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THE EFFECT OF MESQUITE EXTRACT ON THE NUTRITIONAL
QUALITIES OF SINGLE-CELL PROTEIN
by
DARIA LIAN6-KWUN WANG, B.S. in H.E.
A THESIS
IN
FOOD AND NUTRITION
Submitted to the Graduate Faculty of Texas Tech University in
Partial Fulfillment of the Requirements for
the Degree of
MASTER OF SCIENCE
IN
HOME ECONOMICS
Approved
Accepted
August, 1978
(\v-7-{^a.cB
! ^ . . -r> y<-- r
C.r - ^
ACKNOWLEDGEMENTS
I am deeply indebted to Dr. S.P. Yang and Dr D.W. Thayer fo r
the i r guidance, advice and encouragement in the development of th is
pro ject . I also want to express my appreciation to Dr. C.V. Morr
fo r his suggestions and c r i t i c i sms .
I am deeply grateful to my parents, Mr. and Mrs. Kwang-Yuan
Wang, fo r the i r encouragement and support of my education.
The research was supported by a grant from Dodge Johns
Foundation.
n
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
ABSTRACT iv
LIST OF TABLES v
LIST OF FIGURES vi
I. INTRODUCTION 1
II. EXPERIMENTAL 3
III. RESULTS AND DISCUSSION 7
REFERENCES 20
m
ABSTRACT
Cells of Brevibaoterium JM98A were grown in a medium containing
mesquite extract as the sole source of carbon. A 14 l i t e r glass
fermentor with automatic temperature and foam control was used for the
study. Viable ce l l counts, protein production, and reducing sugar in
both 1% and 0.5% mesquite extract were measured. The production of
microbial protein was most e f f i c i e n t in both mesquite extracts during
the f i r s t 24 hours of fermentation. The ce l l masses and to ta l protein
y ie lds were greater with the 0.5% mesquite extract than 1.0% extract .
The nu t r i t i ona l value of microbial cel ls grown on mesquite extract was
determined by amino acid analysis and feeding t r i a l s . The lysine
content of the microbial protein exceeded the FAO/WHO amino acid
pattern and the requirements of in fant and adult humans. The other
essential amino acids, except methionine, met the FAO/WHO amino acid
pattern and the requirements of humans at three stages of development.
Net protein u t i l i z a t i o n (NPU) measurements indicated that the b io log
ica l value of protein in the in tac t single cel ls was i n f e r i o r to that
of casein. There were no s ign i f i can t (P <0.01) differences in weight
ga in, nitrogen d i g e s t i b i l i t y or NPU of experimental rats fed diets
containing mesquite extract-grown cel ls and Trypticase Soy Broth-grown
c e l l s .
IV
LIST OF TABLES
Table Page
1. Data of production of Bvevibaotevivm JM98A grown in 1% or 0.5% mesquite extract for 72 hr 10
2. Composition of Trypticase Soy Broth, mesquite wood, mesquite ex t rac t , Brevibaoterium jn98A grown on TSB, and S. grown on mesquite extract 13
3. Amino acid contents (g/100 g protein) of Trypticase Soy Broth, mesquite wood, mesquite ex t rac t , Brsvihacterium JM98A grown on TSB, B. JM9BA grown on mesquite wood and B. JM98A grown on mesquite extract and FAO/WHO amino acid pattern and amino acid requirements of humans 14
4. Ef fect of feeding young rats for 10 days on an otherv/ise adequate but prote in- f ree d ie t or the same d ie t supplemented with 10% crude protein d ie t 17
LIST OF FIGURES
Figure Page
1. Relationships between period of growth and protein content, viable count and reducing sugar of the cells of Brevibacterivm JM98A grown in 1% mesquite extract 9
2. Relationships between period of growth and protein content, viable count and reducing sugar of the cells of Brevibaoterium JM98A grown in 0.5% mesquite extract 9
VI
CHAPTER I
INTRODUCTION
e
ma
A growing concern fo r the acute food needs of the world's
xploding populat ion, led to an examination of a var iety of unusual
t e r i a l s as potent ia l expanders of the world's food supply (M i l l e r ,
1968). Development of means fo r the large-scale production and
u t i l i z a t i o n of protein from single cel ls offers the best hope for
providing major new protein supplies independent of agr icu l tu ra l land
use.
