United States Patent [191

[111

3,867,555

Newell et al.

[45] Feb. 18, 1975

154]

MANUFACTURE OF YEAST PROTEIN

ISOLATE HAVING A REDUCED NUCLEIC
ACID CONTENT BY AN ALKALI PROCESS

3,681,195
3,718,541
3,725,075

[75]

Inventors: Jon Albert Newell, Webster Groves;

Primary ExaminerJames R. Hoffman

Attorney, Agent, or Firm-Gravely, Lieder &
Woodruff

Ernest Aleck Robbins, High Ridge;

Robert Dudley Seeley, Crestwood,
all of Mo.

[73] Assignee: Anheuser-Busch, Incorporated, St.

[22]

Filed:

Louis, Mo.
Nov. 29, 1972

[ 57]

8/1972
2/1973
4/1973

Suekane et a1. ...................... .. 195/4

Kalina ................................. .. 195/28
Muroi et a1. .......................... .. 99/14

ABSTRACT

This disclosure relates to a process for producing a

yeast protein isolate (lYP) by treating the protein with

alkali under controlled temperature conditions to re

[211 App]. No.: 310,453

[52]

US. Cl .............. .. 426/60, 260/112 R, 426/148,

[51]
[581

Int. Cl. .......................... .. A23j 1/18, A231 1/28

Field of Search ........... .. 426/60, 148, 364, 204,

426/204, 426/212, 426/364

426/212, 279; 260/112 R

duce the nucleic acid (RNA) content of the protein.

The yeast cells are ruptured by homogenization; ex
tracted to remove the protein from the cells; and the

cell wall debris separated by centrifugation. The solu

bles fraction (which contains protein and nucleic
acid) is treated with alkali in either a high temperature
low alkali process (pH 10 to 10.5; temp. 75 to 85C.;
time 1 to 4 hours), or a low temperature high alkali

[56]

References Cited
UNITED STATES PATENTS

3,268,412
3,585,179
3,615,654
3,634,194

8/1966
6/1971
10/1971
1/1972

Champagnat et a1. ................ .. 195/3

Samejima et a1. ................ .. 260/112
Atukawa et a1 ......................... .. 99/9
Frankenfeld et al ................ .. 195/28

process (pl-1 11.5 to 12.5; temp. 55 to 65C.; and time

1 to 2 hours) to hydrolyze the nucleic acid. The pro
tein is acid precipitated and washed to produce a final
lYP of low RNA content.

20 Claims, 1 Drawing Figure

3,867,555
1

MANUFACTURE OF YEAST PROTEIN ISOLATE

HAVING A REDUCED NUCLEIC ACID CONTENT
BY AN'ALKALI PROCESS

to those versed in the art. We prefer to utilize biomass

of Saccharomyces cerevisiae and Candida urilis grown
on food grade nutrients in batch and continuous fer
mentation, but other yeasts are suitable for use in this
process. The main considerations are that the yeast be

BACKGROUND OF THE INVENTION

There has been considerable information published

of food grade and produced in good yield.

meaning connotates the whole cell, in which the pro

tein is contained within the con?nes of the cell wall and
therefore is relatively nonfunctional. The other mean

The biomass is harvested by centrifugation or ?ltra

tion and water washed. When necessary, dilute alkali
may be incorporated in the wash to remove adhering
color and taste bodies. The yeast cells are ruptured by
any of several known methods, such as high pressure

