AMMONIATED LACTIC ACID FERMENTATION
OF FRUIT WASTE SUGARS

Thesis for the Degree of M. S.

MICHIGAN STATE UNIVERSITY

KEITH WILLIAM SINGLETARY
1977

 

 

LIBRA R Y
N'Chigan State ‘
Unwcmty 4" g

ABSTRACT

AMMONIATED LACTIC ACID FERMENTATION
OF FRUIT WASTE SUGARS

BY

Keith William Singletary

Experiments were conducted to determine the feasibility of converting
concentrated pineapple mill waste into a nitrogen-enriched feedstuff for
ruminant animls by means of an amnoniated lactic acid fermentation. Com-
mercially canned pineapple Juice was diluted to sinnlate the waste. Hano-
lactic fermentat ions were conducted with 2—liter batches at constant pH and
temperature (45°C) by use of Lactobacillus delbrueckii NRRL 13—445. The
lactic acid produced was continuously neutralized by ammonium hydroxide.
The maximal sugar conversion rate was highest (9.9 mg/ml h) with yeast
extract (YE) supplementation (10.0 mg/ml) and at pH 6.0, although lactic
acid yields were as great (83-85%) at pH 5.5 as at pH 6.0. Contamination
was less of a problem at pH 5.5, but sterilization of the medium was still
necessary. With the YE supplementation, lactic acid yields increased to
over 80% during fennentations at pH 5.0 and greater. As the initial sugar
level was increased to 55.0 ng/ml, the maximal sugar conversion rate in-
creased in proportion, but remained essentially constant at higher sugar
levels. However, lactic acid yields retained maximal at initial sugar
concentrations up to 116.0 ng/ml. Altogether the results indicated that
the fennentation might be feasible if conducted at pH 5.5 with a sterilized,

supplemented medium containing a high content of fennentable sugar.

AMMONIATED LACTIC ACID FERMENTATION
OF FRUIT WASTE SUGARS

By
Keith WilliaHISingletary

A THESIS
Eiflnfitted to
Michigan.State University
in partial fulfilhment of the requirements
for the degree of
MASTER.OF SCIENCE
Department of MicrObiology and Public Health

1977

ACKNOWLEDGMENTS
I wish to thank Dr. Philipp Gerhardt for his patient

encouragement and for his example of thoroughness and

excellence.

ii

U.

Ike

“

~L

TABLE OF CONTENTS

LISI‘ OF TABLES . .

LISI‘ OF FIGURES

1.

2.

5.

IN'I'FUDUCI'ICN

HISRRICAL BACKHIJND. . . . .....
2.1 The World Food Problen and Ruminants . . .

2.2
2.3
2.4

Anmcnium Salts as a Feed Supplenent for Biminants.

Fruit Waste Utilization
lactic Acid Ferment at ion . .

MATERIAISANDMEI‘IUJS. . . .....

seesaw?
\lmCflhP-OONH

Bacterial Culture

Fermentation Media . . .

Fermentation Equipment and Procedure .
Sanple Preparation ...... . . .
Carbohydrate Determination .

lactic Acid Determination . .
Total Nitrogen Determination . .

RESULTS..

1359:5ka
01.5me

4.6

Typical Fermentation .

Organism Selection .

Effect of pH. . . .
Effect of Yeast Extract Addition . .
Effect of Initial Sugar Concentration
CulturePurity.

DISZUSSICN

BImIocmmY .

iii

Page

iv

LI ST OF TABLES

Table

1. Material balance data for a fermentation of simulated
pineapple mill waste supplenented with yeast extract .

2. Effect of pH and yeast extract on the yield of lactic acid
during fennentation of pineapple juice by L. delbrueckii .

3. Lactic acid yield and sugar utilization at various initial

sugar concentrations during ferment at ion of pineapple
juice by L. delbrueckii

iv

Page

19

25

Figure

LIST OF FIGURES

Change in total carbohydrate (O), lactic acid
(A), and total nitrogen (9) levels during fer-
mentation by L. delbrueckii of pineapple juice at
pH 6.0 and with yeast extract added (10.0 mg/ml) . .

 

Gas chrcuatcgram of initial substrate (bottcm) and
final product (top) fran a fermentation conducted
according to procedures as described in Materials
and Methods; attenuat ions = 16 and 32, respectively.
Synbols: 1, background; 2, unknowns; 3, malic acid;
4, lactic acid; 5, citric acid .......... .

Sugar utilization by L. delbrueckii NRRL B—445 in
Media A (O), B (O), and C (A) at pH 5.5 (refer to
conditions as described in Materials and Methods)

 

Sugar utilization by L. lactis ATCII 4947 in Media A
(G), B (0), and C (A) at pH 5.5 (refer to condi—
tions as described in Materials and Methods) . .

Sugar utilization by L. plantarum strain 3070 in
Media A (O), B (0), and C (A) at pH 5.5 (refer to
conditions as described in Materials and Methods)

Effect of pH on the maximal rate of sugar fermentation
by L. delbrueckii. The fermentation medium contained

 

10.0mgyeast extract and 55.-0 65. 0mg. initial
sugar/ml...... .......... .

Effect of yeast extract addition on utilization of
pineapple juice sugars by L. delbrueckii . Residual
sugar levels after 25 - 30 h of fermentation were
evaluated in media with 10.0 ng yeast extract/m1
(A) and without yeast extract (0). The initial
sugar level was about 60.0 mg/ml . . . . ......

 

Effect of yeast extract addition on the course of a

typical fermentation of pineapple juice at a constant

pH of 6. 0. Symbols. (0) 10. 0 ng yeast extract/ml,
(A) no yeast extract added . . .