Mesquite (Genus Prosopis) is a deep-rooted, sprouting tree with
an extensive root system that enables i t to withstand droughts, severe
competition from grasses, and adverse conditions due to prolonged
over-grazing of range and pasture lands. Mesquite has greatly
hindered the managing and caring for l ivestocks and the use of desir
able range improvement practices (Fisher, 1960). Thayer et a l . (1975),
Chang and Thayer (1975) and Thayer (1976) demonstrated the s u i t a b i l i t y
of mesquite wood as a carbon and energy source for the growth of
selected bacteria to produce a feed supplement or po ten t ia l l y a
complete feed for l ivestocks. The s ing le -ce l l protein produced from
mesquite by Brevibaoterium JM98A exceeded or equaled the amino acid
pattern recommended by FAO/WHO (1973) in seven essential amino acids
and contained more than double the amount of methionine and threonine.
Rat growth experiments showed that the s ing le -ce l l protein produced by
JM98A was acceptable and provided a f a i r l y sat is fac tory growth response
1
2
(Thayer et a l . , 1975; Yang, 1972; Yang et a l . , 1977). The use of
mesquite and/or ce l lu los ic waste materials for the growth of micro
organisms may simultaneously a l l ev ia te environmental po l lu t ion and
provide a source of nutr ients for animal production (Fu and Thayer,
1975).
Pre-treatment of cel lu lose by physical , chemical, and/or enzym
a t i c methods may lead to i t s increased d i g e s t i b i l i t y by microorganisms
(Han et a l . , 1974). Repeated studies in microbial degradation of
cel lu lose have shown that modif icat ion of cel lulose f ibers p r io r to
t he i r u t i l i z a t i o n as substrates is necessary for a successful produc
t ion of s ing le -ce l l pro te in . Cellulose may be d i rec t l y assimilated by
c e l l u l o l y t i c microorganisms, or i t may be hydrolyzed to soluble carbo
hydrates and then u t i l i z e d as substrate for s ing le-ce l l protein
production (Srinivasan and Han, 1969). Obtaining mesquite extract by
bo i l i ng mesquite wood in water is one of the pretreatments of ce l l u
lose. The highly soluble carbohydrates in mesquite extract may provide
a r ich medium for bacter ia l growth. This paper reports the results of
evaluation of protein qua l i ty of Brevibaoterium JM98A grown in a med
ium containing a water extract of mesquite as the sole source of
carbon.
CHAPTER I I
EXPERIMENTAL
The mesquite wood used in th is research was harvested in
October, 1976, and provided by the Department of Range and Wi ld l i f e
Management, Texas Tech Univers i ty . The mesquite wood was chopped to
the size of 0.5-1.0 x 3.0-4.0 cm and stored in a freezer un t i l used.
The mesquite extract was prepared by autoclaving chopped mesquite
wood in d i s t i l l e d water. The residue was separated from the extract
by f i l t r a t i o n through a viscose 200 ]im f i l t e r bag (GAF Corporation)
and a coconut charcoal column. The a l iquot was concentrated by evap
orat ion at 70°C, and then lyoph i l i zed.
Brevibaoterium JM98A was used in th is study. Cultures were
maintained on Trypticase Soy Agar (TSA), provided by Div. Becton,
Dickinson & CO. (BBL), at 4^0 and transferred monthly. The cel ls
were propagated in two 14 l i t e r New Brunswick glass fermentors with
automatic temperature and anti-foam control systems. The fo l lowing
conditions were established at the beginning of each fermentation:
100 g of mesquite extract were dissolved in 2 l i t e r s of d i s t i l l e d
water and then f i l t e r e d through Whatman GF/A f i l t e r paper (934AH) for
removing p rec ip i ta te . Eight l i t e r s of mineral sal ts medium (Thayer,
1976) with 2 l i t e r f i l t e r e d mesquite extract were autoclaved fo r 1 hr
at 121°C. The fo l lowing operating conditions were established during
propagation of the c e l l s : ag i t a t i on , 600 rpm; aerat ion, 1.0 vo l . per
vo l . o f medium per min; temperature, 35 j ^ 0.5°C and pH,6.8-7.0.