ing connotates a protein isolated as a separate entity

from the microbe. In either case, for human nutrition,

sonic disintegration, repeated freeze-thaw cycles, lytic

on the production of microbial protein. The term mi

crobial protein has developed two meanings. One

homogenization, attrition in a sand ' or colloid mill,

enzymes and the like. The main consideration is to rup

the nucleic acid content of the protein product should
be reduced to a lower level, namely below about 9% by 15 ture the majority of cells under such conditions that the
majority of the protein remains in the soluble state, but
weight, if yeast protein is a substantial source of protein
can be harvested in a later step. The ruptured cell sys
in a human diet. The Recommended Daily Allowance
tem chomogenate) may be diluted, warmed and pH ad
of The Food and Nutrition Board, National Research
justed to favor processability.
Council, for protein is 65 grams per day for a 70 kilo
The homogenate is separated by centrifugation and
gram adult male. The Protein Advisory Group of the
/or
?ltration into a cell wall residue and an extract usu
United Nations System recommends that the amount of
ally referred to as the alkali extract. The development
nucleic acid ingested per day from microbial protein
of a yeast gylcan from the cell wall residue is described
should be less than 2 grams. Therefore, the nucleic acid
in a copending application (Yeast Glycan and Process
content of the protein should be less than 6%, if yeast
protein supplies 50% of dietary protein. The nucleic 25 of Making Same, Ser. No. 310,452, filed Nov. 29,
1972).
,
acid should be below about 3% if yeast is the sole
If the protein is recovered by isoelectric precipitation
source of protein in the diet.
at this stage, the protein will contain high levels of nu
The nucleic acid content of yeast cells, such as Can
cleic acid. Such preparations are referred to as a full
dida utilis and Saccharomyces cerevisiae, is about 12 to
15 grams of nucleic acid per 100 grams of crude pro 30 RNA-IYP. However, if the alkali extract is treated with
additional alkali in accordance with the conditions of
tein. Crude protein is calculated for purposes of this ap
our invention, then the protein is relatively free of nu
plication as the Nitrogen (Kjeldahl) content multiplied
cleic acid and may be recovered by isoelectric precipi
by 6.25. The protein isolated from these cells also con
tation. The recovered protein product has a desirably
tain 12 to 15 grams nucleic acid per 100 grams of crude
protein. Thus, the nucleic acid content should be re 35 low level of nucleic acid and is referred to as low RNA
duced several fold before a substantial amount of the

protein is used for human nutrition. The nucleic acid of

yeast is mainly ribonucleic acid or RNA, and in this ap
plication these terms will be used interchangeably.
Heretofore alkali has been used to extract the nucleic

IYP.

'

The separation of the low RNA-IYP results in a pro

tein sludge and the soluble cytoplasmic constituents.

The soluble cytoplasmic constituents contain the nu

cleic acid fragments, protein fragments, glycogen, and

all of the metabolic intermediates. It can be recognized

that the. soluble cytoplasmic constituents constitute a
of the nucleic acid of the cell, so that the nucleic acid
valuable fraction of the total microbial system. The
to protein ratio of the yeast is decreased. One disadvan
tage of this process is the concurrent loss of valuable 45 harvested low RNA-IYP may be water washed to re
move adhering cytoplasmic material. The washed pro
solids, including protein, from the cell with a conse
tein product may be dried with or without in vacuo
quent decrease in protein yield. Another disadvantage

acids from yeast cells, thereby decreasing the content

is that the protein confined by the cell walls remains in

relatively non-functional form.

We have discovered a process by which alkalican be
used to prepare a protein product having reduced nu

cleic acid content in good yield from yeast. The protein

product has desirable functional characteristics. An
other advantage of our process is that the cell walls of
the yeast can be recovered as a valuable product. A still 55

concentration to a powder by spray drying, drum dry

ing, freeze drying and the like.
The recovered low RNA-lYP is substantially cell free
and has the following composition: about 65% to about
85% protein; about 0.5 to 9% RNA (preferably 0.5-5%
and ideally less than about 3% RN); about 7% to about
15% lipid; about 1% to about 5% ash; about 5% to
about 20% carbohydrate; and about 0 to about 1% ti

ber.
further advantage is that the non-protein soluble cyto
Cell rupture, extraction of solubles, and processabil
plasmic constituents of the cell can be recovered and
ity
are affected by pH, temperature, time, solids con
processed to a valuable product.
centration, and homogenizer efficiency. Our usual
Our process is comprised of the following steps: pro
duction of yeast cells, rupture of the cells, separation 60 method of measuring the extent of cell rupture is to de
termine the amount of nitrogen that remains soluble,
of the insoluble cell wall fragments from the soluble cy
i.e., the % N Extractability = 100 X
toplasmic fraction, treatment of the soluble fraction
with alkali, recovery'of the low nucleic acid protein by
precipitation and centrifugation, vacuum concentra
tion, and drying.
65

DETAILED DESCRIPTION
Yeast cells (biomass) is produced by methods known

Candida ulilis biomass after washing has a pH of

4.5-5.5. The biomass is usually chilled, then passed

3,867,555

3
through a Manton-Gaulin homogenizer to a chilled re

TABLE NO. lB-Continued

ceiver. The process is repeated for a total of three

passes. At least three passes are needed to obtain maxi

Effect of Extraction pH. Temperature, Time, Solids Content. and

Homogenizer Ef?ciency Upon the Nitrogen Extractability of

mum cell rupture. in practice, the biomass is homoge

nized at the ambient pH of the yeast, namely 4.5-6.5.

Saccharomyces cerevisiae

Cell rupture can also be achieved at a higher pH, up to

Chilled suspensions of commercial baker's yeast at ambient pH of

at least pH 9.5, but the subsequent separation of the

6-6.5, 7-l0% solids, were homogenized by means of a Manton-Gaulin

homogenizer at a pressure of 8000 psig. The chilled homogenate was
recycled through the homogenizer to give one, two or three passes. The

cell wall residue from the solubles becomes more diffi

cult.

homogenates were diluted with up to two volumes of water and

The effects of pH, solids concentration, and homoge

nizer efficiency upon the N extractability of Candida

adjusted in pH. The diluted homogenates were incubated for 5-60

minutes at 2560C. and centrifuged. The nitrogen contents of the
homogenates and supernates after centrifugation were measured by the

utilis and on Saccharomyces cerevisiae are shown in Ta

bles IA and Table I8.

