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This is to certify that the
thesis entitled

Effectiveness of Different Post Harvest Treatments and
Packaging Methods for Peeled Chestnuts

presented by
Kuo-Chun Yen

has been accepted towards fulfillment
of the requirements for the

MS degree in School of Packaging

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2105 p'JClRC/DateDue.lndd-p.1

Effectiveness of Different Post Harvest Treatments and Packaging Methods for
Peeled Chestnuts
By

Kuo-Chun Yen

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
School of Packaging

2006

ABSTRACT
Effectiveness of Different Post Harvest Treatments and Packaging Methods for
Peeled Chestnuts
By

Kuo—Chun Yen

Almost all the American chestnut trees were destroyed in the early 20th century.
Therefore, there is limited information on chestnuts grown in the US. The purpose of this
study was to evaluate the baseline level of the microbial population and quality of
chestnuts grown in the US and processed using the brulage chestnut peeler (Boema
Company, Italy). The quality of stored, pretreated (hydrochloride and chlorine dioxide
gas) and untreated fresh peeled chestnuts in different packaging material (Nylon, and PE)
under vacuum and atmospheric conditions were evaluated. Chinese chestnuts (Castanea
sativa Mill.) were harvested, peeled by the automatic peeling machine, treated with either
sodium hypochlorite or chlorine dioxide, packaged in low density polyethylene (LDPE)
and nylon/LDPE pouchs, and stored at either 4-5C and lO-lZC for 4 weeks. The texture,
moisture content, color and microbial population of the chestnuts were evaluated
periodically in storage. Packaging methods and materials had no significant effect on
extending the shelf life. Treatment with sodium hypochlorite or chlorine dioxide reduced
the initial microbial population but only extended the shelf life to 14 days when stored at
4-5C. Low temperature storage provided some additional quality preservation. A trained
sensory panel evaluated the appearance, texture, and off- flavor of the product. Sensory
scores indicated that there was no significant different between treated and untreated

samples before and after storage.

ACNOWLEDGEMENTS

First of all, I want to express my sincere gratitude to my major advisor Dr. Bruce Harte
for his guidance and advice. My deepest gratitude goes to Dr. Janice Harte for guidance,
support and help during these past years. Special thanks to Dr. Maria Rubino for serving
my committee and Dr. Dennis Fulbright, Dr. Elliot Ryser, Dr. Muhammad Siddiq for
their guidance and support. I thank the Chestnut Grower Inc. for their support through my
graduate program. Many thanks to faculty staff and graduate students in the School of

Packaging for their help and friendship. Special thanks to Bob Hurwitz.

iii

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................ v
LIST OF FIGURES ..................................................................................... vii
I. INTRODUCTION ..................................................................................... l
2. LITERATURE REVIEW .......................................................................... 3
History of chestnuts ................................................................................... 3
Nutritional value of chestnuts ................................................... . .................... 7
Problems associated with chestnuts during the post harvest period ......................... 12
Non-heat Sanitation methods ....................................................................... l3
3. MATERIALS AND METHODS ................................................................. l8
4. RESULTS & DISCUSSION .................................................... . .................. 26
Baseline levels of microorganisms ................................................................ 26
Contamination from the peeling line ............................................................. 27
Quality of frozen and thawed peeled chestnuts stored at 4 and 10C ........................ 31
Microbial populations of the chestnuts ......................................................... 37
Fresh, Peeled Chestnut Packaging .............................................. . .................. 38
Effectiveness of different sanitizer ............................................................... 45
5. CONCLUSION .................................................................................... 50
6. REFERENCES ..................................................................................... 51
7. APPENDD( .......................................................................................... 56

LIST OF TABLES

Table l. Proximate composition of raw chestnut kernels ......................................... 8
Table 2. Mineral composition of raw chestnut kernels .......................................... 8
Table 3. Amino Acid composition of raw chestnut kernels ...................................... 9
Table 4. Proximate composition of raw chestnut (Colossal) grown in Michigan ............ 10
Table 5. Amino Acid composition of raw chestnut (Colossal) grown in Michigan .......... l I
Table 6. Advantages and Disadvantages of chlorine and chlorine dioxide for

washing fresh fruits and vegetables ...................................................... 14
Table 7. Microbial populations of chestnuts stored at 2°C before peeling .................... 23
Table 8. Microbial populations on chestnuts packed in PE bags from different

locations within the chestnut peeler ..................................................... 25
Table 9. Microbial populations on chestnut from different locations within the

chestnut peeler, packed in PE bags and stored at 4C for l4days ................... 25
Table 10. Microbial populations on chestnuts from different locations within the

chestnut peeler and packed in PE and stored at 10C for l4days .................... 26
Table 11. Microbial populations on chestnuts from different locations within the

chestnut peeler and packed in Nylon bags (day 0) .................................... 26
Table 12. Microbial populations on chestnuts from different locations within the

chestnut peeler and packed in Nylon bags and stored at 4C for after 14 days ...... 27
Table 13. Microbial populations on chestnuts from different locations within the

chestnut peeler and packed in Nylon bags and stored at 10C for 14 days .......... 27
Table 14. The bacteria count (TPC) of chestnuts during storage ............................... 34
Table 15. The yeast count on chestnuts during storage .......................................... 34
Table 16. The moisture content of fresh peeled chestnuts after 14 days storage ............. 35
Table 17. The water activity of fresh peeled chestnuts after 14 days storage ................. 36
Table 18. The L-value of fresh peeled chestnuts after 14 days chestnut storage ............. 37
Table 19. The a-value of fresh peeled chestnuts after 14 days storage ........................ 38
Table 20. The b-value of flesh peeled chestnuts after 14 days storage ........................ 38

Table 21. The total plate count of fresh chestnuts stored at 10-12C after 14 days

storage .................................................................................

Table 22. The total plate count of fresh chestnuts stored at 4-5C after 14 days

Storage ................................................................................

Table 23. The total yeast counts on fresh chestnuts at 10—12C afier 14 days storage.

Table 24. The total yeast counts on fresh chestnuts stored at 4-5C after 14 days

storage ...................................................................................

Table 25. The total mold counts on fresh chestnuts stored at 10-12C after 14 days

storage ...................................................................................

Table 26. The total mold counts on fresh chestnuts stored at 4-5C after 14 days

Storage ...................................................................................

Table 27. Microbial populations on fresh chestnuts treated with different

concentrations of hypochlorite (day 0, 4°C) ........................................

Table 28. Microbial populations on fresh chestnut samples treat with different

concentrations of hypochlorite and stored at 4C and assayed afier 14 days.

Table 29. Microbial populations on fresh chestnut samples treat with different

concentrations of hypochlorite and stored at 4C and assayed after 21 days.

Table 30. The sensory results of chestnut samples after 4 days storage at 4C ............

Table 31. The sensory results of chestnut samples after 15 days storage at 4C ...........

vi

....39

....40
.40

41

41

42

44

.44

..45
....45

....46

LIST OF FIGURES

Figure 1. The American chestnut tree reigned over 200 million acres of eastern
woodlands from Maine to Florida, and from the Piedmont plateau in the
Carolinas west to the Ohio Valley, until succumbing to a lethal fungus
infestation, known asthe chestnut blight, during the first half of the 20th
century. An estimated 4 billion American chestnuts, up to 1/4 of the

hardwood tree population, grew within this ............................................ 5
Figure 2. Schematic of integrated components of brulage chestnut peeling line ........... 17
Figure 3. Flow diagram of Frozen and thawed peeled chestnut quality evaluation ........ 18

Figure 4. Flow diagram of Fresh, Peeled Chestnuts subjected to sanitizer treatments . . . .21

Figure 5. The moisture content of frozen and thawed chestnuts during storage ............ 28
Figure 6. The water activity of frozen and thawed chestnuts during storage ................ 29
Figure 7. The L-value (outer layer) of chestnuts during storage .............................. 30
Figure 8. The L-value in the interior of the chestnuts during storage ........................ 30
Figure 9. The a-value of the chestnuts exterior layer during storage ......................... 31
Figure 10. The a-value on the interior of chestnuts during storage ........................... 32
Figure 11. The b-values of the exterior surface of the chestnuts during storage ........... 33

Figure 12. The b-values of the interior of the chestnuts during storage ...................... 33

vii

l . INTRODUCTION

Before the chestnut blight devastated the American chestnut pepulation in the early 20th
century, the chestnut was a major forest component in the eastern United States. Today,
production of edible chestnuts is making a comeback. Michigan State University and
Chestnuts Grower Inc. are working together to re-establish the chestnut industry in
Michigan by producing added value pre-peeled chestnuts.

The minimum processed fruit and vegetable market has grown rapidly in the past 10
years. Retail sales in the US in 1994 were 5.8 billion (Hodge, 1995) and had reached 19
billion by 2003 (Greenleaf, 1999). Valued-added minimal processed chestnut products
have great potential for the fresh produce industry.