4
Hodag F-1 and SAG 471 antifoam agents were used for foam cont ro l . A
500 ml inoculum in baf f led 2,800 ml Fernbach f lask was prepared by
inoculat ing 1 ml of Brevibaoterium JM98A grown on BBL Trypticase Soy
Broth (TSB). The inoculum was incubated at 35°C, for 24 hr and ag i
tated at 250 rpm and then transferred to the vessel. The microbial
ce l ls were incubated fo r 48 hr , and then harvested by centr i fugat ion
with a Cepa-Schnell-Zentrifuge. Harvested cel ls were lyophyi l ized and
then stored at -16°C.
The cultures were sampled at 0, 12, 24, 48 and 72 hr. The
number of viable cel ls per ml of culture was determined by the pour
plate technique with f ive repl icate TSA plates per d i l u t i o n . Colonies
were counted a f te r maximum development at 30°C. The protein content
was determined by the method of Lowry et a l . (1951). Reducing sugar
was assayed with the d i n i t r o s a l i c y l i c acid reagent of M i l l e r (1960).
Brevibaoterium JM98A grown in 0.5% mesquite extract medium was also
studied for comparison. Brevibaoterium JM98A was propagated in TSB
as desdribed above. The cel ls were harvested a f te r 16 hr incubation.
Cell mass of Brevibaoterium JM98A, grown on mesquite wood, was
col lected fo r amino acid analysis. Tv/enty-eight hundred ml shaking
f lask containing 500 ml of the mineral sal ts medium, yeast extract
and 10.0 ground mesquite, and then incubated for 72 hr at 35°C and
agi tated at 250 rpm. The ce l l mass was col lected by f i l t r a t i o n through
a cheese c loth and Whatman GF/A f i be r glass f i l t e r papers and cen t r i
fugat ion , washed twice with d i s t i l l e d water, and lyoph i l i zed .
Standard procedures were followed for determining of the
chemical compositions of TSB, mesquite wood, mesquite ex t rac t , and
microbial ce l ls grown on TSB and mesquite extract (AOAC, 1975).
Moisture content was determined by drying the sample in an a i r oven
at llO^C for 24 hr. Ash was determined by inc inerat ing the samples at
650OC for 6 hr. The crude l i p i d content was determined by extract ion
wi th anhydrous ethyl ether in a Soxhlet apparatus. The crude protein
content (N x 6.25) was determined by micro-Kjeldahl. DNA and RNA
contents of the microbial ce l l were determined by the method of
Herbert and Strange (1971). The amino acid composition of the sample
was determined by a column chromatographic method using a Beckman
model 116 amino acid analyzer (Beckman, 1969), and the cystine content
was determined by the cysteic acid procedure (Moore, 1953).
Forty male albino rats of the Sprague-Dawley s t ra in were
assigned to four experimental d ie ts : p ro te in- f ree , casein, ce l l masses
grown on TSB and ce l l masses grown on mesquite ext ract . The animals
were housed ind iv idua l l y in wire-mesh cases. During the 10-day
experimental per iod, water and food were supplied ad libitum. A l l
d iets contained 10% crude protein except the prote in- f ree d ie t . The
standard basal d ie t contained the fol lowing ingredients in percent:
cerelose, 80; corn o i l , 8; vitamin-starch mixture (AOAC, 1975), 1;
Jones-Foster sal ts (Jones and Foster, 1972), 5; non n u t r i t i v e f i b e r ,
1; water, 5. A l l modif ications of the experimental diets were made
at the expense of cerelose.
A l l of the rats were sacr i f i ced by in jec t ion of 14 mg of sodium
pentobarbital in 0.5 ml of water a f te r a 10-day experimental period.
Each carcass was placed in a mason j a r , autoclaved at 121°C for 4 hr ,
and then ground with an Omni blender. Representative duplicate
samples were taken for the determination of nitrogen content by a
macro-Kjeldahl procedure (AOAC, 1975). The net protein u t i l i z a t i o n
was determined by the procedures of Bender and Doell (1975).