Page

17

18

20

21

27

Figure Page

9. Effect of initial sugar level on maximal rate of
agar f ennent at ion by L. delbrueckii . Ferment at ions
were conducted at a constant pH of 6.5 and supple-
mented with yeast extract (10.0mg/m1) . . . . . . . . . 29

vi

1 . INTRODUCTION

Waste utilization in the fruit processing industry has received
increased attention due to imposition of governmental pollution limita-
tions. In the Hawaiian pineapple canning industry, for example, although
efforts 1mve been made to maximally utilize by-products, a large volume
of liquid waste still is channeled into municipal sewers for ocean
outfall and subsequent environmental shock (17, 25, 47). Of the
4.4 million tons of pineapple produced in the world in 1973, about 50%
was destined for processing (5). In Hawaii alone an estimated 800,000
tons of fresh pineapples were canned in 1971 (30). Asaresult, in a
single Hawaiian plant an average of 527 gallons of waste—water with high
biological oxygen demand (ED) are generated per ton of pineapples
processed (12). The total flow of waste per day in a large cannery dur—
ing processing season can exceed 2. 5 million gallons of high-strength
effluent, having a E1) of about 27,000 lbs. (F. Aona. Chief, City and
County of Honolulu, Department of Public Works, Division of Sewers. 1976.
Personal carmmication. ). The sugar level in the composite liquid waste
is fairly low (5.0 mg/ml); but tm by-products in particular, centrifuge
underflow and mill screening fines, are high in carbohydrates and account
for mnst of the H1). Several million pounds of each of these acidic,
semisolid wastes are discarded yearly (61). Should a practical and
inexpensive use for these concentrated wastes be found, pollution caused

by the composite mill waste could be significantly decreased.

2

Rather than reducing the HI) of pineapple composite mill waste by
standard water-treatment techniques , a bacterial fenmentation to produce
a ruminant animal feedstuff from the concentrated waste might be exploited.
Extensive prior work (43, 67, 74) on the conversion of whey, a waste
product from cheesemking, to a ruminant feedstuff can be used as a model.
In the batch process, whey lactose is converted by Lactobacillus bulgari—
cus almost carpletely to lactic acid, which is continuously neutralized
with ammonia (67). The fermentation can be conducted without putrefaction
at constant pH (5.5) and temperature (43°C). 0f the 55% crude protein in
the product, 75-77% is contributed by ammonium lactate, 17% by residual
whey proteins, and 6—8% by bacterial cells. The process sequesters am—
monia nitrogen in a form that is nontoxic, stable, and nutritious for
ruminant consurptiom. Extensive feeding trials have shown that ammonium
salts of lactic, propionic, and butyric acids are equivalent to soybean
meal as a nutritious feed ingredient and superior to urea as a cannercial
nonprotein nitrogen (NPN) supplement (2) . Since the cost of chemically
synthesized ammonium salts prohibits large-scale use, their alternate
production by fermentation of waste substrates has attracted attention.

Because of the increasing demand for animal feeds that are non-
competitive with human food, as well as the pressure to reduce industrial
pollution, I undertook to determine the feasibility of converting fruit
wastes, as exemplified by pineapple mill waste, into a ruminant fewstuff.
The United States pineapple canning industry is concentrated in one area,
Hawaii. Because Hawaii has a limited agricultural economy, recycling of
waste from the pineapple industry into a feed for the cattle industry has
special significance. In developing countries, such as the Philippines,

India and Malaysia, competition for food between Moms and animals is

3

more apparent. Recycling of the waste from their expanding pineapple
canning industries to a cattle feed would be highly desirable.

To determine the feasibility of this conversion, I examined the
applicability of batch fermenting the sugars in pineapple juice into
ammonium lactate, at constant pH and temperature. Several lactobacilli
were carpared for fermentative efficiency, and the need for medium steri—
lization was considered. The effects of adding yeast extract and of
changing pH and initial sugar concentration were studied with respect to
maximal sugar conversion rate , lactic acid yield, and residual sugar

concentrat ion .

2 . HI STORICAL BACKGROUND

2.1 The World Food Problem and Ruminants

With the world population expected to reach over 6 billion by the
year 2000, enhanced food production as well as efficient utilization of
present resources are essential. In the developing countries, where 86%
of the population growth is occurring (80), grain is the staple diet of
a majority of the people and can not be diverted for animal feed. This
is not so in the U.S. where cattle consumption of grain is not presently
campetitive with human requirements (21). The situation in the U.S.
could change , however , should food shortages develop .

On a world level, the outstanding human food need is for high quality
protein to supplement grain diets. About two thirds of the world's people
consule food that is deficient in energy , protein and other essential
nutrients (76). The world ruminant population, which could make a major
contribution in meeting this protein deficit, is far from meager, however.
For every 3 people there are 2 ruminants , 60% of which exist in developing
countries (21) . A partial solution lies in increasing the productivity of
the present ruminant population without diminishing food suitable for
human consumption.

Compared to other animals ruminants are unique in their ability to
consume cellulosic materials that are not efficiently digested by humans.
Not only can ruminants scavenge on land unsuitable for crop product ion,
but they also can digest many cellulosic agricultural and industrial by-:
products that are presently destined for disposal (21) . Materials that

have been studied with respect to ruminant nutrition include citrus pulp ,

4

5

came molasses, newsprint, pineapple bran, and feedlot waste (16, 22, 34,
55, 59, 60, 63, 70).

The unique fermentative capacity of the ruren allows not only for
digestion of cellulose wastes, but also for the conversion of nonprotein
nitrogen to microbial protein, which is then assimilated by the animal for
host-protein needs. A lack of protein is frequently the major factor
limiting the use of by—products for feeding (59). Replacing portions of
a more expensive protein feed supplement with NPN still allows for adequate

ruminant performance (15) .