However, quality parameters effecting browning, growth of microorganisms and texture
are major problems for the minimal processed fruit and vegetable industry. Many
methods have been reported as beneficial to maintain quality including chemical dips
(Gomy et al., 1999; McHugh and Senesi, 2000; Dong et al., 2000), control atmosphere
packaging ( Gomy et al., 1999; Lakakul et al., 1999), modified atmosphere packaging
(Hotchkiss and Banco, 1992), high pressure processing (Boynton, 1999), and irradiation
(Prakash, 2000) are reported to reduce the number of microorganisms on fruits and
vegetables. However, there is no recent published research related to pre-peeled chestnuts.
Microbial contamination can occur during transportation, storage, and processing. Cross-
contamination usually occurs during cutting or shredding (Grag et al., 1990). Soil and
water present on the surface of the produce can also support the growth of
microorganisms, including pathogens such as L. monocytogenes (Al-Gahazali and Al-

Azawi, 1990). The automatic chestnut peeling line is the first of its kind in the US and

there is no research related to quality of chestnuts produced by this peeling machine, nor

the number of microorganisms.

The objectives of this research are:

1. Determine baseline levels of microorganisms for the chestnuts in the field prior to
harvest, during storage at 4-5C and 10-12C, just prior to peeling and post peeling prior to
freezing.

2. Determine microbial count at various points along the peeling line where the peeling
process may introduce micro flora.

3. Determine the effect of different packaging materials and closure methods, and
different storage temperatures on the shelf life of fresh peeled chestnuts.

4. Compare the efficacy of sodium hypochlorite and chlorine dioxide gas in inactivating

yeast, mold and bacteria on chestnuts.

2. Literature Review

History of Chestnuts

Chestnuts (Castanea) have played an important role in the human history of Europe, Asia,
and North American (Smith, 1953). In China, Chestnuts have been used for food and
timber since ancient times (Payne et al., 1983). The American chestnut was one of the
most important forest trees from Maine South to Florida, and from the Piedmont West to
the Ohio valley (Fig 1). In 1904 a fungus was discovered which was attacking the trees in
the Bronx Zoological Park in New York City (Merkel, 1906). It killed those chestnuts
and spread quickly. Detailed accounts of the origin, progression, and consequences of the
blight epidemic have been described in several reviews (Anagnostakis, 1987; MacDonald
and Fulbright, 1991; Fulbright, 1999). By 1950 the most plentiful tree on over nine
million acres was virtually extinct —- almost every single one of them a victim of the

blight (Anagnostakis, 1982).

Chestnut blight, or chestnut bark disease, is caused by an introduced fungus,
Cryphonecm'a parasitica (Murrill) Barr, (formerly Endothia parasitica [Murrill]
Anderson & Anderson). The fungus enters wounds, grows in and under the bark, and
eventually kills the cambium all the way around the twig, branch, or trunk (Metcalf,

1912).

The Native American chestnut was almost entirely destroyed by the chestnut blight.

Breeders have produced new cultivars of blight-resistant chestnuts by hybridizing C.

dentata with C. mollissima (Jaynes 1979) and backcrossing. Significant progress in

controlling blight has been made over the last 20 years (Griffin 1986).

Michigan is outside of the natural range of the American Chestnut. But Michigan
provides a unique opportunity for recovering populations and producing of the American
chestnut because the blight-resistant gene spread naturally in Michigan (Fulbright wt al.,
1983). Michigan farmers are just beginning to plant and grow Asian and European hybrid
varieties of chestnuts (Fulbright & Mandujano, 2000). For the North American market,
these domestically grown, in-shell chestnuts are fresher than imported nuts, and with
improved post harvest handing, increased production should be available for longer

periods.

The chestnuts industry

Current chestnut worldwide production is over 500,000 tons. China is the leading
producer with 40%, followed by Korea with 15%, Italy, and Turkey, and Japan with 10%
each. The US grows less than 1% (Olsen, 2000). The annual consumption of chestnuts in
the US is less than 0.02 Kg/person (Burnett, 1987). This low consumption rate is not due
to lack of interest in chestnuts, but to the availability of good chestnuts, and to the fact

that consumers are not familiar with chestnuts products.

 

 

Fig l. The American chestnut tree reigned over 200 million acres of eastem woodlands from Maine to
Florida, and from the Piedmont plateau in the Carolinas west to the Ohio Valley, until succumbing to a
lethal fungus infestation, known as the chestnut blight, during the first half of the 20th century. An
estimated 4 billion American chestnuts, up to 1/4 of the hardwood tree population, grew within this
range.(The American Chestnuts Foundation, http://www.acf.org/Chesmut_history.htm)

At present there is a very small U.S. chestnut (Castanea spp.) industry. Certain conditions
must be met for the industry to develop to even a modest level of production. First, trees
of high quality cultivars with resistance to chestnut blight must be made available in
quantity. It will be necessary to develop or improve mechanical harvesting, shelling, and
peeling. Since many Americans alive today have never seen a chestnut, a marketing effort
to familiarize them with this "exotic" nut and its uses will be needed. Established markets
for exported nuts exist in Japan and other countries where consumers are familiar with
the chestnut. Since removal of the "skin" (pellicle) from the kernel is too time-consuming
for most homemakers, peeled kernels should be marketed (Stebbins, 1990) to take

advantage of the demand.

The USDA Foreign Agricultural Service reported that 4,293 metric tons of chestnuts
were imported in fiscal 1986-87, and 3,441 tons in 1987-88, at a value of at 6.5-6.7
million dollars. The value of imported chestnuts was estimated to be 10 to 15 million in
2000 (Olsen, 2000). The primary exporter of chestnuts to the US. has been Italy with
about 84% of the volume, followed by China, Spain, Korea and eight other countries. In
addition, unknown quantities of dried chestnuts have been imported. Wholesale prices are
generally around $2.10 per pound ($4.62 per kg) for the larger size nuts. In Italy, nuts are
sterilized, brought here in sea containers, and fumigated using methyl bromide in port as
required by the USDA Animal and Plant Health Inspection Service (APHIS). Fresh nuts

should be refrigerated (Stebbins, 1990).

U.S. producers could displace imports and expand the US. market for in-shell chestnuts,
and a much larger tonnage could be marketed if a peeled product were made available
(Miller 1988). It is unlikely that many homemakers will want to peel chestnuts as it is a

time consuming task.

Currently, the majority of chestnuts marketed in the United States are imported from Italy
and the wholesale value is $20 million to $40 million per year. Fresh Michigan-grown
chestnuts are able to provide a better quality product for consumers than Italian imports.
Michigan and the United States in general have great potential for increased plantings of
chestnuts. With only a small increase in domestic consumption, the industry could be

worth $600 to $800 million annually (Vossen, 2000).

Nutritional value of chestnuts

Unlike most other tree nuts, chestnuts are low in protein and fat but high in carbohydrate
(McCarthy and Meredith, 1988). Nutritional data on chestnuts grown outside of the US
have been reported for Europe by Dudek and Elkin (1984) and Beaubatie (1979); for
China by Harris et a1. (1949); for Japan by Ha et al. (1982) and Rhee and Kim (1982).
McCarthy and Meredith (1988) have reported in detail the nutritional data of the
American (Castanea dentate) and Chinese chestnuts (Castanea mollissima) grown in the
US and compared this data to that of imported Italian chestnuts (Castanea sativa) (table 1,
2 and 3). Nutritional data on chestnuts grown in Michigan also have been reported (Harte,
2003) (Table 4 and 5). Chestnuts are also a source of essential fatty acids (linoleic and

linolenic acid) (Kunsch et al., 1999).

Table l. Proximate composition of raw chestnut kernels (amount in 100g, edible portion)a.
Chestnuts, raw

 

 

 

 

American Chinese European
Proximates Mean (g) SE. Mean (g) SE. Mean (g) S.E.
Water" 43.70 43.95 54.88
Protein (151885.30) 4.838 0.06 4.20f 0.01 1.988 0.00
Fat° 1.328 0.04 1.11f 0.05 1.63" 0.03
Carbohydrate, totald 48.57 49.07 40.28
Crude fiber 1.91e 0.01 1.64f 0.04 1.368 0.04
Ash 1.58f 0.02 1.678 0.00 1.238 0.00
a Duplicate determination
b One analysis
c our analyses for American chestnuts
(1 Calculated by difference

e—g Means with different superscripts differ (p<0.01)

(McCarthy and Meredith 1988)

Table 2. Mineral composition of raw chestnut kernels (amount in 100g, edible portion)a.