Kjeldahl method. % N extractions were calculated.

TABLE lA

15

Effect of Extraction pH, Solids Level, and Homogenizer

Efficiency Upon Nitrogen Extractability of Candida ulilis

Chilled suspensions of Candida miiis at pH 5.0-5.5. 7-10% solids
were homogenized by means of Manton-Gaulin homogenizer. The
chilled homogenate was recycled through the homogenizer at a
pressure of 8000 psig repeatedly to give one, two, three or four pass
homogenate. The homogenate was diluted with up to 2.0 parts of
water, and adjusted in pH. The diluted homogenates were incubated
for 30 min. at 50C, and then centrifuged. The nitrogen contents of
the diluted homogenate, and of the supernate were measured by the

Kjeldahl method. % N extractions were calculated.

Separation
pH of
Extraction

Solids
Content

7
8

2.5
2.5

3
3

2.5

10
ll

2.5
2.5

3
3

Good
Good

84

Good

82
80

Good
Good

9.5

2.5

70

Good

9.5
9.5
9.5
9
[0
ll
[2
9
10
1l
12

2.5
2.4
2.5
2.4
2.4
2.4
2.4
6.9
6.9
6.9
6.9

2
3
4
3
3
3
3
3
3
3
3

83
89
91
83
82
78
85
64
59
54
41

Good
Good
Good
Good
Medium
Medium
Medium
Medium
Poor
Poor
Poor

Time

C.

No. of

7: Nitrogen

Content

(min. )

Temp.

Passes

Extractability

5.0
6.0
7.0
8.5
9.5
6.0
6.5
7.5
8.5
9.5

3-4
34
3-4
3-4
3-4
3-4
3-4
3-4
3-4
3-4

30
30
30
30
30
60
60
60
60
60

25
25
25
25
25
60
60
6O
6O
6O

3
3
3
3
3
3
3
3
3
3

36
79
93
93
96
42
33
30
73
90

The data of Tables IA and IB show that extraction of

25 the soluble nitrogenous materials can be carried out at

least over the pH range of about 5.5 to about 1 1. Pro

cess considerations further limit the extraction pH to
the range of about 7 to about l0, with pH about 9.5

No. of 7: Nitrogen of Cell Wall

Passes Extracted Residue and
Solubles
76
74

Solids

pH

considered the optimalbalance between extraction and

30 subsequent separation of the cell wall residue from the

solubles. Extraction is best at a low solids content, but

again a consideration of process rated led to the adop

tion of a solids content of about 2.5% to about 47%. Ex
traction time can be varied between about 5 and about

35 60 minutes at extraction temperatures of about 0C. to

about 60C., preferably 2_5-60C. The best process }

rate of the subsequent separation of the cell wall resi~

due from the solubles were obtained when the extrac
40

tion was done at 60C. for 5 to 20 minutes, at' pH 9.5. _

With Candida utilis and Saccharomyces cerevisiameach.

pass through the homogenizer improved, the nitrogen

extractability presumably by rupturing more'cells, but,

again, process rates and equipment must becorisid
TABLE NO. lB

ered. From 1 to 5 passes can be, used, but we prefer a

45 three pass system as a balance between vefficiency and

economy. The pressure of the homogenizer is about

5000 to about l5000 psig. The temperature is 0 to
about 50C. and preferabiy 0 to 10C.

Effect of Extraction pH. Temperature, Time, Solids Content. and

Homogenizer Efficiency Upon the Nitrogen Extractability of

Sar'clmromyces cererisiae

Taking N extractability and process requirements

Chilled suspensions of commercial baker's yeast at ambient pH of

6-6.5. 7-107: solids, were homogenized by means of a Manton'Gaulin
homogenizer at a pressure of 8000 psig. The chilled homogenate was
recycled through the homogenizer to give one, two or three passes. The

into considerations, the optimal process to produce an.

extract of the soluble part of the yeast is: (1) growing
a food grade yeast on a nutrient media, (2) harvesting

homogenates were diluted with up to two volumes of water and

adjusted in pH. The diluted homogenates were incubated for 5-60

minutes at 25-60C. and centrifuged. The nitrogen contents of the

and washing the yeast cells, (3) rupturing the yeast

' homogenates and supernates after centrifugation were measured by the

Kjeldahl method. 71 N extractions were calculated.
55
.

Solids

Time

C.