2.2 Ammonium Salts as a Feed Supplement for Runinants

In 1949 loosli et al. (51) reported that large quantities of essen—
tial amino acids were produced with a ruminant diet containing urea as the
sole nitrogen source. Since then , extensive investigations have been con—
ducted on the efficacy of substituting nonprotein nitrogen for protein
in ruminant rations. Belasco (8) evaluated an array of nonprotein
nitrogen compounds as substitutes for urea. He observed that inorganic
and organic ammonium salts, especially ammonium succinate and lactate,
exhibited higher rates of nitrogen utilization than urea. Diets with arm--
monium salts or urea as the only nitrogen source have been sufficient to
meet requirements not only for ruminant maintenance metabolism but also
for adequate milk production (78), although growth was significantly
reduced (62) . Subsequent studies have indicated that amonium salts of
acetic , propionic , lactic and butyric acids were superior to urea and
comparable to soybean meal as a supplerental nitrogen source in cattle
rations (2, 26, 35, 77). The expense of chemically synthesized organic
ammonium salts makes large—scale use impractical (35). However, produc-

tion of these salts by neutralization of waste fermentations with ammonia

6

provides a practical alternative (26). A successful example of this is
the nonaseptic, harolactic fermentation of whey into ammonium lactate (67 ).
The product is superior to urea and equal to soybean meal as a protein

supplement (36) . The application of this process to other sugary wastes
appeared worth consideration , since tentative costs and supplies of pro—

tein additives may make the use of alternate NPN feedstuffs essential .

2.3 Fruit Waste Utilization

 

Most studies on solid and liquid fruit wastes have dealt with treat-
ment rather than utilization. Liquid wastes from the fruit canning in-
dustry generally have 10 times stronger HID values than darestic effluent
(4). Over 60% of all cannery wastes are presently being treated in
municipal facilities to reduce HI) (49) . Rural operations, however, have
had to implement their own treatment measures. Methods for reducing KI)
have been numerous, including activated sludge treatment (23, 27, 52),
anaerobic digestion (9), lagooning (7, 27, 64), spray irrigation (3, 32,
38), and composting (42, 46, 71). Much work has been done on the carplete
treatment and utilization of the trerendous volume of citrus waste gen-
erated annually by the Florida Citrus industry. Besides the use of the
aforementioned standard techniques ( 3, 18, 23) , citrus by-products such
as wet and dried pulp, seed meal, and citrus molasses also have been suc-
cessfully used in animal rations (ll, 16, 45).

Few practical processes for fruit waste utilization have been devel-
oped and successfully implemented. Economical utilization of processing
wastes depends on lower utilization costs , demand for the recovered
product , and minimal investment for research and developrent (49) . A1-
cohol and yeast production from pear wastes (l, 29, 81) and lactic acid

and vinegar production from citrus by-products have been attexpted (41,

7

54, 58). Using a lactobacillus selected from citrus peel juice, Kagan
et a1. (41) achieved over 90% conversion of sugar to lactic acid. The
fermentation lasted 6 days and required high levels of nitrogenous nutri—
ents for maximum yield. Although it was predicted that over 1.5 x 108
lbs. of crude lactic acid could be produced from Florida's annual citrus
waste, industrial application apparently has not occurred.

The corplete use of all pineapple waste generated has been less ex-
tensive than that for citrus wastes. The processing of pineapple for
reroval of edible flesh uses about 60 to 70% of the whole fruit. Use of
the retaining residue varies considerably depending on the area of culti-
vation. In Southeast Asia and India, reuse has been negligible (40, 72),
whereas in Hawaii extensive efforts have been made toward by-product re—
covery. Methods presently in use in developing countries include vinegar
and alcohol fermentation, and silage production (20, 33, 40, 68, 72, 73).

Wastes from large Hawaiian canneries are basically of two types.

The solid waste consists of the outer shell and core of the fruit which,
after mill juice extraction and syrup recovery (28), are ground and dried
for use as a ruminant feed called pineapple bran. Since initial produc-
tion in 1923, the bran has been a successful livestock feed in Hawaii and
has proven economical because of the relatively high cost to import com-
parable feedstuffs (37). Although the bran is high in total carbohydrates,
it is low in crude protein (4.3%) so that supplerental nitrogen must be
added for effective use (19, 37). In contrast to Hawaii, the Australian
pineapple industry generates wet sol id waste which cannot be economically
dried to a bran. Instead, the residue, supplerented with protein, is fed

in a wet form (48).

8

The other major waste from pineapple canneries is liquid effluent ,
amounting to about 15 million gallons per day during peak processing
season. About 80% of the waste is cooling water which is directly dis—
charged to nearby streams. The reraining 20% consists of corbined waste
from trimming, slicing, packing and juice centrifuge flows, and is higher
in ED. This composite pineapple mill waste is channeled to municipal
sewers and then pumped to the ocean. Although acute marine pollution
was recognized as early as 1944, little work was conducted to alleviate
the problem (47 ). Investigations to date have dealt with treatment by
activated sludge as well as anaerobic digestion (12, 47, 53) . Treatment
by activated sludge was found to be inapplicable on an industrial scale.
The anaerobic digest ion method reduces the chemical oxygen derand (01))
by 66% only under carefully controlled conditions (53) . Centrifuge
underflow, the major HID contributor to the corposite mill effluent, has
shown promise as a valuable ruminant feedstuff (61). Due to its low pro-
tein content , it must be used in conjunction with a protein supplement.