 

 

 

 

Chestnuts, raw
American Chinese European
Minerals Mean (g) SE. Mean (g) SE. Mean (g) S.E.
Calcium 24 0 18 2 23 1
Iron 1.52" 0.11 1.41" 0.02 0.718 0.01
Magnesium 79" 0 84" 2 32c 0
Phosphorus 96" 0 96" 2 :50c 1
Potassium 504 32 447 15 378 15
Sodium 3 0 3 0 3 0
Zinc 1.16 0.21 0.87 0.09 0.44 0.01
Copper 0.390" 0.004 0.363" 0.002 0.208d 0.003
Manganese 2.164 0.296 1.601 0.403 0.563 0.019

 

a Duplicate determination
b-d Means with different superscripts differ (p<0.01)

(McCarthy and Meredith 1988)

Table 3. Amino Acid composition of raw chestnut kernels (g of amino acid per gof nitrogen)

 

 

Amino acids American Chinese European
Tryptophan 0.059 0.062 0.059
Threonine 0.209 0.228 0. 195
Isoleucine 0.212 0.205 0.209
Leucine 0.329 0.341 0.306
Lysine 0.295 g 0.292 0.318
Methionine 0.098 0.128 0.125
Cystine 0.118 0.139 0.168
Phenylalanine 0.23 1 0.275 0.225
Tyrosine 0.174 0.194 0.152
Valine 0.285 0.282 0.289
Arginine 0.418 0.557 0.388
Histidine 0.135 0.160 0.148
Alanine 0.271 0.254 0.369
Aspartic acid 0.886 1.076 0.904
Glutamic acid 0.681 0.681 0.684
Glycine 0.249 0.232 0.286
Proline 0.235 0.206 0.270
Serine 0.228 0.23 1 0.260

 

a Mean values

(Kunsch et al., 1999)

Table 4. Proximate composition of raw chestnut (Colossal) grown in Michigan

Analysis

Calories

Calories from Total Fat

Total Fat
Saturated Fat
*Polyunsaturated Fat
*Monounsaturated Fat

Cholesterol

Sodium

Total Carbohydrate
Dietary Fiber
Sugars

Protein

Vitamin A

Vitamin A

Vitamin C

Calcium

Iron

* = Non—mandatory label declarations

gm = grams
mg = milligrams

IU = International Units
RE = Retinol Equivalents
< = Less than

10

per loogm

1—4
\l
O

l—Jt-‘Hl—‘l—‘Nrbmwl—‘AOOOl—‘ko

(voluntary)

gm
gm

gm
mg
mg
gm
gm
gm
gm
IU
RE
mg
mg
mg

(18.8%)
(52.3%)
(28.9%)

Table 5. Amino Acid composition of raw chestnut (Colossal) grown in Michigan

Fatty Acid

 

Saturated Fatty Acids

C12:
C14:
C15:
C16:
C17:
C18:
C20:
C21:
C22:
C23:
C24:

0
0
0

00000000

Lauric Acid
Myristic Acid
Pentadecylic Acid
Palmitic Acid
Margaric Acid
Stearic Acid
Arachidic Acid
Heneicosanoic Acid
Behenic Acid
Tricosanoic Acid
Lignoceric Acid

Monounsaturated Fatty Acids
C16:1

C18:
C20:

1
1

Palmitoleic Acid
Oleic Acid
Gondoic Acid

Polyunsaturated Fatty Acids

C18:

2

Linoleic Acid

C18:3 Linolenic Acid
C20:2 Eicosadienoic Acid

C20:

4

Arachidonic Acid

11

% of Total

FattyAAcids

 

l

OOOOOl—JOAOOOCD

l\) N
Odom

52.
44.
.94
.17
.28

.75
.18
.36
.18
.89
.25
.02
.51
.18
.67
.07
.44

.93
.43
.61
.89

32
93

Problems associated with chestnuts during the post harvest period

As new disease-resistant varieties of chestnut trees mature, the domestic grower will be
able to compete favorably with imported chestnuts for the fresh chestnut market. Also,
peeled fresh chestnuts add convenience and thus have the potential to be very successful
in several market categories. However, peeled flesh chestnuts have been found to have a
limited shelf life because of color change and microbial growth during storage. Dooley et
al. (1980) stored European chestnuts, in air. Fungal infection was 10-15% in the air stored
nuts.

Chestnuts have higher water activity (aw) level, moisture content and starch and lower
protein level than most other nuts (Beuchat, 1978). Consequently, they have the potential
to support the growth of a broad spectrum of fungi and bacteria, which are responsible for
significant losses during storage. Fungal colonization of the nuts may occur at any stage
flom flowering through harvest, storage, sorting or transport via damage of the outer shell.
Early studies on fimgal spoilage were published by Wright (1960) and Wells and Payne
(1975a) for Castanea mollissima, and Lanza (1950) and Riccardo (1963) for Castanea
sativa. These studies reported that Penicillium spp. were most flequently (40.7%)
isolated flom infected chestnuts. Next, in order of frequency of occurrence, were
Rhizopus, Altemaria and Aspergillus; each comprising about 17% of the total mycoflora
isolated. Washington et a]. (1997) reported that the fungi most commonly isolated flom
rotted chestnuts in Australia comprised Penicillin»: spp., Bohytis cinema, Phomopsis
castanea, F usarium spp., Mucor spp., T runcatella spp. and Cytospora spp. Overy et al.
(2003), in a market survey of Canadian grocery stores, isolated three dominant

mycotoxigenic fungal species; Penicillium cmstosum, Penicillium glabrum and

12

Penicillium discolor at flequencies of 67. 1%, 18.6% and 17.7%, respectively. In the same
study, the presence of the mycotoxins penitrem A, chaetoglobosin A and C, emodin and
ochratoxin A was confirmed in extracts prepared flom nut tissue. Thus, chestnut spoilage
by fungi not only affects the aesthetic value of the nut due to the presence of fungal
mycelium, but infected nuts may contain mycotoxins if they are not properly handled
during post harvest operations.

Contamination of the tissue also occurs while peeling the pellicle of the chestnut flom the
kernel. About 80% of the labor in processing is needed in the peeling process, which is
mostly done by hand using a knife. This results in the high cost of chestnut

confectioneries (Harris and Smith 1988).

Non-heat Sanitation methods

A. Irradiation

The application of irradiation to food has been approved by FAO/WHO for more than 50
types of food (Mertens and Knorr, 1992). The technology uses ionizing radiation for
inactivating microorganisms (Thayer, 1994). Cobalt-60, x-ray, Beta electrons, IR etc. are
common sources of Irradiation processing. Sterilization of packaging materials for aseptic
packaging by irradiation has been done for several years. The package materials that can
be used with irradiated foods are formed in the US Code of Federal Regulation title
21.1495. However, irradiation is not widely used because acceptance by the public is

limited.

l3

B. Sanitization (Chlorine, chlorine dioxide)

Chlorine is widely used in the washing and sanitizing of flesh produce and is highly

effective in reducing the bacteria and mold population on product surfaces (Beuchat,

1998). Chlorine sanitization usually can reduce bacteria populations by 1 to 2 log

(Pirovani et al., 2000; Sapers wt al., 1999; Beuchat et al., 1998). Typically, chlorine is

applied at a concentration of 200ppm or less. Some commercial products contain sodium

hypochlorite, buffer, and surfactant to provide a high concentrate of hypochlorous acid

(Tenzer, 1997; Wartanessian, 1997). Chlorine dioxide is also used as an anti-microbial

agent and has been approved for use on raw products (21CFR173.25, 2000).

Table 6. Advantages and Disadvantages of chlorine and chlorine dioxide for washing

 

 

 

 

flesh fluits and veggables.
Agent Used Level Advantages Disadvantages
(ppm)
Easy to apply Decomposed by organic
Inexpensive matter
Effective against all Reaction products may be
. microbial form hazardous
Chlorine 50200 Not affected by hot water Corrosive to metals
Easy to monitor Irritating to skin
FDA approved Activity pH-dependent
Population reductions
limited to 1-2 logs
More potent than chlorine Must be generated on-site
Activity not pH dependent Explosive at high
Fewer chlorinated reaction concentration
Chlorine products formed than Not permitted for out
1-5 with C12 fluits and vegetables
dioxide Effective against biofilms Population reductions
FDA approved limited to 1-2 logs
Residual antimicrobial
action
Less corrosive than C12 or
Ozone

 

 

 

 

l4

(Novak et al, 2003)

 

C. High Pressure processing

With increasing consumer demand for light-processed flesh-like food, several non-
thermal food processing methods have been deveIOped such as pulse electrical fields,
irradiation, and high pressure processing (HPP). HPP is a novel food preservation method
that can produce food products with improved shelf-life, nutritional quality, flavor,
texture, reduced enzymatic activity, and decreased microbial risk (Cheftel, 1995). HPP at
100-800 MP8 is commercialized in the USA, Europe and Japan for processing products
such as fruit juices, guacamole, tomato-based salsa and flesh oysters. Processing of pre-
packaged foods is typically done using a batch-type system in concert with flexible
packaging. Flexible packaging films are made by single layer or multi-layers using
coated or uncoated polymeric materials. Ideally, HPP should not affect packaging
material properties, however, recent research has showed that changes occurred in the
barrier properties of PET and EVOH films after HPP. This occurred because both PET
and EVOH showed crystallinity changes when exposed to high pressure (Lambert et al.
2000; Caner et a1, 2000).