No. of

7: Nitrogen

pH

Content

( min. )

Temp.

Passes

Extractability

9.5
9.5
9.5
9.5
9.5
9.5
9.5
9.5
9.5
9.5
9.5
9.5
9.5
4.0

9.l
4.8
3.1
3.1
3 l
3-4
3-4
3-4
3-4
3-4
3-4
3-4
3-4
3-4

30
30
30
30
30
5
20
30
60
5
20
30
60
30

25
25
25
25
25
50
50
50
50
60
60
60
60
25

3
3
3
2
l
3
3
3
3
3
3
3
3
3

83
84
92
80
63
91
93
96
96
93
94
9]
90
33

cells at a temperature of 0-l0C., (4) handling'the

ruptured cells at pH 9.5, at 60C. for 20 minutes, (5)
removing the yeast insolubles at a temperature of about
60C. The extract containing the soluble part of the
yeast is called the alkali extract. Under the optimal
conditions, 85-90% of the Kjeldahl N of the homoge
nate is obtained in the alkali extract. One wash of the
cell wall residue increases the extraction yield to
90-95%.
if the alkali extract is adjusted to pH 4.5 at this stage,

65

then the protein product recovered has the same ratio

of nucleic acid to protein that exists in yeast, that is 13
to 15 grams of nucleic acid per 100 grams of crude pro

tein. The recovered protein product constitutes a full

3,867,555

6 .

on the Hydrolysis of Nucleic Acid & The Protein

RNA~1YP. The crude protein nitrogen recovered after
Recovery from the Soluble Fraction of Yeast
the pH 4.5 isoelectric precipitation of the alkali extract
has routinely amounted to 75-80% of the crude protein
The soluble fraction of Candida utilis (alkali extract)
nitrogen present in the alkali extract. The full RNA
IYP from bakers yeast has averaged 81.5% crude pro 5 was prepared as set forth in Example 1. Sodium hy
tein, l 1.7% RNA (dsb); that from Candida utilis has av

droxide was added to aliquots of the alkali extract. The

eraged 74.6% crude protein, 10.7% RNA (dsb).

The RNA is calculated by the following method

aliquots were incubated at 60, 80, and 100C. for 1

to 4 hours. The samples were cooled, adjusted to pH
4.5, and centrifuged to yield a protein product and a

RNA determination: About 50 mg. W? is digested

with 5 ml. of 0.2 N KOH for 30 minutes at 100C. The

supernate called acid whey. The samples were analyzed

to determine the percent hydrolysis of the nucleic acid

digest is acidi?ed with 5 ml. HCIO, citrate reagent (0.4

M citrate buffer, pH 2.2 containing 1.7 ml., 70% HClO4

and the amount of protein recovered.

TABLE 11A
% Hydrolysis of Nucleic Acid 1"

Incubation Temperature
NaOH [N]

1hr.

60C.
2hr.

3hr.

1hr.

80C.
2hr.

4hr.

1hr.

100C.
2hr.

36

0.025

52

67

80

75

63

78

94

0.05
0.075

6
X

23
X

46'
X

69
83

86
86

81
86

90
90

97
93

100
100

0.10

53

82

88

X i

0.15

79

87

91

'

4hr.

'4 Nucleic Acid Hydrolysis = 100 X Total Am, of Acid Whey of Sample/Total Aw, of Acid Whey of an aliquot
of the alkali extract heated for 30 min.; 100C; in 0.2N KOH.

TABLE H13
92 Precipitation Yield 2
,

60C .

NaOH [N]

0
0.025

0.05

1hr.

2hr.

80

81

8 0C.

3hr.

81

lhr.

2hr.

100C.

4hr.

lhr.

2hr.

X'

4hr.

82

78

74

' 74

74

65 -

79

77

74

73

68

69

62

48

48

0.975

68

65

58.

56

' 52

52

0.10

71

66

64

0.15

67

63

60

' X

' X

X '

n 1'" 71 Precipitation Yield = 100 X kjeldahl N content of Alkali Extract-N Content of Acid Wh'ey/Kjeldahl N
Content of Alkali Extract

"" Theoretical yield: a full RNA-1Y1 is obtained in 8091 precipitation yield and contains 10.7 RNA. 74.6% crude

protein. 11 all of the RNA has been hydrolyzed. and is not precipitable, then the precipitation yield cannot be
greater than 686; .

per 100 ml.). The residue is removed by centrifugation. 45 Temperatures greater and lesser than those given in
The AM of the suitably diluted supernatant is mea-'
Table II have been investigated. At 30C.,incubati0n
for 2 hours at pH 10, 10.5 and 1 1.0 did not present suf
sured. The extinction coefficient of 31.7 A260 ml/mg. is
ficient RNA hydrolysis. Incubation at 120C. for .1 hour

used to calculate RNA. The RNA content is corrected

at pHs 4.5, 5.5, 6.5, 7.5, 8.5 and 9.5, followed by pre

for the A260 contribution of protein fragments in the hy

drolysate as measured by the Lowry method.