The ammonia-neutral ized lactic acid fermentation would not be appl i-
cable with pineapple mill effluent due to the low sugar content (5.0
mg/ml) of the waste. However, the fermentation could be used with a more
concentrated residue such as the centrifuge underf low or the sol id waste
before being dried as bran. Both wastes are low in protein, and even a
partial conversion of the carbohydrate to ammonium lactate and bacterial
cells could significantly elevate the crude protein content . Enhancing
the use of centrifuge underf low, which is presently being discarded,

would markedly lower the 3]) of the composite liquid effluent.

2.4 Lactic Acid Fermentation

Sugar fermentations to produce lactic acid have been utilized since
1881. Whor substrates containing sucrose or glucose have been used, the
Intofermentative Lactobacillus delbrueckii has been the organism of
choice. In lactose fermentations, for sample the production of lactic
acid from whey, L. bulgaricus comonly has been used. Commercial fer-
mentations have required only limited asepsis, because possible contami-
nants are inhibited by the relatively extreme growth conditions tolerated
by the aciduric, thermoduric lactobacilli (14).

Several factors limit the fermentation. The concentration of un-
dissociated acid has been identified as a limiting factor in the lactic
acid fermentation with Streptococcus lactic (69). A parallel effect was
noted during a continuous lactic acid fermentation of whey using L. bul-
garicua . The undissociated acid that accumulated permitted the fermenta-
tion to be free of contamination at a pH higher than that which would
normally restrict competing organisms (43). The reroval of lactate during
growth of L. delbrueckii in a dialysis culture system enhanced acid
production and maximum cell concentration (31) .

Maintenance of a controlled pH has also been shown to increase acid
production (50). Not only lactic acid yield and rate of production, but
also optimal levels of required growth supplements have been affected by
variations in pH (44).

The lactobacilli are also noted for having fastidious nutritional
requirements. Vitamins, amino acids and substantial amounts of manganese,
potassium, and phosplate ions are frequently necessary for optimum growth
and acid production (65, 75).

3. MATERIALS AND METHODS

3.1 Bacterial Culture

Lactobacillus delbrueckii NRRL B—445 (Northern Regional Research
Laboratory, Peoria, Ill.) was used throughout the study. This organism
was selected as a result of several fermentation trials using L. del-
brueckii NRRL B-445, L. lactis A'III? 4947 and L. plantarum strain 3070.
L. delbrueckii is generally the organism of choice in glucose or sucrose
fermentations. Cultures for inoculation were transferred daily into
tubes of diluted comercial pineapple juice.

Cell counts were conducted with a Petroff Hausser Bacteria Counter
(C. A. Hausser 8: Son, Philadelphia, Pa. ). After appropriate dilution of
dense cultures, cells in 15—20 squares were counted and cells/ml was

calculated .

3 . 2 Fermentation Media

 

Concentrated pineapple mill waste was simulated by the use of canned,
unsweetened, Spartan brand Hawaiian pineapple juice (distributed by
Spartan Stores, Inc. , Grand Rapids, Mich. ). The juice, which contained
about 120.0 mg sugar/ml, was diluted with distilled water to the appro-
priate initial sugar concentration called for by each fermentation. The
pH was adjusted to the appropriate initial value using ammonium hydroxide
(28.8%) and/or hydrochloric acid (6N). Yeast extract (Technical grade;
Difco laboratories, Detroit, Mich.) was added (10.0 mg/ml), as needed,

. before autoclaving. lower levels of yeast extract (1.0 mg/ml) have typi-

cally been used in lactic acid fermentations. The 10.0 mg/ml concentration

10

11

was chosen in this study so that yeast extract would not be limiting with
respect to initial sugar levels.

All media used during organism selection consisted of 1:2 diluted
pineapple juice at pH 5.5. The media were not autoclaved before inocula-
tion. One medium (Medium A) was not supplerented. Medium B was supple—
mented with 5.0 g yeast extract/l. Medium C was supplemented with the
follOWing (g/l): MgSO4'7HzO (0.6), FeSO4°7H20 (0.03), MnSO4-H20 (0.03),
KHZPO4 (0.5), K2HPO4 (0.5), yeast extract (5.0), and trypticase (5.0)
(M, Cockeysville, Md.).

During organism selection trials, each inoculum consisted of cells
harvested from 200 ml of a 12 h culture grown in trypticase soy broth
(BBL, Cockeysville, Md.) with 1.0 mg yeast extract and 1.0 mg glucose/ml
added.

The medium used for routine culture transfer contained pineapple
juice that was diluted 1:2.4 with distilled water, adjusted to pH 6.0

with ammonium hydroxide, and autoclaved.

3 . 3 Fermentation Egriprent and Procedure

 

Fermentations were conducted with 2 liters of medium in a 5-1iter
bench-top fermentor equipped with autorat ic terperature control (Micro-
ferm Model MF 105, New Brunswick Scientific Co. , New Brunswick, N.J.)
and pH control (Model pH-22, and also Model pH—40, New Brunswick
Scientific Co. , New Brunswick, N.J.).

A 5% inoculum of cells grown 12-15 h at 44°C in unsupplerented
1:2.4 diluted pineapple juice was used. Each batch of medium was auto-
claved in the fermentation assembly at 114°C, 12 1b pressure for about
20 min. Other than this initial sterilization, precautions were not

taken to maintain asepsis. Before inoculation the fermentation medium

12

was equilibrated at a temperature of 45°C. A thermometer was used to
regularly check the terperature. The unadjusted pH of the pineapple juice
was usually 3.7, so ammonium hydroxide was added to elevate the pH to the
appropriate initial level . The pH of fermentation samples was periodically
checked against a pH meter (Beckman Zeroratic II, Beckman Instruments,
Inc. , Fullerton, Calif. ). A nitrogen blanket was maintained over the
medium throughout the fermentation and agitation was constant at 400 rpm.