In addition to mechanical considerations, HPP may affect sorption parameters (Matsui et
a1. 1992). This could affect the organoleptic and nutritional quality attributes of packaged
products (Gavara et al. 1996). Sorption refers to the uptake of volatile components flom a
food product to plastic materials. Sorption, including flavor scalping, can occur in foods
such as juice and fluit—flavor beverages. Sorption may cause the degradation of flavor
quality of packaged food during storage or processing which causes a reduction in shelf-
life (Fukamachi et al. 1996). Generally, polyolefins such as PE and PP absorb larger

quantities of aroma compounds than the polar polymers such as PET. It is likely that

15

enzyme activity would be reduced and the microbial population of flesh peeled chestnuts

would be lowered by HPP treatment.

D. Modified Atmosphere Packaging

A general definition of modified atmosphere packaging (MAP) is that the package
atmosphere is different flom the normal composition of air (21% Oxygen, 0.03% Carbon
dioxide, 78.97% Nitrogen)(Mathlouthi, 1994). Using this definition, several packaging
techniques can be included such as vacuum packaging, control atmosphere packaging and
modified atmosphere packaging. Vacuum packaging is the most common method of
modifying the internal packaging atmosphere. In vacuum packaging, the product is
placed in a package of low oxygen permeability, the air is evacuated and the package
sealed in the absence of any other gas. Typically, an atmosphere with elevated levels of
carbon dioxide develops as the food product and contaminating microorganisms consume
any residual oxygen and produce carbon dioxide (Devlieghere and Debevere, 2000).
Control atmosphere packaging includes systems to maintain a certain atmosphere around
the product. This technique tends to be limited to storage in the warehouse or in bulk
packs. Modified atmosphere packaging is established by gas flushing or by allowing air-
packed products to generate an internal atmosphere as a result of respiration.

Modified atmosphere packaging can extend the shelf life of food by inhibiting chemical,
enzymatic and microbial spoilage. This allows preservation of the food without heat and
chemical treatment. The gases used in most MAP applications include carbon dioxide,
oxygen and nitrogen, and different products need different combinations of gases. Carbon

dioxide is used primarily to inhibit the growth of microorganisms. Carbon dioxide

16

typically inhibits the growth of gram negative bacteria which usually grow rapidly and
produce off flavors associated with spoilage of many foods (Devlieghere and Debevere,
2000). The amount of oxygen in the package depends on the product. Oxygen is used
with gas packs of flesh meat to maintain desired color. The level of oxygen needs to be
carefully determined, because if the level is too low, it may result in anaerobic respiration
and produce off flavors. Nitrogen is an inert gas usually used as a headspace filler. Many
types of food products are currently being packed by modified atmosphere packaging,
and one has to be careful to select a suitable gas combination. Fresh peeled chestnuts can
be packed under modified atmosphere in order to decrease the microbiological risk.
Again, a suitable gas composition must be used.

Many researches have reported that the modified atmosphere packaging can inhibit the
growth of microorganisms in order to extend product shelf life. Simon et a1 (2005)
reported that modified atmosphere packaging with C02 levels of 2.5% and 02 levels of
10-20 % had a beneficial effect on sliced mushrooms at 4C. Modified atmosphere
packaging inhibited the growth of bacteria, improved slice appearance, and reduced the
incidence of bacterial blotches. No microbial spoilage was observed in packages of
blueberry stored under modified atmosphere with 6% 02 and 4% C02 at 4C after 12

weeks (Day et al, 1990).

17

3. MATERIALS AND METHODS
Chestnuts
Chinese chestnuts (Castanea sativa Mill.) were harvested flom several local orchards in
southwest Michigan (Benton Harbor) and northwest Michigan (Traverse City) in October
2003 and 2004. The chestnuts were washed, drained and transported to Michigan State
University and stored at -2 2t 2C. The chestnuts were then transported to the pilot plant in
Jackson, M1 for peeling and processing. The peeled chestnuts were then sent back to

Michigan State University for further treatment and evaluation.

Chestnuts Peeler — Project 1

A new machine, called a Brulage chestnut peeler pilot plant (Boema) (Figure 2), was
imported flom Italy in 2001 . The shelling equipment is the first of its kind in the United
States. The chestnut peeler is made up of four parts: I. a double burner which burns off
the relatively thin chestnut shell and pellicle. 2. a thrasher which uses centripetal force to
remove any remaining shell or pellicle. 3. a steamer that moistens and cleans the
chestnuts. and 4. a brusher/washer which removes the remaining dirt, shell or pellicle.
Chestnuts with the shell on are metered into the burner; conveyed through the oven
within a screw auger cage to make the peel brittle; passed into a thrasher containing
rubber-ended paddles moving against steel rods which break away the peel and all or part
of the pellicle; conveyed to steamer which is a closed screw conveyor one—half filled with
water and heated with steam to 70—80°C (~158-l 76°F) to loosen any remaining shell or
pellicle; then finally moved onto a brusher/washer which removes loose pellicle and shell

with counter-rotating pairs of rollers, followed by cleaning rinse.

18

Burner
1 Steamer BrusherMIasher

Thrasher l

 

 

 

 

 

 

I

 

Figure 2. Schematic of integrated components of brulage chestnut peeling line

Chestnut samples used for microbial analysis

Chestnut (with the shell) microbial assays were preformed at 0 time, 2weeks, 2 months
and 4 months storage. Chestnut shells were removed by hand in a laminar flow hood and
a sanitized knife was used for removing the shells.

The baseline microbial populations of microorganisms for the four different parts of the
peeler (burner, thresher, steamer, and brusher/washer) were also determined. 15|bs of
chestnuts were collected flom each part and divided into 3 sets. The first sets were used
to determine the real time microbial populations. The second sets were packed in Nylon
bags (225g), vacuum sealed and flozen at -17.6C. After 2 weeks storage, the samples
were pulled out and thawed at 25C for 1 hour. The chestnut samples were then stored at
4-5C and 10-12C for 2 and 4 weeks and then assayed. The third set was packed in PE

bags (2253) and stored at 4-5C and 10-12C for 2 and 4 weeks and then assayed.

l9

 

Chestnuts pulled from burner,
thrasher, steamer and brasherlwasher

1

Frozen for 2 weeks

I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Thawed Peeled Chestnuts
Packed in Nylon bags
4-5C 10-12C

 

 

 

 

 

 

 

 

 

I

Evaluation after 0, 2, 4 weeks

I

Aw, Moisture content, Microbial test, Color L, a, b value

 

 

 

 

 

 

 

 

Fig 3. Flow diagram of Frozen and thawed peeled chestnut quality evaluation

Packaging Materials

Three different packaging materials, Nylon/Polyethylene (Koch Supplies Inc., Kansas
City, MO), Polyethylene (V WR International, Batavia, IL) and Porous Polyethylene (12
0.2in diameter holes on the bag and covered by Tyvek®) , were used in this study. For the
Nylon/Polyethylene bags (12in x 8in, 3mil), vacuum and atmospheric packaging
processes were employed. Vacuum packaging was accomplished using the MULTIVAC A

300/16 machine (MULTIVAC INC., Kansas City, MO) set at 800 mbar. For the

20

Polyethylene bags(121n x 8in, 2mil), punctured and non-punctured bags were used.
Punctured PE bags were fabricated by placing 9 evenly distributed holes (about 0.5 cm
diameter) on both sides of the pouch. These were then covered by Tyvek®. All bags,
excepted the vacuum packed bags, were sealed using the SENCORP SC-12 impulse heat
sealer (Sencorp System Inc., Hyannis, MA). Tyvek® is a bound polyethylene which

allows flee flow of gases such as oxygen.

Microbial assays

The total microbial count for- each sample was determined using the APHA Standard
Plate Count Method (Busta et al., 1984). 25g of chestnuts were taken and shaken together
with 225ml of phosphate buffer (pH 7) containing 0.1% peptone water. A 10'1 sample
dilution was made by aseptically transferring 1 ml of the previous buffer solution into a
tube containing 9 ml of sterile phosphate buffer solution and mixing well. Sample
dilutions of 10’2 to 10'8 were used for microbial enumeration. A 0.1 ml sample was taken
flom each dilution to determine the microbial numbers for the different microorganisms
(bacteria, yeast, mold, E. Coli), using standard pour plate techniques. All tests were done
in duplicate. Media and incubating conditions for each type of microbial group were as
follows:

Total bacteria count: Trypticase Soy Agar with 0.6% Yeast Extract (TSA-YE, Sigma,
MA), incubated at 23C for 48-72 hours.