Feces of each r a t , accumulated during the 10-day experimental
per iod, were col lected in a 250 ml f lask containing 50 ml 20% HCl.
Af ter autoclaving for 2 hr and cooling to room temperature, the fecal
sample was poured through a sieve and made to 250 ml with d i s t i l l e d
water. Duplicate 10 ml al iquots were used fo r the nitrogen determin
a t i on .
Analysis of variance was used to test the differences of the
nu t r i t i ona l value of the experimental d ie ts . Duncan's New Mul t ip le
Range Test ( L i , 1974) was used to determine the differences among the
means of various treatments.
CHAPTER I I I
RESULTS AND DISCUSSION
In the 72-hr batch cul ture studies of Brevibaoterium JM98A,
the number of viable ce l ls increased from 0.6 x 10^ to 15.1 x 10^
colonies/ml, the protein content increased from 0.6 to 0.81 mg/ml,
and the reducing sugar decreased from 1.80 to 0.36 mg/ml in the 1%
mesquite extract medium. With the 0.5% mesquite extract presented in g
the medium, the viable ce l l count increased from 0.6 x 10 to 7.3 x 9
10 colonies/ml, the protein content in ce l ls increased from 0.04 to
0.53 mg/ml and reducing sugar in the medium decreased from 0.88 to
0.17 mg/ml. In each of these two extract concentrations, the increase
in protein level was roughly constant a f ter 48 hr of growth. The
viable ce l l count reached i t s maximum point at 48 hr of growth, and
then decreased gradually. The reducing sugar was u t i l i z e d by the
cul ture most rapid ly during the f i r s t 12 hr period (Figs. 1 and 2) .
Af ter 72 hr growth, 80% of the reducing sugar, present in the medium,
was used by the organism. The production of microbial protein was
most e f f i c i e n t in both concentrations during the f i r s t 24 hr of
fermentat ion, amounting to 0.025 g/1 per hr in the 1.0% mesquite
ex t rac t and 0.018 g/1 per hr in the 0.5% mesquite ext ract . The rates
of prote in production in 1.0 and 0.5% mesquite extract media during
the next 48 hr were 0.003 and 0.002 g/1 per hr respectively (Table 1).
Even though the protein y i e l d decreased considerably a f te r the f i r s t
24 hr of growth, a 48 hr batch fermentation was used fo r th is study
8
1.0
0.8 .,
- 0.6
CD
s
+->
o ct 0.4
0.2 --
0.0
16 -r 2.0
-14
-12
0 12 24 36
Time (hrs)
48 64 72
o
- 1.5
-4 C^ "0.5
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cr — J
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ty)
*—^
6 >" 1 — »
o
O-c o . . ^ I
1.0c2 to c
CQ
a» -s
3 U3
±0.0
Figure 1. Relationships between period of growth and protein content, viable count and reducing sugar of the cells of Brevibaoterium JM98A grown in 1% mesquite extract.
0.6 i 10
OT E
E
•̂ -cu +-> o s_
CL
0.3
0.2
- 8
« 1.0
VO
0 12 24 36 48
Time (hrs)
64 72
--0.8
«=: — J .
Ql
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X
1—' _
o
- 0.6
- 0 . 4
:z3 ro a. c cin
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^ 3
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—0.2
-LO.O
Figure 2. Relationships between period of growth and protein content, viable count and reducing sugar of the cells of Brevibaoterium JM98A grown in 0.5% mesquite extract.
10
Table 1. Data of production of Brevibaoterium JM98A grown in 1% or 0.5% mesquite extract for 72 hr.
