulosic substance, are dried in a dryer 4 and are used as such or combined with other proteins that are produced in the process.
The ultraEiltrate containing the dissolved sugars is passed to a tank 5 and nutrient salts and any other substances that are required to produce a culture medium upon which the preselected yeast is to be grown are added thereto and mixed therewith. This culture medium is then passed to the fermentor 6 in which it is inoculated with a culture of the preselected yeast, which is then aerobically grown therein.
After conclusion of the fermentation in the fermentor 6, the ; brew is passed into a separator 7 in which the-yeast cells are separated from the accompanying liquor and are eventually washed with water. The slurry of yeast cells which has a dry-solid con-tent between 16 and 20% by weight is then collected in the tank 8.
From this tank the slurry is then passed to a dryer 9 in which the solids are dried and the dried solids are then packaged into sacks or other containers.
Although alpha-amylase was used in Example 2, it is to be understood that other known starch-hydrolyzing enzymes may be sub-. ' 1 stituted therefor.
Furthermore, instead of using an enzyme, the hydrolysis may be effected by cultivation in the slurry of a microorganism that is known to produce an enzyme such as alpha-amylase that is cap-able of hydrolyzing starch, such as microorganism of the genus Endomycopsis, such as, for example, the species Endomycopsis fib-uligera~ The microorganism is then separated from the hydrolyzate before the hydrolyzate is subjected to ultrafiltration or is separ-ated with the other solids during the ultrafiltration step.
Other acids such as orthophosphoric and sulfuric ac:ids can be substituted for the hydrochloric acid that was used for the hydrolysis step of the process in the Eoregoing Examples 1, 3, 4 and 5.
When enzymes such as amylases are used in the hydrolysis step, none or at most only small quantities of the proteins that are present in wheat and other cellulosic substances containing starches and proteins are converted to products that are soluble in the hydrolyzate. Acids have a tendency to degrade proteins to amino acids.
; 20 Instead of separating the solids from the liquid portion of the hydrolyzates by ultrafiltration and fermenting only the ultra-filtrate, as illustrated in the foregoing examples, the entire hydrolyzate as such containing the desired sugars as well as the ~, proteins and other slids can be fermented. The proteins that are originally present in the cellulosic substance are practically not effected or converted during the fermentation and can be recovered together with the yeast cells in the subsequent ultrafiltration step. The~ultrafiltrate then contains only dissolved salts that have been washed out of the solids, which solids are rPtained on the ultrafilter membranes. Such ultrafiltrates have a biochemical . .

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1 oxidation demand of only 1000 parts per million or less.
When the cultivation or fermentation is conducted in a con-tinuous manner that is by continuously withdrawing brew and adding fresh culture medium, a residence period of the brew in the ferment-er of approximately 4 to 5 hours must be a]lowed for completion of the fermentation with yeasts, whereas a residence period of 2 hours at most is required in a bacterial fermentation.
When bacteria axe used instead of yeasts, the fermentation is completed after a period of 20 hours at most.
The processes of the present invention are advantageous since the amino acids that are present in -the yeasts and in the original protein-containing cellulosic substances are an extremely valuahle combination from a physiological and nutritional standpoint. Thus, the amino acids that are present in the proteins of the starch and protein-containing cellulosic substances may compensa-te for in-adequate amounts of methionine and cystine that are supplied by the yeast. On the other hand, the yeasts may supply asparagine and other amino acids that may be present in inadequate amounts or may be absent in the proteins of the starch and protein-containing cellulosic substances.
If comminuted plants such as wheat which has not been pre-liminarily threshed, or grains or cereals which still include large portions of fibrous substances in the Eorm of chaff husks, cobs, or bran, are to be used as the starting starch and protein-contain-in~ cellulosic substance in the processes of the present invention, such f~brous substances can be separated preliminarily from the starch or protein-containing portions of the cellulosic substances by con~entional straining or centrifuging procedures. On the other hand, if the starch and protein-conkaining cellulosic substances include fibrous substances that can be hydrolyzed to pentoses, such .~ '; ' ~S~

1 fibrous substances can be included along with the other components of the cellulosic substance that is to be hydrolyzed in the pres-ence of an acid and be subse~uently fermented together with the other sugars or hexoses that are present in -the hydroly~ate to produce additional ~uantities of yeast and need not be preliminarily sep-arated from the other fibrous substances.
Hydrolysis of starch may take place in the presence of an organic acid, such as citric acid o~r acetic acid.

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Claims (11)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the production of proteins from a starch and protein-containing cellulosic substance which comprises first sub-jecting the starch and protein-containing cellulosic substance to hydrolysis to hydrolyze the starch contained therein, subjecting the hydrolyzate containing sugars to ultrafiltration to separate the solids from the liquid portion thereof and aerobically growing yeasts in the liquid ultrafiltrate and recovering the yeast cells thus grown from the resulting beer.

2. A process as defined in claim 1 in which the starch and protein-containing substance is a cereal.

3. A process as defined in either claim 1 or claim 2 in which the hydrolysis of the starch is effected in the presence of a dilute mineral acid.

4. A process as defined in either claim 1 or claim 2 in which the hydrolysis of the starch is effected in the presence of an organic acid.

5. A process as defined in either claim 1 or claim 2 in which the hydrolysis of the starch is effected at an elevated temp-erature.

6. A process as defined in either claim 1 or claim 2 in which the hydrolysis of the starch is effected by the action of an enzyme.

7. A process as defined in either one of claims 1, 2, or 6 in which the hydrolysis of the starch is conducted only to the stage in which dextrins or disaccharides free from monosaccharides are the principal hydrolytic products.

8. A process as defined in either one of claims 1, 2, or 6 in which the solids in the hydrolyzate are separated from the liquid by ultrafiltration.

9. A process as defined in claim 6 in which the starch-hydrolyz-ing enzyme is produced in situ by cultivation of a microorganism that is capable of producing the said enzyme in a slurry containing the starch and protein-containing cellulosic substance, the said microorganism being separated from the hydrolyzate before the hydro-lyzate is subjected to the subsequent ultrafiltration step.

10. A process as defined in claim 6 in which the starch-hydrolyzing enzyme is produced in situ by cultivation of a micro-organism that is capable of producing the said enzyme, the said microorganism being separated from the hydrolyzate in the subsequent ultrafiltration step.

11. A process for the conversion of a cellulosic starch and protein-containing substance to a nutriment or concentrate richer in proteins and recovering the original proteins therein as a sep-arate fraction which comprises dispersing in water the cellulosic substance in comminuted form, subjecting the starch in said disper-sion to hydrolysis by means of a starch-hydrolyzing enzyme to convert the starches therein to fermentable sugars, separating the protein-containing solids from the remaining sugar-containing liquid in the resulting hydrolyzed dispersion by ultrafiltration, aerobically cultivating in the said sugar-containing liquid a yeast or a bacterium that is capable of fermenting the sugar-containing liquid and utilize the sugar therein for growth, and subsequently recov-ering from the fermented liquid the protein-containing yeast or bacterium that was grown therein.