Yeast and Mold: Rose Bagel Agar (Sigma, MA), incubated at 23C for 5 days.

E. Coli: Petri film (3M, MN), incubated at 37C for 24-48 hours.

Colonies flom each plate were counted and calculated as the average colony forming

21

units (CFU) per gram chestnuts.

The following general methods were used when determining the bacteria, yeast and mold
population levels. A chestnut sample weighing ~25 grams was removed and soaked in
225 m1 phosphate buffer and mixed using a pulsifier for 1 min. The yeast/mold and
bacteria populations were determined by plating the buffer solution on rose bagel agar
and trypticase soy agar containing 0.6% yeast extract (T SAYE), respectively and

incubating at the appropriate temperature. All of the microbial assays were duplicated.

Water Activity and Moisture Content

The water activity of chestnuts was determined at 25C using an electronic dew-point
water activity meter, Aqualab Series 3 model TE (Decagon Devices, Pullman,
Washington, USA). For each determination five replicates were obtained and the average
reported. Moisture content was measured by loss in weight after heating at 105 °C in the

vacuum oven for 6 hours.

Frozen and thawed peeled chestnut - Project 2

Approximately 50 pounds of peeled chestnuts were stored at -20C in cooler and then
thawed at 25C for 1 hour after 2 weeks. The thawed samples were packaged in 3mil
nylon bags and the filled packages (225g) were vacuum-sealed and stored at 4-5C and
10-12C. The moisture content, color (Hunter Color Difference), water activity and
microbial (bacteria, yeast and mold) counts of the chestnut samples were determined.

Triplicate analyses were done for all of the tests.

22

 

Fresh Peeled Chestnuts

 

 

 

 

 

l ,1 l

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Inhibitor Sanitizer Control
LDPE Nylon
4-5C 10-12C

 

 

 

 

 

 

 

 

 

l

Evaluated after 0, 2weeks

l

Aw, Moisture content, Microbial test, Color L, a, b value

 

 

 

 

 

 

 

 

Fig 4. Flow diagram of Fresh, Peeled Chestnuts subjected to sanitizer treatments

Fresh, Peeled Chestnut treated with sanitizing agents — Project 3

Approximately 100 pounds of peeled chestnuts which harvested and peeled in October,
2004 were used in this study. Two thirds of the chestnuts were dipped into a 0.1%
solution of potassium sorbate or commercial chlorine sanitizer (80 ppm) and then drained
for 5 minutes. All of the control and treated samples were packaged using three different

bag materials, low-density polyethylene (LDPE) 3mils thick, nylon 3 mils thick and

23

micro-porous PE bags 3mils thick. All filled packages (225 g) were weighed, heat-sealed
to closure and stored at 4-5C or 10-12C for 14 days. Evaluation of chestnut moisture
content, color (Hunter Color Difference, L, a, b values), water activity and microbial
population (bacteria, yeast and mold) was made. Triplicate analyses were done for all

tests.

Fresh peeled chestnuts with alternate sanitizers — Project 4

Fresh peeled chestnuts were immersed in distilled water containing sodium hypochlorite
(100, 200ppm) (SC Johnson, US) then pulled out and drained, or boxed in plastic boxes
with holes and exposed to chlorine dioxide gas using a novel Z-series 2000G solid release
chlorine dioxide delivery system (ICA TriNova LLC, GA). Distilled water dipping of the
chestnuts without sanitizer served as the control. After 5 and 10 minutes of hypochlorite
solution immersion, or 12 hours chlorine dioxide exposure, chestnut samples were
assayed for bacteria, yeast and mold populations. The remaining treated samples were
vacuum packed in Nylon pouches and held at 4-5C or 10-12C for 2 and 4 weeks storage.

Sensory evaluation and microbial count assays were then preformed on the chestnut.

Sensory evaluation
A eight-member trained sensory panel evaluated the quality of the chestnuts. A nine-
point scale was used for all attributes except texture. All sensory testing was conducted in

the Sensory Laboratory at Michigan State University. Panelists were MSU faculty and

24

students who were selected on the basis of their ability to detect specific product

attributes and their availability. Eight panelists participated in the training sessions before
every sensory evaluation, at which the training samples varied in color (1 = dull chestnuts,
9 = flesh peeled chestnuts), flavor (1= water, 9 = strong chlorine flavor, 200ppm) and
texture ( l= cooked chestnuts, 9 = dried chestnuts) in order to give the panelists a range of

attribute intensities.

Statistical Analysis
Statistical analysis of all of the data was performed using an Analysis of Variance and
Duncan multiple-range test using the statistical software program, SAS version 8.01.

Statistical significance was defined as p<0.05.

25

4. RESULTS & DISCUSSION

Baseline levels of microorganisms

Chestnuts harvested at the end of September 2003 flom several orchards in Benton
Harbor, M1, were transported and stored at -2C 2k 2C at Michigan State University. The
initial microbial populations of these flesh chestnuts were determined after removing the
shells by hand under sterile conditions. Results showed high bacteria (total plate count)
populations of Log 107 CFU/g chestnut. After 2 months storage, the bacterial populations
increased to Log 109 CFU/g. After 4 months storage, the microbial populations
decreased slightly to 1.5x108 CFU/g (Table 4). Yeast, mold, and E. coli were not detected
or populations were very low. These results indicate that the chestnuts naturally harbor
large populations of bacteria prior to the peeling process. Also, because of the chestnut
metabolism, the starch is converted to glucose which is easy to use by spoilage
microorganisms even in low temperature storage (Kawano et al., 1984). Kawano et al.
(1984) indicated that the shelf life of flesh chestnut (in the shell) is 3 months at 1C, 2

months at 5C, and 1 month at 10C storage.

Table 7. Microbial populations of chestnuts stored at 2°C before peeling." 2

 

 

 

 

 

Amount CFU/ g 0 Day 2 weeks 2 months 4 months
Bacteria 151107 1x107 2x109 15x108
Yeast - 1.5x10' - 2:1102
Mold - 2.510‘ — 411102
E. coli - - - -

 

 

 

 

 

 

 

(-) indicates no colonies detected
2 Results are the average of duplicate analyses

26

Contamination from the peeling line

Chestnuts were removed flom sections of the peeling line including the burner, the
tangential thrasher, the steamer and the washer/brusher, packed in PE (3 mil) bags and
stored at 4C and 10C. Initial bacterial counts flom the different peeler positions were Log
107, Log 107, Log 106, and Log 108 CFU/g (Table 5), respectively. After 2 weeks storage,
samples taken flom the burner, thrasher, and steamer had bacterial populations of
approximately Log 108 CFU/g. The samples taken flom the washer/brusher had bacteria
populations > Log 10H CFU/g (Table 6 and 7). The results showed that the steamer
reduced log populations by one log, but after chestnuts passed through the washer/brusher,
bacterial numbers again increased. This may have been due to brush contamination.
Yeast and mold were not found or were less than 102 CFU/g, except for the yeast
population of the chestnuts stored at 10C after l4days. This was probably due to
contamination during sampling or microbial testing. The E. coli populations were less
than 1 CFU/g before and after storage. The results showed that the initial populations of
yeast, mold and E. coli were at very low levels.

Frozen chestnut samples flom the 4 different parts of the peeling line were packed in a
commercial barrier material (LDPE/Nylon, 3mil) and tested immediately after thawing
and again after 2 and 4 weeks storage at 4 and 10°C. The initial bacteria counts were
approximately 105 CFU/g (Table 8). After 2 weeks storage at 4 and 10°C, the bacteria
populations had increased to approximately 1x107 and 1x108 CFU/g, respectively, with
populations of >1x10'2 CFU/g on samples taken flom the washer/brusher (Table 9 and
10). Yeast, mold and E. coli were less than 102 CFU/g which indicates that the initial

yeast, mold, and E. coli counts were low. The results also indicate that most of the

27

contamination was flom the washer/brusher. Chestnuts packed in both packaging
materials had results about the same, and thus neither material had any significant effect

on controlling growth of microorganism.