1% Mesquite 0.5% Mesquite Extract Extract
Operating volume (1) 20.0 20.0
Protein produced per hr (g/1) 0.0113 0.0074
Protein produced at f i r s t 24 hr (g/1) 0.025 0.018
Protein produced at next 48 hr (g/1) 0.003 0.002
Reducing sugar consumed (g/1) 1.44 0.52
Protein yield per g of sugar consumed (% W/W) 0.56 0.73
Protein yield per g of cell mass (% W/W) 0.41 0.40
Yield (g/g substrate) 0.22 0.26
11
in order to obtain a larger quantity of protein. The yields of cell
mass and total protein were greater with the 0.5% mesquite extract
than the 1.0% extract. In order to obtain a larger amount of protein
from each fermentation, the 1% mesquite extract was used. Fu and
Thayer (1975) performed a similar fermentation study using mesquite
wood, while the mesquite extract was used as the sole carbon source
in the present study. At the end of 72 hr, the amount of protein
produced in 1.0% mesquite wood was the same as that in 0.5% mesquite
extract. The protein produced with 1% mesquite extract was 1.5 times
higher than that in 1% mesquite wood. The advantages of mesquite
extract as substrate would be: (1) in collecting the solubilized
wood products, (2) by increasing the soluble carbohydrate, (3) by
decreasing the cellulose and the lignin contents, (4) by eliminating
most insoluble materials such as resins, tannins, waxes, gums and
fats, (5) by simplifying the harvesting process, and (6) by purifying
the cell masses without residual cellulose. However, the yield of
converting mesquite wood to mesquite extract solids is 8.2%. The
improvement in cell production would be switched to semicontinuous
method. Fu and Thayer (1975) compared the batch method with semi-
continuous method for growing Brevibaoterium JM98A on mesquite wood.
They concluded that the protein production of the semicontinuous
culture resulted mainly from maintenance of the culture in the loga
rithmic phase of growth. The ratio of the amount of protein produced
by the batch culture was 2.97. Therefore, the semicontinuous culture
from mesquite extract may be more efficient, more economical, and less
time consuming than the batch culture.
12
Table 2 shows the approximate chemical composition of Trypticase
Soy Broth (TSB, BBL), mesquite wood, mesquite extract and Brevibaoterium
JM98A grown on TSB and mesquite extract. Comparing mesquite wood with
mesquite extract shows increased nitrogen, protein and ash contents,
and decreased lipid and carbohydrate contents in the latter. TSB
contained a higher level of nitrogen and less carbohydrate when compared
with either mesquite wood or mesquite extract. Brevibaoterium JM98A
grown on TSB had higher protein, RNA and lipid contents, and lower DNA,
ash and carbohydrate contents than B. JM98A grown on mesquite extract.
The total amino acid contents of JM98A grown on both TSB and mesquite
extract (42.4 and 35.7% respectively) reflected total protein content
more accurately. It is noticeable that crude protein levels of
Brevibaoterium JM98A grown on both TSB and mesquite extract (60.4 and
52.7% respectively) obtained from the Kjeldahl nitrogen determination
were over-estimated. Kihlberg (1972) has previously pointed out that
when a single-cell protein sample was tested in nutritional studies
at the conventional dietary nitrogen level of 1.6%, the quality of the
protein was less than 10% due to the lower true dietary protein
content.
Table 3 shows the amino acid compositions of TSB, mesquite wood,
mesquite extract and Brevibaoterium JM98A grown on TSB, mesquite wood
and mesquite extract, FAO/WHO amino acid pattern, and the amino acid
requirements of humans (FAO/WHO, 1973). Total sulfur and aromatic
amino acids in TSB were higher than in either mesquite wood or
mesquite extract. The amino acid compositions of mesquite wood and
mesquite extract were very similar. The lysine content of three cell
13
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15
masses ranged from 7.4 to 5.7% and exceeded the FAO/WHO amino acid
pattern and the requirements of infant and adult humans; only the cell
mass grown on TSB had enough lysine to meet the requirement of growing
children. The total sulfur amino acid contents of Brevibaoterium JM98A
grown on TSB far exceeded the FAO/WHO amino acid pattern and the requ
irements of humans at all three stages of development. The cell mass
grown on mesquite wood showed only marginally lower sulfur amino
acids than the FAO/WHO pattern but met the requirements of infant and
adult subjects. The sulfur amino acid content of cell mass grown on
mesquite extract was low and only met the requirement for adults. The
other essential amino acids of three cell masses met both the FAO/WHO
pattern and the requirement of infant, child and adult subjects. It
appears that changing the growing environment and the materials avail
able for amino acid production does affect the bacterium's ability to
produce needed amino acids when these are not sufficiently available
in media such as mesquite wood or mesquite extract. The great
deficit of sulfur amino acids in cell mass grown on mesquite extract
could explain the low net protein utilization observed in the rat
growth experiments. Kihlberg (1972) stated that the analysis of the
amino acid contents of microbial cells gives valuable information
about the potential nutritional value of the protein. However, the
amino acid pattern obtained after acid hydrolysis will not always
correctly reflect the pattern of the physiologically available amino
acids.