The crude protein is calculated by the Kjeldahl nitro

50

gen method. The total nitrogen of the IYP is measured

and multiplied by a factor of 6.25.

cipitation at pH 4.5 and recovery of the protein gave

14.1, 12.2, 7.8, 5.6, 4.3, and 2.6% RNA (dsb) in the

protein product respectively. These data and the data

of Table [I show that alkalinity, time, and temperature

are all related in the recovery of a low RNA-protein

The alkali extract was treated with additional alkali
up to 0.15 M sodium hydroxide, at temperatures up to 55 product in good yield.
The separation of the protein from the solubles frac

120C., (preferably about 50C. to about 120C.) and

tion is initiated by the acidi?cation of the incubated

for incubation times sufficient to hydrolyze the nucleic
product to a pH of about 2 to about 6 (preferably about
acids of the nucleoprotein, thereby allowing the recov
3.5 to about 5.5) at a temperature of about 0-100C.
ery of a protein product containing a reduced content
of nucleic acid. This time preferably is less than about 60 This causes the protein to precipitate. The solubles
fraction is then separated by centrifugation or ?ltration
4 hours. The pH is maintained at about 95m about
at approximately the conditions of pH and tempera
12.5. The amount of protein recovered was measured
to allow the determination of the balance between the

nucleic acid content of the protein and the protein

yield. The data are presented in Tables 11A and IIB. 65
TABLES 11A and B

Effect of NaOH Concentration, Temperature, & Time

ture.

DESCRIPTION OF DRAWING
The drawing is a schematic ?ow sheet of the process
of this invention.

3,867,555

The following are detailed examples of this invention.

EXAMPLE 2

'

' Preparation of Low RNA-Protein Product from "

EXAMPLE 1

Candida utilis by the HTLA Process

5

Preparation of a Low RNA-Protein Product from

Candida utilis by the LTHA Process

'

Alkali extract was prepared in accordance with the

procedure set forth in Example 1. The alkali extract
was adjusted to 0.025 N NaOH with a resulting pH of
10l0.5. The extract was incubated for four hours at

176F. (80C.) with gentle agitation. Sufficient 85%

Candida utilis Biomass was produced in continuous 10 phosphoric acid was then added to adjust to pH 4.5
This is called the High Temperature Low Alkali
(HTLA) process. In the HTLA process the tempera

fermentation upon a molasses substrate supplemented

with a source of nitrogen and phosphate. The biomass
was harvested by centrifugation and washed three

ture is 75 to 85C.; the pH is 10 to 10.5 and the time

times with water.

is l to 4 hrs.

Fifty gallons of a suspension of Candida utilis biomass

containing 32.6 pounds of yeast solids, which consisted
of 16.1 pounds of crude protein and 2.2 pounds of nu

'

The digest was centrifuged into the protein product

and acid whey. The protein product was washed once
and spray dried. About 6.1 pounds of washed protein
solids were obtained having the composition (as-is):
4.3% moisture, 70% crude protein, 1.0% nucleic acid,
11.3% lipid, and 2.6% ash.

cleic acid, were cooled to 45F. and subjected to ho

mogenization at a pressure of 8000 psig whereafter it
was cooled to 45F. The homogenization was repeated
for a total of three passes. The homogenate was diluted

EXAMPLE 3

to 110 gallons with water and adjusted to pH 9.5 with

sodium hydroxide. The material was agitated for 15

Preparation of Low RNA-IYP from Saccharomyces

cerevisia by the HTLA Process

minutes, heated to 140F., and centrifuged. The 140F. 25

temperature facilitates the separation of the insoluble
Commercial bakers yeast was used as a source of
Saccharomyces cerevisiae. The biomass was given three
cell walls and the solubles (called the alkali extract).
water washes and thickened by centrifugation to 16.1%
The separation yielded 12.5 pounds of cell wall solids
solids.
and 18.1 pounds of alkali extract solids.
The alkali extract was adjusted to 0.1 N is sodium hy 30 The alkali extract and the low RNA-protein product
was prepared in accordance with the procedure set
droxide by the addition of 10 N NaOH. The pH was
forth in Example 2. The composition of the spray dried
about 12. The extract was incubated for 1 hour at
protein product from bakers yeast: 5.6% moisture,
140F. (60C.) with gentle agitation. Sufficient 85%

73.6% crude protein, 3.3% nucleic acid, 11.3% lipid,

phosphoric acid was then added at 140F. to adjust to
pH 4.5. This is called the Low Temperature, High A1 35 5.6% ash, and 4.0% carbohydrate by difference.
The effect of the nucleic acid reduction process on
kali (LTHA) process. The LTHA process treats the al
the ?avor of the protein product was generally assessed
by comparing the taste ofa 5% W/W, pH'7 suspension

kali extract at a temperature of about 55 to 65C. for

about 60 to about 120 minutes at a pH of about to
about 11.5 12.5.