Table 8. Microbial populations on chestnuts packed in PE bags flom different locations
within the chestnut peeler (day 0). 1’

 

 

 

 

 

 

 

 

 

 

Amount CFU/g Burner Thrasher Steamer Brusher/Washer
Bacteria 2x107 2x107 1x10" 3x108
Yeast - - - -
Mold - - - 4x102
E. Coli - - - -

 

 

(—) indicates no colonies detected
2 Results are the average of duplicate analyses

Table 9. Microbial populations on chestnut flom different locations within the chestnut

peeler, packed in PE bags and stored at 4C for l4days."2

 

 

 

 

 

 

Amount CFU/ g Burner Thrasher Steamer Brusher/Washer
Bacteria 2x10" - 2x107 >1012
Yeast - - - -
Mold - - - -
E. Coli - - - -

 

 

 

 

 

T( —) indicates no colonies detected

2 Results are the average of duplicate analyses

28

 

Table 10. Microbial populations on chestnuts flom different locations within the chestnut

peeler and packed in PE and stored at 10C for l4days. " 2

 

 

 

 

 

 

 

 

 

 

 

Amount CFU/ g Burner Thrasher Steamer Brusher/Washer
Bacteria 6x108 4x107 3x108 >10l2
Yeast - 3x107 - >10‘2
Mold - - - -
E. Coli - - - ..
1 ( —) indicates no colonies detected

2 Results are the average of duplicate analyses

Table 11. Microbial populations on chestnuts flom different locations within the chestnut

peeler and packed in Nylon bags (day 0). l' 2

 

 

 

 

 

Amount CF U/g Burner Thrasher Steamer Brusher/Washer
Bacteria 1:1105 1:1105 1.5x104 3.5x105
Yeast - - - 2x10‘
Mold - - - 1x102

E. Coli - - - -

 

 

 

 

 

 

2 Results are the average of duplicate analyses

(——) indicates no colonies detected

29

 

 

Table 12. Microbial populations on chestnuts flom different locations within the chestnut

peeler and packed in Nylon bags and stored at 4C for after 14 days. 1’ 2

 

 

 

 

 

 

Amount CFU/ g Burner Thrasher Steamer Brusher/Washer
Bacteria 1.4x107 1x107 1.2x107 >10”
Yeast - - - -
Mold - .. .. -
E. Coli - - - -

 

 

 

 

 

I (—) indicates no colonies detected

2 Results are the average of duplicate analyses

Table 13. Microbial populations on chestnuts flom different locations within the chestnut

peeler and packed in Nylon bags and stored at 10C for 14 days. "2

 

 

 

 

 

 

 

 

 

 

Amount CFU/g Burner Thrasher Steamer Brusher/Washer
Bacteria 2.5x108 3x10" 4.5x108 >10”
Yeast - - - -
Mold - - - -
E. Coli - - - ..

 

1(—) indicates no colonies detected

2 Results are the average of duplicate analyses

30

 

 

Quality of frozen and thawed peeled chestnuts stored at 4 and 10C

The moisture content and water activity of flozen and thawed peeled chestnuts stored at 4
and 10 8C are shown in Figures 5 and 6. The storage period and the storage temperature
had no significant effect on moisture content and water activity. The highest moisture
value was determined on day 0 (packing day) and decreased slightly during the storage
period at both 4 and 10 8C. The water activity of the chestnuts did not very significantly

between 0 and 28 days at 4 and 10 8C.

 

59
58 -
% 57 *-
56 ,- --
55 l
54 ' ‘ ‘
l

l

 

 

 

 

 

 

Days

Figure 5. The moisture content of flozen and thawed chestnuts during storage

31

[-rm_ ___ ___ 1,-.1 ___ ___._r __.___.____ L -, _.__ .L.. L .22, 1

Water Activity 1

 

 

 

 

 

 

 

 

 

Days j

Figure 6. The water activity of flozen and thawed chestnuts during storage

Analysis of the color values showed that there was a significant difference in the “L”
(white) values (P<0.05) for the outside and inside of the flozen and thawed peeled
chestnuts after 28 days storage at 4-5 and 10-12C. The “L” (white) values for the outside
surface of the chestnuts were higher than the inside values (Figure 7 and 8). There was no
significant difference between the outside and inside of the chestnuts during storage at
10-12C. However, the chestnuts became darker after 28-days storage at 4~5C. The
darkening of chestnuts likely occurred because of PPO activity even in low temperature

storage (Xu, 2005).

32

 

 

 

 

 

 

30 1 I l l J

 

 

Days __ _ 3._ j

 

Figure 7. The L-value (outer layer) of chestnuts during storage

 

 

 

 

 

 

 

 

 

 

30 l I l l l

 

 

Figure 8. The L-value in the interior of the chestnuts during storage

33

The “a” (red) value for the interior of the chestnuts was higher than that for the outside,
(Figure 8 and 9) at day 0. During storage, the a-value decreased on the inside of the
chestnuts, but the a-value increased on the outside of the chestnuts. The “a” (red) values
of the inside and outside of the flozen and thawed peeled chestnuts did not significantly

differ after 28 days storage at 4-5 and 10-12C.

F T“ M W ”“7"“ MET—W" “TM—m —Tl

l a value (outside)

 

 

 

 

 

 

 

 

_-_ __ __. . _.__.._._—-.__

Figure 9. The a-value of the chestnuts exterior layer during storage

34

 

 

 

 

 

 

 

 

 

Figure 10. The a-value on the interior of chestnuts during storage

The “b” (yellow) values for the outside and the inside of the frozen and thawed peeled
chestnuts were significantly different (Figure 11 and 12). However, there were no
significant differences in b values between the outside of the chestnuts stored at 4-5 and
10-12C. There were, however, significant differences in b values in the interior of the
chestnuts. The b values of chestnuts stored at 4-5C decreased during storage (Figure 12),

but the b values of chestnuts stored at 10-12C were essentially the same.

35

 

r.rr_r.r_c r_-_r-_-__ , , Lyric LLLLL
. b value (outside) 1
l 25[

 

 

In

 

15 "

I. —- . 1:109

 

 

 

 

 

 

5
0

 

 

 

I 0 5 10 15 20 25 30 ;
l Days J

 

Figure 11. The b-values of the exterior surface of the chestnuts during storage

 

 

 

 

 

 

 

 

Figure 12. The b-values of the interior of the chestnuts during storage

36

Microbial populations of the chestnuts

Mold did not grow on the samples probably because of the vacuum packaging and lower
storage temperature. Bacteria and yeast numbers increased very quickly in the packaged
products stored at 10-12C (Table 11 and 12). The data show that the lower storage
temperature (4 —- 5C) inhibited the growth of microorganisms. There were significant
differences between chestnuts stored at the different temperatures after l4—days storage.
Bacteria counts increased to 1012 and 106 at 10-12C and 4-5C storage, respectively. Based
on microbial numbers, the results also indicate that the shelf life of the chestnuts is about

2 weeks when stored at 4-5C and less than 2 weeks at 10-12C storage.

Table 14. The bacteria count (TPC) of chestnuts during storage1

 

 

 

 

 

 

 

0 days 14 days 28 days
4C-5C 1.2x 10‘ 5.4x 10" 3.8x 10“
10— 12C 1.2 x104 7.5 x 10‘2 3.7 x109

 

I Results are the average of duplicate analyses

Table 15. The yeast count on chestnuts during storageI

 

 

 

 

 

 

 

0 days 14 days 28 days
4C—5C 4.5x 103 6.5x 10" 2.4x 10“
10 — 12C 4.5 x103 2.5 x 10'2 5.4 x109

 

I Results are the average of duplicate analyses

 

 

Fresh, Peeled Chestnut Packaging

Chestnuts harvested and peeled in October, 2004 were used in this study. Two thirds of
the chestnuts were dipped into a 0.1% solution of potassium sorbate or commercial
chlorine sanitizer (80 ppm). All of the control and treated samples were packaged using
three different bag materials, low-density polyethylene (LDPE) 3mils thick, nylon 3 mils
thick and micro-porous PE bags 3mils thick. The moisture content and water activity did
not change significantly during storage. The different treatments, packaging materials or
storage temperatures did not have any significant effect on chestnut moisture content and
water activity after 14 days storage (table 13 and 14). The moisture content and the water
activity of the flesh peeled chestnuts were about the same as for flozen, thawed peeled
chestnuts. Therefore, the moisture content and water activity did not change significantly

during flozen storage.