The growth data obtained from young rats fed for 10 days an
16
otherwise adequate but prote in- f ree d ie t or the same d ie t supplemented
with the 10% crude protein (N x 6.25) are summarized in Table 4. As
expected, the casein d ie t showed the highest weight gain, nitrogen
d i g e s t i b i l i t y , and net protein u t i l i z a t i o n . There were no s ign i f i can t
(P <0.01) differences in weight gain, nitrogen d i g e s t i b i l i t y or net
prote in u t i l i z a t i o n between the animals fed the d iet containing TSB-
grown ce l ls and mesquite extract-grown c e l l s , although net protein
u t i l i z a t i o n , nitrogen d i g e s t i b i l i t y , and weight gain were lower with
the mesquite extract-grown ce l l d ie t . The high fecal nitrogen
contents in both TSB and mesquite extract groups lowering the nitrogen
d i g e s t i b i l i t y indicated that the microbial ce l l wall was hard for the
rats to digest. Han and Call ihan (1974) found that there was a l inear
re la t ionship between the dietary Cellulomonas level and the fecal
nitrogen content of the ra ts . They thought that the high fecal nitrogen
content was probably due to resistance of the ce l l wall of Cellulomonas
to d igest ion. Yang et a l . (1977) studied cel ls of Pseudomonas JM127
grown on mesquite wood and indicated that the nitrogen d i g e s t i b i l i t y
and net protein u t i l i z a t i o n were s ign i f i can t l y improved a f ter the
ce l ls were mechanically homogenized. Ribbons (1968) suggested that
an i n tac t and non-digestible ce l l wall may prevent access of digestive
enzymes to the digestable cytoplasmic mater ia ls; i t may thus be
essential in some instances to break the ce l l wall of the microbial
ce l l before i t is en t i re l y susceptible to digest ion.
A high nucleic acid content may have an adverse e f fec t on
growth of ra ts . Hedenskog and Mogren (1973) showed that an increase
of the n u t r i t i v e value alone without a decrease of the content of
17
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18
nucleic acid is not sufficient in yeast. Yang (1976) studied the
Brevibaoterium JM98A grown on 2% mesquite extract. While the nucleic
acid content was decreased from 29.0 to 13.0%, the fecal nitrogen
values of the rats fed cell masses, grown on mesquite extract with
reduced nucleic acid content, were lower than those fed the cells
with normal nucleic acid content. The nitrogen digestibility and net
protein utilization were improved when the nucleic acid content in
the cell was reduced.
Rats fed the JM98A microbial protein did not reveal d.ny adverse
effect during 10-day experimental period. The acute toxicity of patho
genicity of Brevibaoterium JM98A for mice was investigated by Thayer
et al. (1975). Their results showed that the cell mass was neither
extremely toxic nor pathogenic for mice. However, the result can
neither be applied to other animals, nor implies non-toxicity in cell
mass grown from mesquite extract. Some harmful chemicals such as an
tifoam silicone and materials such as mesquite wood particles might
retard the rat's growth, but not appear in such a short period.
In summary, our studies have demonstrated that the mesquite
extract can be used as a substrate to grow Brevibaoterium JM98A.
Chemical analysis indicated a high protein content and an adequate
amount of lysine, methionine and other essential amino acids. Some
techniques may improve the efficiency of single-cell production.
Collecting cell masses by semicontinuous culture method may increase
the production of protein. In the rat growth experiment, cells
grown on either TSB or mesquite extract showed similar results in
weight gain, nitrogen digestibility and net protein utilization. To
19
improve the nu t r i t i ona l value of s ing le -ce l l p ro te in , a reduction of
nucleic ac id , rupture of ce l l walls and supplementation with methi
onine in the d ie t may be suggested.
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20
21
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