' to that of a full RNA-protein product made at the same

40

time. The protein product made by the LTHA process

The digest was centrifuged into an insoluble protein

usually had a greater ?avor impact than that of the full
product fraction and a solubles fraction, called the acid
RNA-protein product. The protein product made. by
whey: The protein product was washed once with wa
the HTLA process usually had the same or less ?avor
ter. About 9.5 pounds of washed protein solids and 2.5
impact than the full RNA-protein product. In all prod
pounds of wash solids were obtained. The washed pro 45 ucts, the ?avor impact could usually be reduced by in
tein product was then spray dried. The composition of
vacuo concentration.

spray dried protein product (as-is) was: 5.0% moisture,_

The effect of the nucleic acid reduction processes on

68.3% crude protein, 1.9% nucleic acid, 6.7% ash,

12.2% fat. 0.3% crude fiber, and 6.1% carbohydrate.

the nutritional quality of the protein product is'shown

in Table 111.
TABLE Ill

Nutritional Quality of Unfractionatcd Candida utili.~ and of isolated Yeast Protein (lYP)
Isolated yeast proteins were produced from Candida ulilix in accordance with the process set forth in
Examples 1 and 2. In addition, a full RNA-IYP was prepared according to Example 1 except that the
nucleic acid reduction step was omitted. The isolated yeast proteins and the unfractionated yeast were

spray dried products.

Product Composition

Level of

7: dsb

Corrected

Nucleic Acid

Test No.
l
2

Material

Reduction
Step

Corrected
Protein

Nucleic
Acid

Protein in
the Diet

PER

C. utilis
Unfractionated
C. utilis

None

41.6

7.6

10.0

1.47

Homogenate

None

33.4

5.4 -

10.0

1.63

None

42.0

6.7

10.0

1.70

None
None

40.5
62.4

6.0
10.2

10 .0
10.0

1.62
1.60

C . utilis

C . utilis

Homogenate
Full RNA-IYP

Homogenate

3,867,555
9

10

TAB LE 111- Continued

Nutritional Quality of Unfractionated Candida mills and of Isolated Yeast Protein (lYP)
Isolated yeast proteins were produced from Candida utilix in accordance with the process set forth in
Examples 1 and 2. In addition, a full RNA-lYP was prepared according to Example 1 except that the
nucleic acid reduction step was omitted. The isolated yeast proteins and thc unfractionated yeast were

spray dried products.

Test No.

Nucleic Acid
Reduction
Step

Material

Product Composition
7: dsb
Correctedz
Nucleic
Protein
Acid

Level of
Corrected
Protein in
the Diet

PER"'

6
7
8
9
10
11
12

Full RNA-IYP
Low RNA-1Y1
Low RNA-IYP
Low RNA-IYP
Low RNA-IYP
Low RNA-IYP
Low RNA-lYP +

None
MSE
LTHA
LTHA
LTHA
LTHA

65.4
67.4
68.0
68.0
68.0
70.0

11.5
1.4
1.6
1.6
1.6
2.0

10.0
10.0
10
12.5
15.0
10

1.55
1.64
0.51
0.71
0.83
0.41

13
14

Meth. (4)
Low RNA-IYP
Casein (ANRC)

LTHA
HTLA
X

70.0
71.2
X

2.0
2.0
X

10
10
7.5

1.87
0.71
2.40

15

Casein

16
17

Casein
Casein

10.0

2.50

X
X

X
X

X
X

12.5
15.0

2.31
2.10

' In accordance with our copending application Newell ct al. A Process 01' Making Yeast Protein Isolate Having Reduced
Nucleic Acid Content". This process uses malt sprout extract (MSE) to hydrolyze the nucleic acid in the alkali extract before

the protein is separated. This produces an IYP having low RNA content.
"-" ('orrccted protein = 6.25 (Total N by Kjcldahl - ,1 Nucleic Acid Content/6.13)
'l" The Protein [Equivalence Ratio (PER) was measured by WARF. Inc. calculated to a PER = 2.5 for ANRC casein at 10%

level in the diet.

" (1.2; methionine was incorporated into the diet.