For each fermentation during organism selection, the pH was main—
tained at 5.5 and the temperature at 45°C (except for L. plantamm, which
required 37°C).

With all fermentations, 10 ml samples were removed at regular in—
tervals and stored at -18°C for later analyses. Fermentations were

generally terminated after 25—30 h.

3 . 4 Sample Preparation

 

For analyses, fermentation samples were first centrifuged for
0.5-1.0 h at 48,000 X g in order to rerove bacterial cells and plant
material that could interfere with the assays. Analyses (except for

total nitrogen) then were conducted with the supernatant fluid.

3 . 5 Carbohydrate Determination

 

Total carbohydrate (predominantly sucrose , but some glucose and
fructose in the pineapple juice) was determined by the anthrone method
of Morris (57) . Samples were diluted to contain 50-300 pg equivalents
ofglucose. Toalmlportion ina25mmtest tubewasadded3mlof
distilled water, followed by rapid addition of anthrone reagent and
mixing. Anthrone reagent was made by dissolving 2 g of anthrone crys-

tals (The Matheson Co. , Inc., Norwood, Ohio) in 1 liter of 95% sulfuric

13

acid. After standing at room temperature for 15 min, the tubes were
assayed in a Spectronic 20 spectrophotometer (Bausch 8; Lamb, Inc. ,
Rochester, N.Y.) at a wavelength of 540 nm. A standard curve was made
by use of a series of tubes containing 100, 200, and 300 pg glucose/m1.
A 2 mg glucose/ml solution was diluted 20 times as needed daily for
preparation of standards. A blank and a set of standards were included
with each determination .

The method of Dubois et a1. (24) as modified by Montgomery (56) was
used only during preliminary trials to select a lactobacillus. The un-
centrifuged fermentation samples were diluted to contain 5-50 pg carbo-
hydrate/ml. A 1 mg sucrose/ml stock solution was diluted to 100 pg/ml
and then used to make 3 working standards of 10, 20 and 40 pg/ml. Then
2 ml portions of standards and diluted samples were transferred to a
20 mm test tube, followed by addition of 1 m1 phenol reagent and 5 ml
concentrated sulfuric acid. Phenol reagent was prepared by addition of
25 g phenol crystals to 500 ml distilled water. After vortexing, the
tubes were allowed to stand for l h at room temperature. The optical
density then was read at 485 mm in a Spectronic 20 spectrophotameter.

Maximum fermentation rates were determined graphically fram plots

of sugar utilization vs. time.

3 . 6 Iact ic Acid Determination

 

Lactic acid was determined by two methods. The first analysis was
the simplified gas chromatographic procedure of Carlsson (13). A 1 m1
sample was applied onto 1 m1 of packed cation-exchange resin (Dowex
50W-X8 , 100—200 mesh hydrogen form, washed in water; Bio-Rad laboratories ,

Richmn, Calif.) on glass wool in a Pasteur pipette. After the sample

14

drained through the resin, the resin was washed twice with 0.5 ml of
distilled water. All the fluid from the pipette was collected in a test
tube and a 2 p1 aliquot was analyzed in a gas chromatograph (Model 810,
hydrogen flame detector, F & M Scientific Corp. , Avondale, Pa.). The
column was 6 ft by 1/4 in 0.D. and 2 mm I.D. coiled glass (HP 810, septum
to normal FID, Supelco, Inc. , Bellefonte, Pa. ). The column was condi-
tioned overnight at 250°C and then run isothermally at 220°C. The
nitrogen carrier gas was maintained at 10 ml per min, while the hydrogen
and air pressures were 7. 5 and 11 .0 psig, respectively.

The second lactic acid assay was conducted according to the method
of Barker and Summerson as modified by Pryce (66). The centrifuged fer-
mentation sample was diluted to contain a final lactic acid concentration
of 10-200 pg/ml. A standard solution of 400 pg lithium lactate/ml was
diluted to 50, 100 and 200 pg/ml from which fractions were removed for
analysis. The remainder of this determination used the following reagents:

Copper reagent - 10.0 g sodium tungstate was dissolved in 800 m1
of distilled water. To this was added 22 m1 of 85% analytical-
reagent grade orthophosphoric acid , fol lowed by 5. 0 g copper
sulfate (CuSO4-5H20). The solution then was made to 1 liter with
distilled water.

Color reagent - 1 . 5 g p-hydroxybiphenol (p—phenylphenol) was
dissolved in 100 ml dimethylformemide.

Sulfuric acid - analyt ical-reagent grade.

To 0.2 m1 of the sample was added 3.8 m1 of copper reagent, followed
by mixing. Then 1 ml was transferred to a 25 mm pyrex tube, and 6 ml of
sulfuric acid was rapidly added by syringe. The solution was allowed to

stand 1-2 min. After 0.1 ml of color reagent was added, the tubes were

15

mixed and allowed to stand 10 min. Samples and standards were then heated
for 90 sec in a steam chamber, cooled in ice, and measured in a Spectronic
20 spectrophotometer at a wavelength of 565 nm.

The lactic acid procedure of Pryce was used routinely for quantifi-
cation, while the gas chromatographic method of Carlsson was used for

product quality determination.