Table 16. The moisture content of flesh peeled chestnuts after 14 dgys storagel

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Moisture Content %(10-12C) Moisture Content %(4-5C)

Initial (day 0) 59.14% 59.14%
i-pe 59.68% 58.87%
i-ppe 58.13% 60.78%
i-ny 58.42% 57.81%
s-pe 57.01% 55.09%
s-ppe 55.57% 58.17%
s-ny 57.07% 59.72%
c-pe 55.80% 59.55%
c-ppe 56.24% 59.22%
c-ny 58.87% 59.50%

 

i = Inhibitor, s = Sanitizer,

c = Control, pe = PE bag, ppe = Porous PE bag, ny= Nylon bag

lResults are the average of duplicate analyses

38

 

Table 17. The water activity of fresh peeled chestnuts after 14 days storage '

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Water activity(10-12C) Water activity(4-5C)
Initial (day 0) 0.935 0.935
i-pe 0.883 0.878
i-ppe 0.878 0.884
i-ny 0.890 0.919
s-pe 0.923 0.943
s-ppe 0.879 0.900
s-ny 0.894 0.877
c-pe 0.886 0.909
c-ppe 0.885 0.909
c-ny 0.889 0.902
1 = Inhibitor s = Sanitizer c = Control
pe = PE bag ppe = Porous PE bag ny= Nylon bag

Results are the average of duplicate analyses

39

No significant change was observed in the L-value and b-value of the chestnut samples.
The a-value increased during storage. This shows that the chestnuts were found to darken
during storage. Polyphenol oxidase is responsible for the enzymatic browning reaction,
which is triggered during the handling, storage and processing of fluit and vegetables
(Macheix etal., 1990). It is also known that flee phenolics in contact with PPO, cause
substantial browning in plant materials. However, the phenolic content in chestnuts was

not significant different between flesh and 6 months stored chestnuts at 4C (Xu, 2005).

Table 18. The L-value of flesh peeled chestnuts after 14 days chestnut storage ‘

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample L-value (10-12C) L-value (4-5C)

Initial (day 0) 46.45 46.45
i-pe 42 41.55

i-ppe 41.65 39.9

i-ny 43 43.6
s-pe 42.75 41.35
s-ppe 42 41.75
s-ny 41.85 42.35

c-pe 42.5 43.3
c-ppe 42 41.95

c-ny 42.5 42.5

i = Inhibitor s = Sanitizer c = Control
= PE bag ppe = Porous PE bag ny= Nylon bag

Results are the average of duplicate analyses

40

 

Table 19. The a-value of flesh peeled chestnuts after 14 days storagel

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample a-value (10-12C) a-valuev(4-5C)

Initial (day 0) 1 1.55

i-pe 3.15 2.9
i-ppe 3.9 3.25
i-ny 2.45 2.55
s-pe 2.65 2.85

s-ppe 3.5 3.8

s-ny 2.45 2.9

c-pe 1.6 1.8
c-ppe 3.85 3.15

c—ny 2.45 3.1

i = Inhibitor s = Sanitizer c = Control
= PE bag ppe = Porous PE bag ny= Nylon bag

Results are the average of duplicate analyses

Table 20. The b-value of flesh peeled chestnuts after 14 days storage ‘

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample b-value (10-12C) b-valuc (4-5C)
Initial (day 0) 21.15 17.7

i-pe 18.3 16.1
i-ppe 17.25 16.05
i-ny 19.6 16.75
s-pe 18.55 16.75
s-ppe 18.85 16.55
s-ny 19.5 16.6
c-pe 19.9 17.5
c-ppe 18.65 17.25
c-ny 18.65 16.6

i = Inhibitor s = Sanitizer c = Control

pe = PE bag ppe = Porous PE bag ny= Nylon bag

Results are the average of duplicate analyses

41

 

 

The different packaging materials (PE/Nylon), sanitizer treatments, and storage
temperature did not have a substantial effect on the growth of bacteria and yeast. This
was probably because the initial loading of the chestnuts was so high; it overshadowed
any possible benefit flom the packaging system. The mold, however, only grew in the
micro-porous PE bags stored a 10-12C probably because this bag allowed flee flow of
oxygen. Storage at 4—5C mostly inhibited the growth of the mold on chestnuts stored

under these conditions.

Table 21. The total plate count of flesh chestnuts stored at 10-12C after 14 days storagel

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Initial (CPU/g) After storage
i-pe >2x10" 4.0x109
i-ppe >2x10" >10“
i-ny >2xl06 6.0xlo9
s-pe >2x106 7.5x109
s-ppe >2x10" >10”
s-ny >2x10" 6.0x109
c-pe >2x106 7.5x109
c-ppe >2x10" >10”
c-ny >2x106 1.0x1010
1 = Inhibitor s = Sanitizer c = Control
pe = PE bag ppe = Porous PE bag ny= Nylon bag

Results are the average of duplicate analyses

42

Table 22. The togplate count of fresh chestnuts stored at 4-5C afier 14 days storage'

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Initial (CFU/g) After storage
i-pe >2x106 2.2x l 010
i-ppe >2x10" 7.4x10‘°
i-ny >2xlo6 1.1x10lo
s—pe 1.18xlo5 6.0x109
s—ppe 1.18x105 6.2xlo'O
s-ny 1.45x105 15x1010
c-pe 1.45x10" 3.1x1010
c-ppe 1.45x105 1.7x10‘0
c-ny 1.45x105 1.5x10'°

i = Inhibitor s = Sanitizer c = Control

pe = PE bag ppe = Porous PE bag ny= Nylon bag

Results are the average of duplicate analyses

Table 23. The total yeast counts on flesh chestnuts at 10-12C after 14 gag storageI

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Initial (CF U/g) After storage
i-pe 1.25xlo5 2.051109
i-ppe 2.0x109 >10ll
i-ny 8.5x10‘ 7.051109
s-pe >10“ 4x109
s-ppe 4.5x104 >10ll
s-ny 7.0x109 7.051109
c—pe 5.6x10‘ 7.0x109
c-ppe 4x109 >10ll
c-ny 7.2x104 5.5x109
i = Inhibitor s = Sanitizer c = Control
pe = PE bag ppe = Porous PE bag ny= Nylon bag

Results are the average of duplicate analyses

43

 

 

Table 24. The total yeast counts on flesh chestnuts stored at 4-5C after 14 days storage‘

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Initial (CPU/g) After storage
i-pe 151105 2.9x10‘°
i-ppe 2.9x10lo 5.4x10‘0
i-ny 7.95th4 1.31th10
s-pe 5.4111010 1.5x10‘°
s-ppe 4.5x10“ 7.51110lo
s-ny 1.3x10lo 1.05110lo
c-pe 5.2x104 3.4x10‘"
c-ppe 1.5x10l0 1.6x10‘"
c-ny 4.355th5 1.7x10‘0

1 = Inhibitor s = Sanitizer c = Control
pe = PE bag ppe = Porous PE bag ny= Nylon bag

Results are the average of duplicate analyses

Table 25. The total mold counts on fresh chestnuts stored at 10-12C after 14 days storageI

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Initial (CFU/g) After storage
i-pe <1 <1
i-ppe 4.551103 >10”
i-ny 1x103 <1
s-pe <1 <1
s-ppe 1.5x102 >10ll
s-ny <1 <1
c-pe <1 <1
c-Ppe <1 >10“
c-ny <1 <1
i = Inhibitor s = Sanitizer c = Control
= PE bag ppe = Porous PB bag ny= Nylon bag

Results are the average of duplicate analyses

 

 

Table26. The total mold counts on flesh chestnuts stored at 4-5C after 14 days storagel

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Initial (CFU/g) After storage
i-pe 2.5x l 05 <1
i-ppe <1 <1
i-ny 1.551103 <1
s-pe <1 <1
s-ppe <1 <1
s-ny <1 <1
c-pe 2x103 <1
c-ppe <1 <1
c-ny 2x103 <1
i = Inhibitor s = Sanitizer c = Control
=- PE bag ppe = Porous PE bag ny= Nylon bag

Results are the average of duplicate analyses

Effectiveness of different sanitizer

Hypochlorite solutions were prepared using sodium hypochlorite (5% active chlorine, SC
Johnson, USA) and distilled water. Chlorine solutions of 100 and 200 ppm were prepared
by appropriate dilutions. The 100 and 200 ppm chlorine solution reduced the bacterial
populations by 5 logs. The results showed that the sodium hypochlorite was more
successful with chestnuts than flesh apples because the sodium hypochlorite only reduced
1-2 logs on flesh apples (Beuchat et al., 1998). The bacterial populations on chestnuts
floated with 200 ppm chlorine solution went up to Log 107 and Log 10” CFU/g after 2
weeks storage at 4C and 10C, respectively. The chestnuts treated with 100 ppm chlorine

had population levels of Log 108 and > Log 10” CFU/g after 2 weeks storage at 4C and

4S

10C, respectively. The results showed that hypochlorite effectively reduced the number of
bacteria on the chestnuts after peeling, but then a more rapid increase in numbers (T able
24 and 25) occurred. Thus, the flesh peeled chestnuts had a shelf life of about 2 weeks
when stored at 4C and less than 2 weeks at 10 C.

The bacterial counts on chestnuts previously treated with 200 ppm chlorine solution
increased to 107 and 1011 CFU/g after 2 weeks storage at 4 and 10°C, respectively. The
100 ppm chlorine treated samples had population levels of 10° and >1011 CFU/g after 2
weeks storage at 4 and 10°C, respectively. Although hypochlorite reduced bacterial
populations on the peeled chestnuts, these chestnuts only had a shelf-life of 2 weeks
when stored at 4°C.