The reproducibility of the nutritional quality of the

of the FAO reference pattern except for methionine
materials is apparent from the comparisons among
and cystine. The essential amino acid requirements for
Tests Nos. 2, 3 and 4; between 5 and 6; and between 30 the growing rat are also met'or exceeded except for me
8 and 11. The nutritional quality of the full RNA-lYP
thionine, cystine, and lysine. The available lysine con
is equivalent to the yeast homogenate from which the
tent is equal to that of casein.
W? is made. Test No.. 7 shows that a low RNA-IYP
Alkali effects on the nutritional quality of proteins is
can be made without reduction in nutritional quality.
not understood completely, but may be the result in
The alkali treatments to reduce the nucleic acid con

35 part of modi?cations involving cystine, lysine, arginine

and possibly serine, and to the racemization of threo
nine. The available lysine content of the low RNA-[YP
(Test No. 8) is identical to that for the full RNA-IYP
(Test No. 5) and stands in the same relationship of the

tent does reduce the nutritional quality, but the nutri-_

tional quality was improved by the addition of methio
nine.

'

Table IV shows the essential amino-acid composition 40 total lysine. Threonine was not affected as evidenced ,
of the isolated yeast protein products. The full RNA
by the comparison of column and mba values. Cystine,
lYP (Tests No. 5 and No. 6) and the low RNA-lYP

but not methionine, content was decreased by the

made by our Malt Sprout Extract process (Test No.v 7) ; _

LTHA and HTLA treatments. Because methionine can
are especially rich in all of the essential amino acids ex

replace cystine in the diet, the addition of methionine

cept for the sulfur containing amino acids. Except for
45 .to the low RNA-IYP would be expected to increase'the
methionine and cystine, the contents of the essential
nutritional quality. The additionof 0.2% methionine to
amino acids exceeds that cited for the FAO reference
the diet (2.0% methionine on a protein basis) did im
protein (FAO Committee on Protein Requirements
prove
the nutritional quality (Test No. 12,Tab1e 111) of
(1957b) FAO Nutritional Studies No. 16') and meets
the low RNA-IYP made by the LTHA process. The sul

or exceeds that needed for the growing rat.

50 fur containing amino acids can be supplied as the iso

The low RNA-lYPs made by theHTLA and LTHA

processes also exceed the essential amino acid content

lated amino acid, or as a mixture with a protein rich in

cystine or methionine, such as wheat protein.

TABLE IV
Essential Amino Acid Composition of Isolated Yeast Protein from Candida utilis I

Amino Acid

Method
of
Detn.

Lysine

Grams of Amino Acid per 100 Grams Corrected protein

Method of RNA Reduction
None
MSE"-
LTHA
HTLA
ANRCL"
o.
No.
No.
No.
No.
No.
Casein
5
6
7
8
11
13

FAQ)

Amino Acid)
Pattern
Reqd by the
Growing Rat

Column

8.46

9.56

8.70

9.60

8.12

7.86

8.0

4.2

9.0

Lysine
Lysine
Methionine
C stine

mba
Availab1e"
mba
mba

9.05
7.95
1.35
0.68

X
X
1.32
1.28

X
X
1.36
1.34

9.00
7.92
1.67
0.25

X
X
1.41
. 0.56

X
X
1.30
0.42

X
X .
2.84
0.52

X
X
2.2
2.0

X
X
3.4

T reoninc
Threonine
Tryptophan
Valine

Column
mba
Column
Column

5.25
5.40
1.37
6.41

5.42
X
1.35
6.46

5.40
X
1,53
6.60

5.00
4.84
1.59
6.29

4.95
X
1.47
6.38

5.05
X
1.39
6.34

3.52
X
1.27
7.12

2.8
X
1.4
4.27

5.0
X
1.1
5.5

1.6

3,867,555

11

12
TABLE IV Continued

Essential Amino Acid Composition of Isolated Yeast Protein from Candida urilis
Grams of Amino Acid per 100 Grams Corrected Lrotein
Method of RNA Reduction
Methodm

of

Amino Acid

Phenylalanine

Leucine

None

No.

Detn.

cam};

Column

'

. MSE

No.

No.

533

5.26

55157

9.29

9.48

9.68

LTHA

No.

No.

11

5.36 ' 5.46

9.41

9.34

Amino Acid)

HTLA

ANRCi

No. .

Casein

13

" 5.32

FAO

' -

'

5.02

Pattern

V. -

Reqd by the
Growing Rat

'28

"

452'

9.26

9.65

4.8

7.0

lsoleucine

Column

575

6.06

6.05

5.88

5.70

5.80

4.85

4.2

5.5 '

Tyrosine
Histidine

Column
Column

6.39
2.51

4.59
3.20

4.85
2.67

4.43
2.80

4.42
2.54

4.74
2.38

5.55
3.20

2.8
X

3.0
2.5

"The amino acid contents were determined by WARF. Inc. by the amino acid analyzer (column), by microbiological assay (mba). and by USP XVlll, 947 (1970)

(Available).
mMalt Sprout Extract - See footnote No. l of Table 111.