3. 7 Total Nitrogen Determination

 

Total nitrogen was determined by the micro~Kjeldah1 procedure (39) .
Fermentation samples (without centrifugation) were accurately weighed
into 10 ml volumetric flasks so that 0—200 ppm nitrogen were present.
This was followed by addition of 0.4 g of Pope Kjeldahl salt mixture and
then 1 m1 of concentrated sulfuric acid, plus a boiling head. The
Kjeldahl salts consisted of a 15.0 g K2314 -— 0.7 g HgO bulk granular
mixture (Pope Testing Laboratories, Dallas, Tex. ). Samples then were
digested on a block digester as the temperature was slowly increased.
This was continued until foaming ceased and dense white fumes appeared.
Digest ion was prolonged for 15 min past clearing of the solution. The
flasks were removed and allowed to cool. Samples were then diluted with
distilled water to the calibration mark, mixed, and further cooled to
near roan terperature. The liquid level was again adjusted to the mark,
if needed, and samples were poured into screw—cap tubes for storage until
analysis with a Technicon AutoAnalyzer II (Technicon Instruments Corp. ,

Tarrytown, N.Y.) after the method of Wall and Gehrke (79).

4 . RESULTS

4. 1 Typical Fermentation

Figure 1 illustrates the changes in sugar, lactic acid and total
nitrogen during a typical lactic acid fermentation of simulated pine-
apple mill juice by L. delbrueckii NRRL B-445. The conversion was tomo-
1actic, as evidenced by gas chromatography (Figure 2) and material balance
data (Table 1). Figure 2 also indicates that malic acid in the pineapple
juice was metabolized, presumably to lactic acid. Continuous neutrali-
zation of the lactic acid with ammonia, in combination with protein syn—
thesis contributed to greater than a 4—fold increase in total N per unit
wet weight of juice. The increase in total nitrogen is actually greater
than this, because it was necessary to raise the pH of the juice from
3.7 to 6.0 with ammonium hydroxide before the fermentation could commence.
The adjustment of three factors , pH, presence of yeast extract , and ini-
tial sugar concentration, affected the rate of carbohydrate fermentation,

and the yield of lactic acid.

4 . 2 Mien Select ion

 

L. delbrueckii was selected because of fewer contamination problems.
The two themoduric lactobacilli, L. delbrueckii and L. Zactis, fermented
the enriched media to about equivalent residual levels (Figures 3 and 4).
In those fermentations exhibiting contamination, the desired lactobacillus
constituted about 80% of the cell population and lactic acid was still the
predominant product as evaluated by gas chromatography . The two thermo-
duric lactobacilli exhibited about equivalent fermentation times, 15 h and

21 h, to reduce the sugar levels in Media C and B, respectively, to
16

TOTAL SUGAR (mg/m1)

17

 

 

 

 

 

 

 

I I I I I “71‘
60 . ,
0 o
(D (D
A
50 I- O ‘A-
.2; .
B 1
£40 II f ’ "‘
G
H
0
<2 ‘1
8 ;
S
20 , ..
10 " u
o , ‘9
0 l l 1
15 20 25

 

TIME ([1)

Fig. 1. Change in total carbohydrate (O), lactic
acid (A). and total nitrogen (C) levels during
fermentation by L. delbrueckii of pineapple juice at pH
6.0 and with yeast extract added (10.0 tug/m1).

1.0

0.8

0.6

0.4

0.2

0.0

TOTAL NITROGEN (Z)

 

 

 

 

 

 

VA ——

Fig. 2. Gas chromatogram of initial substrate (bottom)
and final product (top) from a fermentation conducted accord-
ing to procedures as described in Materials and Methods;
attenuations = 16 and 32, respectively. Symbols: 1, background;
2, unknowns; 3, malic acid; 4, lactic acid; 5, citric acid.

19

Table 1. Material balance data for a fermentation of simulated pineapple
mill waste supplerented with yeast extract.

 

 

Fermentat iona A B A+B
time (h) Sugar (mg/m1) Lactic acid (mg/m1) Sum (mg/m1)b
0.0 59.2 0.00 59.2
3.5 57.6 0.0 57.6
5.5 53.1 1.6 54.
7.0 56.4 2.3 58.7
10.0 48.8 9.6 58.4
12.0 36.5 18.3 54.8
13.0 30.8 23.8 54.6
14.0 23.9 30.1 54.0
15.0 21.2 35.4 56.6
16.0 13.1 40.6 53.7
17.0 9.1 42.6 51.7
18.0 6.4 50.3 56.7
28.0 6.4 50.3 56.7

 

a’Fermentations conducted at pH 6.0 in a medium containing 60 mg sugar and
10 mg yeast extract/ml.

bMean = 56.0; standard deviation = 2.2.

CThe gas chroratogram (Fig. 2) of a 0.0 h fermentation sample indicates
the absence of lactic acid, while the Pryce lactic acid assay indi-
cates 3.3 mg/ml, which is due to high sugar concentrations causing
false lactic acid readings. Therefore, 3.3 mg/ml was subtracted from
the 0.0, 3.5, 5.5, and 7.0 h values for lactic acid.

TOTAL SUGAR (mg/ml)

20

 

>’}c

60
A
50 lfi
O
i
40 ..

 

 

 

 

 

0' .-
20 h u
- -
10 f- '-
C
‘ ' A A
A
l l l .1
0 10 20 30 40
TIME (h)

Fig. 3. Sugar utilization by L, delbrueckii NRRL B-445 in
Media A ((3), B ((3), and C (ll) at pH 5.5(refer to conditions
as described in Materials and Methods).

 

TOTAL SUGAR (mg/m1)

6O

50

40

30

20

10

21

 

 

 

 

 

C
F" q
A
.- \’ -
o‘
F’ ‘-
O
P - -
‘ .k
‘r.—___.__A_______A .
l l I I
10 20 30 40
TIME (h)

Fig. 4. Sugar utilization by L, lactis ATCC 4947 in Media

A (‘3), B (CD), and C (‘5) at pH 5.5 (refer to conditions
as described in Materials and Methods).

22

5.0-6.0 mg/ml. L. plantarum was eliminated as a possible organism because
not only did it require a longer fermentation time than the other two
lactobacilli to reach a sugar concentration of 5.0—6.0 mg/ml in Medium B
(Figure 5), hit it also had an optimum temperature of 37°C, which was

less restrictive for contaminants than was 45°C.

4.3 Effect of pH

 

A change in pH within the range of 4.5—7.5 had a pronounced effect
on the maximum rate of fermentation (Figure 6). A maximum fermentation
rate of 9.9 mg/ml h occurred at approximately pH 6.0 in supplerented
media; however, the yield of lactic acid was fairly constant at pH values
of 5.0 and above (Table 2). Extreme pH values of either 4.5 or 7.5 ef-
fect ively depressed the rate of carbohydrate utilization .

The inf luence of pH on the yield of lactic acid was greatest in
unsupplerented pineapple juice. As indicated in Table 2, an increase in
pH from 4.5 to 5.5 with this medium resulted in over a 100% increase in
lactic acid. In a medium supplexented with yeast extract a similar pH
adjustment caused only a 41% increase. The yield of lactic acid, however,
was much higher in the supplemented medium as corpared to the unsupple—
mented one. In both media the highest yield occurred at pH 5.5.

4.4 Effect of Yeast Extract Addition

 

Sugar utilization was significantly improved by the addition of yeast
extract (Figure 7). Without yeast extract, fermentations were incorplete
after 25—30 h. However, upon addition of yeast extract , residual sugar
levels below 10.0 mg/ml were obtained.

In Figure 8 is illustrated the course of a fermentation with and

without a nitrogenous supplerent . There was over a 5—fold increase in

TOTAL SUGAR (mg/ml)

23

 

60

50

40

3O

20

10

 

 

 

 

TIME (h)

Fig. 5. Sugar utilization by L, plantarum strain 3070 in
Media A (‘3), B ((>), and C ((3) at pH 5.5 (refer to conditions
as described in Materials and Methods).

24

 

 

 

 

 

 

U 41 l
8 I— d
O

A O
I: p- u
'3
a * 3
5
Z 6 P- .1
O
H
E-I
<3
E
m - -
E
[.11
In
[14
O
a: 4 " .1
[-0
a
A

2 L. .—

n- «I:
0 I l l L
4 5 6 7 8
pH

Fig. 6. Effect of pH on the maximal rate of sugar
fermentation by L. delbrueckiL. The fermentation medium
contained 10.0 mg yeast extract and 55.0 - 65.0 mg
initial sugar/m1.

25

Table 2. Effect of pH and yeast extract on the yield of lactic acid
during fermentation of pineapple juice by L. delbrueckii.

 

Lactic acid yield (%)

 

 

With Without Crude
PH yeast extracta yeast extract protein (%)b
4.5 60.0 21.6 3.5
5.0 83.5 30.3 4.6
5.5 85.0 45.4 4.8
6.0 83.7 33.5 5.1
6.5 82.1 31.0 4.7
7.0 80.4 -- 5.4

 

aMedium contained about 60.0 mg initial agar and 10.0 mg yeast
extract/m1.

bCalculated after completion of the yeast extract supplemented

ferment at ion .

 

fl, I, fll
50 F- uni
40 '- "‘

 

S
B
\
on
5 30 T- 1‘ -
a
O
:2
a)
S!
:3
c:
5&3
10 I- d

 

 

pH

Fig. 7. Effect of yeast extract addition on utilization of
pineapple juice sugars by L, delbrueckii. Residual sugar levels
after 25 - 30 h of fermentation were evaluated in media with

10.0 mg yeast extract/ml (150 and without yeast extract ((3).
The initial sugar level was about 60.0 mg/ml.

 

TOTAL SUGAR (mg/m1)

27

 

 

 

 

 

I I I I I
60 ."" o '-
‘ .1 ~o
A . , A
50 .. o q
a ‘3 [L
40 - ..
A
30 - ._
e
20 - . ..
10 - - ..,
w s
0 1 1 l J 1
0 5 10 15 20 25
TIME (h)

Fig. 8. Effect of yeast extract addition on the course
of a typical fermentation of pineapple juice at a constant pH
of 6.0. Symbols: (0) 10.0 mg yeast extract/m1, (A) no
yeast extract added.

28

the rate of fermentation in supplemented pineapple juice as corpared to
the unsupplerented medium. This of course caused a significant reduction
in fermentation time in the enriched medium to 16 h.

A comparison of Figures 3 and 8 indicated that L. delbrueckii more
efficiently utilized unsupplemented pineapple juice during organism
selection trials than in later fermentations. The difference in inocula
and operating pH between the two fermentations would not be expected to
cause such a change in sugar utilization. The reason for the discrepancy

was not known and was not investigated further.

4.5 Effect of Initial Sugar Concentration

 

Between initial sugar levels of 5.0-55.0 mg/ml, the maximum rate of
fermentation increased in proportion to increases in sugar concentration
(Figure 9). Above initial sugar levels of 60.0 mg/ml at pH 6.5, the
maximum rate of utilization was less dependent on sugar concentration.
Fermentations conducted in media with 77.0 to 116.0 mg sugar/ml plus
yeast extract (Table 3) still produced final carbohydrate concentrations
of 5.0—10.0 mg/ml and lactic acid yields of approximately 82-85%. The
fermentation time was extended to only 19-24 h. Throughout the studies
with yeast extract supplemented media, residual sugar levels of 5.0-10.0

mg/ml were usually obtained.