'Amino Acid analysis of ANRC casein.

u'FAO Committee on Protein Requirements (1957 b) "FAO Nutritional Studies" No. 16.
'RB. Ramo Rao, H.W. Norton, and B. Connor Johnson. J. Nutrition. 82. p 88 ( 1964).

chloric or phosphoric acid to pH about 4.0 to about pH

What is claimed is:

5.0 at a temperature between about 50C. and about

1. A process for producing a yeast protein product
20
comprising the steps of:
80C.
a. Rupturing yeast cells,
13. The process according to claim 12 wherein the

precipitated protein is recovered by centrifugation at

b. Separating a solubles fraction containing the nu

cleic acid and protein from an insoluble cell wall

debris fraction,
0. Hydrolyzing the nucleic acid with alkali at a pH of
about 9.5 to about 12.5 and a temperature of about
50C. to about 120C. for less than 4 hours,

pH about 4.0 to about 5.0 at a temperature between

25 about 50C. and about 80C.

14. The process of claim 13 wherein the recovered

yeast protein contains less than about 5%.RNA.

15. The process of claim 13 wherein the recovered

yeast protein contains less than about 3% RNA.
a pH of about 2 to about 6 and a temperature of
16. The process according to claim 1 wherein the
30
-l00C., and
precipitated protein is recovered by centrifugation at
e. Separating the insoluble protein produced from
pH about 4.0 to about 5.9 at a temperature between
the nucleic acid containing solubles part.
about 0 and about 100C.

d. Precipitating the protein by the addition of acid to

2. The process of claim 1 wherein the yeast cells are

ruptured by homogenization below about 50C.

3. The process of claim 1 wherein the ruptured yeast

35

17. A process according to claim 16 wherein the re

covered protein is concentrated and deflavored in
vacuo.

cells are extracted at a pH between about 5.5 and about

11 and a temperature between about 25C. and about
60C. for about 5 to about 60 minutes.

'

>

18. A process accordingto claim 17 in which the

food grade yeast is selected from the strain of Saccharo

myces cerevisiae. and Candida utilis.

4. The process of claim 3 wherein the ruptured yeast
19. The process of claim 1 including the step of re
cells are extracted at pH 9.5 at a temperature between 40
covering
a yeast protein product comprising:
25C. and 60C. for 5 to 60 minutes.
about 65% to about 85% protein;
5. The process of claim 3 wherein the ruptured yeast
about 0.5% to about 9% RNA;
. . '
cells are extractedat pH 9.5, 60C., for 30 minutes.
about
7%
to
about
15%;
lipid;
about
1
to
about
5%
1 6. The processrof claim 1 wherein the insoluble cell. 45

wall material is separated by centrifugation.

ash; about 5 to about 20% carbohydrate; and about

7. The process of claim 1 wherein the solubles frac

tion after the separation of the cell wall debris is treated
with alkali at a pH between about 9.5 and about 12.5,
a temperature between about 50C. and about 120C.,

0% to about 1% ?ber.

for up to 4 hours.

'

>

'

20. A process comprising the steps of:

a. Growing a food grade yeast on nutrient media,
50

8. The process of claim 7 wherein the solubles frac

tion is treated with alkali at a pH of 10.0 to 10.5, a tem
perature of 7585C., for a time of 1 to 4 hours.
9. A process according to claim 7 wherein the solu
55
bles fraction is treated with alkali at a pH of 1 l.5-12.5,
a temperature of 55-65C., for a time of l to 2 hours.
10. A process according to claim 7 wherein the solu
bles fraction is treated with alkali at pH 9.5 at
l l5l20C. for not greater than 1 hour.
11. A process according to claim 1 wherein the pro 60

tein product is precipitated by the addition of acid to

a pH between about 3.5 and about 5.5, at a tempera
ture between about 5C. and about 90C.
12. A process according to claim 11 wherein the pro

tein product is precipitated by the addition of hydro

b. Harvesting and washing the yeast cells,

c. Rupturing the yeast cells at a temperature between
0 and 50C.,

'

d. Holding the ruptured cells at a pH between about

7.0 and about 11.0 at a temperature not greater
than about 70C. for a time not greater than about

5 minutes,
e. Removing the yeast insolubles at a temperature not .

greater than about 75C.,

f. incubating the yeast solubles'at a temperature of
about 80C. and at a pH between about 10.0 and
about 10.5 for a time sufficient to decrease the nu
cleic acid content of the harvested protein, 2

g. Precipitating the protein by the addition of a food

grade acid to pH about 4.0 to pH about 5.0, and
h. Removing the yeast protein at a temperature be
tween about 0C. and about 80C.
*