4 . 6 Culture Purity

 

The initial objective of this study was to accorplish the pineapple
waste conversion in a completely nonaseptic manner. However, repeated
problems with contaminants were encountered. Although contamination was
less of a problem at pH 5.5 than at higher pH values, sterilization of all

media was still necessary.

29

 

MAXIMAL RATE OF FERMENTATION (mg/ml h)

 

 

 

0 1 l 1 1 I

 

o 20 40 60 80 100
INITIAL SUGAR (mg/ml)

Fig. 9. Effect of initial sugar level on maximal rate of
sugar fermentation by L. delbrueckii. Fermentations were
conducted at a constant pH of 6.5 and supplemented with yeast
extract (10.0 mg/ml).

 

Table 3. Lactic acid yield and sugar utilization at various initial
sugar concentrations during fermentation of pineapple

juice by L. delbrueckii.

 

 

Initial Fermentat iona Residual Lactic acid

smear (me/ml) time (h) sugar (mg/ml) yield (%)
31.0 18 5.0 77.4
56.0 17 5.0 84.9
77.0 19 6.0 83.8
97.0 20 6.0 82.9
116.0 24 9.6 81.9

 

aFermentations were conducted at pH 6.5; 10.0 mg yeast extract/ml

was added.

5 . DISCUSSION

Application of the armoniated lactic acid fermentation for the
processing of fruit wastes requires that the fermentation is operated
in an efficient and inexpensive manner and that the product is useful as
a ruminant feedstuff. In the present study the lactic acid yield, the
rate of fermentation and the residual sugar retained almost constant in
supplerented media containing 55.0—116.0 mg initial sugar/ml. This
indicates that higher concentrations of sugar might be efficiently fer-
mented. In contrast, initial lactose levels of up to only 70.0 mg/ml
can be fermented efficiently by L. bulgaricus grown at pH 5.5 in whey
(67 ). With an initial concentration of 115.0 mg lactose/ml in whey,
there remained 40.0 mg/ml after 16-24 h, indicating incomplete fermenta-
tion. Because L. delbrueckii can utilize high levels of initial sugar,
the product from the fermentation of pineapple waste might be attractive
as a ruminant feedstuff if a material high in fermentable carbohydrate
is used as a substrate. The product , due to its higher content of
armonia nitrogen, would be more suitable for use as an NPN feedstuff,
and would require less concentration to reach a nitrogen level comparable
to that in other crude protein supplements. Liquid effluent from pine—
apple canning plants is low in carbohydrate (less than 10.0 mg/ml),
which would not make a lactic fermentation worthwhile. However, if

either semisolid centrifuge underflow waste or ground pineapple shells

31

32

and cores could be partially fermented with ammonia neutralization, a
more nutritious nitrogen enriched feedstuff would be produced.

The lack of certain nutrients required for the growth of L. del-
brueckii in simulated pineapple mill waste significantly depressed lactic
acid production. An additive rich in growth factors such as yeast ex-
tract was required for efficient lactic acid production. The maximum
yield of lactic acid in yeast extract supplerented and in unsupplerented
pineapple juice was 85% and 45%, respectively, at pH 5.5. In corpari-
son, the lactic fermentation of yeast extract enriched wheat grit mash
with the same strain of L. delbrueckii gave a lactic acid yield of 80%
occurring at pH 4.6 (44). The optimum yield in unsrpplemented mash was
70% at pH 6.0. Supplerentation permitted satisfactory yields of lactic
acid at low pH, which was not true in the present investigation, where
optimum yields occurred at pH 5.5 regardless of nutrient addition.
locating a readily available and less expensive growth supplement than
yeast extract is a limiting factor in practical application of this fer-
mentation process. During fermentation of ground pineapple shells or
cull fruit, some, but not all, of these nutrient requirexents might be
supplied by components in the skin .

Limiting factors in the use of fermented pineapple waste as a rumi-
nant feedstuff would be the cost of ammonia and the cost of initial
sterilization. Drying the product would significantly increase the
crude protein content but would also increase the cost . For the simu-

lated pineapple waste (60.0 mg sugar/ml) fermented at pH 5.5, the final
crude protein level was 4.8%. A 10-fold concentration would yield a

33

material with over 40% crude protein. This is an appreciable amomt,
since the concentrated product from the amnoniated lactic acid fermenta-
tion of whey has 55% crude protein (67 ).

In summary, the process of crude protein enriclrrent of pineapple
mill waste by an ammoniated lactic acid fermentation might have potential
use if several conditions are met . The fermentation should be conducted
at constant low pH (5.5) and high temperature (45°C). Although the
maximal rate of sugar utilization by L. delbrueckii in supplemented
pineapple juice occurred at pH 6.0, the fermentation should be conducted
at pH 5.5, since the lactic acid yield is slightly higher and contamina-
tion is less of a problem at the lower pH. Initial sterilization may be
necessary , however. A waste with a high content of carbohydrate and
supplemented with a cheap source of organic nitrogen growth factors
should be used. Concentrating the product, if economically allowable,
would significantly enhance the total nitrogen level.

In developing countries, such as India or the Philippines, where
growth of the food processing industry coincides with depletion of
cattle feeds (6, 10), the procedure holds promise as a means of both
reducing environmental pollution and producing a protein enriched
ruminant feed suppleient. This supplerent in conjunction with a suitable
energy feed could partially substitute for more expensive protein addi—
tives, and could also improve animal productivity in areas where protein
supplerentat ion is presently inadequate .

In Hawaii, with an economy limited mainly to pineapple and sugar cane
production, the recycled pineapple waste could reduce the dependence of

the growing cattle industry on expensive imported protein supplerents.

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10.

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12.

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