The initial microbial populations on peeled chestnuts treated with C102 gas and vacuum
packed were reduced about 2 logs after 12 hrs exposure. After 2 weeks storage at 4 and
10°C, the microbial counts increased to 109, which is higher than the results flom
hypochlorite treatment.

No significant difference in sensory results (day 4) were found for any of the chestnut
samples in appearance/color (1 = worst, 9 = best), off-flavor (1 = no off flavor, 9 = strong
off flavor), texture (1 = very soft, 9 = very hard), and overall quality (1 = worst, 9 = best).
After 15 days storage, the results showed no significant difference between any of the
samples. Therefore, the chlorine concentration and treatment time did not affect the

appearance/color and texture, nor the overall sensory qualities.

46

Table 27 . Microbial populations on flesh chestnuts treated with different concentrations

of hypochlorite (day 0, 4°CL " 2

 

 

 

 

 

 

 

 

 

 

 

 

as)? 13...... ”5°32.“ 83:82" 22°22 213.1%? 8'02
Bacteria 1.5x107 3x10} 4.9x103 6.7x10° 5.8x103 8.4x10’
Yeast -. - - - - 3.5:th2
Mold - - — - - -
E. coli - - - - - _

 

(—) indicates no colonies detected

2 Results are the average of duplicate analyses

Table 28. Microbial populations on fresh chestnut samples treat with different

concentrations of hypochlorite and stored at 4C and assayed after 14 days. " 2

 

 

 

 

 

 

 

 

 

 

 

 

Bacteria >10" 1.14x10" 1.45x10° 2.4x107 1.55x107 2.951109
Yeast - - — - - 7.5x102
Mold - - - - - -
E. coli - - .. - _ _

 

I (——) indicates no colonies detected
2 Results are the average of duplicate analyses

47

 

 

Table 29. Microbial populations on flesh chestnut samples treat with different

concentrations of hypochlorite and stored at 4C and assayed after 21 days. 1’ 2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Amount 100ppm, 100ppm, 200ppm, 200ppm,
CFU/g Control 5min 10min 5 min 10min C102
Bacteria >10“ 3.221109 6.2x109 7.2x10‘" 8x10” 9x109
Yeast - - - - - -
Mold - - - - - -
E. coli - - - - - -
(-—) indicates no colonies detected
2 Results are the average of duplicate analyses
Table 30. The sensory results of chestnut samples after 4 days storage at 4C
. 100ppm, 100ppm, 200ppm, 200ppm,
“mm” 10min 5min 10 min 5min C102 °°“"°‘
Slirniness 3.38s 2.00ab 2.00ab 2.63ab 1.63b 1.25b
APP“ “we 6.35a 6.208 5.76a 5.40a 5.51a 5.85a
/color
Off flavor 2.19a 3.48a 3.56a 3.24a 3.16a 3.28a
Texture 5.65a 5.51a 5.24a 5.24a 5.23a 5.38a
Overall 7 .03a 6.44a 6.43a 6.01a 6.13a 5.80a

 

 

 

 

 

 

 

 

a, b means within the same row with different superscript letters are different (p < 0.05).

48

 

 

Table 31. The sensory results of chestnut samples after 15 days storage at 4C

 

 

 

 

 

 

 

 

 

 

 

 

 

Attribute ‘OOPPm’l ‘00 ppm, 20° PP”, 200 ppm. 0102 control
0mm 5mm 10 mln Smln
Sliminess 3.718 3.86a 3.578 3.298 3.868 2.578
”mm“ 6.018 6.078 5.0911 4.368 5.41s 4.648
/color

Off flavor 3.608 3.83a 3.568 4.018 4.438 3.398
Texture 5.838 4.848 5.418 5.068 4.708 4.618
Overall 5.518 5.838 5.018 5.148 4.418 5.468

 

a, b means within the same row with different superscript letters are different (p < 0.05).

49

 

Conclusion

These results indicate that chestnuts naturally harbor large populations of bacteria prior to
the peeling process. Also, because of chestnut metabolism, the starch will be converted to
glucose which is easy to use by spoilage microorganism even at low storage temperature.
The steamer reduced microbial populations by one log but after chestnuts passed through
the washer/brusher, bacterial numbers were found to have increased. This may have been
due to brush contamination. Chestnuts packed in both packaging materials had similar
results and neither material had any significant effect on inhibiting the growth of
microorganism. Mold did not grow on the chestnut samples probably because of vacuum
packaging and lower storage temperature. Bacteria and yeast numbers increased very
quickly in the packaged products stored at 10-12C. Therefore, low storage temperature
can inhibit microbial growth. The shelf life of the chestnuts packed by different
packaging materials and stored at 4-5C or 10-12C was less than l4days. The different
sanitizer treatments did not significantly inhibit microbial growth. None of the treatments,
packaging materials or storage temperatures had any significant effect on chestnut
moisture content and water activity afler 14 days storage. To extend the shelf life, MAP
or CAP can probably be used and fitrther research is needed to find suitable gas
compositions. Other sanitation methods might be used such as ozone treatment or

pasteurization methods such as high pressure processing.
s

50

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55

Appendix A
Name Date

 

 

Please look at the chestnuts and answer the questions:

1. Please evaluate the appearance/color of the chestnuts on the following scale.

+ ----------------------------------------------------------- +

l 9
dull, or mottled, atypical appearance typical appearance
2. Is there any presence of mold or decay? Yes _ NO

If yes, please rate the amount on the following scale and describe;

+ ----------------------------------------------------------- +

1 9
large amount mold/decay on surface no mold/decay on surface
(>50%)

3. Overall appearance quality?

+ ----------------------------------------------------------- +
l 9
Very poor Very high

Please taste the samples:

4. Please rate the texture of the samples:

+ ----------------------------------------------------------- +
1 9
very soft or chewy typical texture very hard or tough

5. Do you detect any off flavor in the samples?

+ ----------------------------------------------------------- +
1 9
No off flavor Strong off flavor

56

6. Please rate the Overall quality afier tasting the chestnuts:

+ ——————————————————————————————————————————————————————————— +

1 9

Very poor Very high
Comments:

THANK YOU FOR YOUR PARTICIPATION

57

Appendix B

Do you eat chestnuts or want to try them? Participate in a
quality evaluation of chestnuts!

We are looking for panelists who consume and/or are familiar with chestnuts to
participate in sensory evaluations of chestnut whole and peeled quality. Approximately
4-8 evaluations will be held over a 4-month period, beginning in October 2004. Each
evaluation will take about 20 minutes of your time. For research purposes, we ask that
you participate for all scheduled evaluations when possible. We will make every effort to

accommodate your schedule.

We will give you a food treats for

participating each time.

102 TROUT FOOD SCIENCE

& HUMAN NUTRITION

(N. W. comer of Wilson Rd. and Farm Lane)

For any questions contact Janice Harte in the Department of Food

Science and Human Nutrition Building, harteia@msu.edu

 

Or 355-8474, ext. 105

58

Appendix C
Consent Form

Microbial populations on peeled chestnuts and their inhibition
Dear Participant:

Several Michigan State University researchers are investigating the quality of chestnuts. We
would like you to take about 20 minutes (including the time you spend reading this letter) to help
us samples of flesh and frozen chestnuts. We are asking for volunteers, over the age of 18, who
eat or cook with chestnuts.

We are asking that panelists participate in an evaluation of chestnuts that will be

conducted at intervals over a 4-month period starting October, 2004. Training will
consist of approximately 1 session of 30-60 minutes. After training, evaluations will be
scheduled up to eight times. You will be consulted prior to scheduling the tests in order
to accommodate your schedule and availability. It is important for this research that we
have the same panelists participate for each evaluation when ever possible. However, if
you cannot attend any evaluation or continue as 8 panelist, please inform the researchers
when contacted. Your signing this consent form will indicate your agreeing to participate
when possible. You will be given a coupon or food treat worth less than $5 for your

participation and completion of the questionnaires.

If you have a known food allergy to chestnuts or other tree nuts please do not participate in this
study.

If you believe there is a potential of an allergic reaction upon sniffing and tasting, notify the
on—site sensory evaluation coordinator and/or principle investigator immediately. You will be
released from participating in this study. Please note if you are injured as a result of your
participation in this research project, Michigan State University will assist you in obtaining
emergency care, if necessary, for you research related injuries. If you have insurance for medical
care, your insurance carrier will be billed in the ordinary manner. As with any medical insurance,

any costs that are not covered or in excess of whatever are paid by your insurance, including

59

deductibles, will be your responsibility. Financial compensation for lost wages; disability, pain or
discomfort is not available. This does not mean that you are giving up any legal rights you may
have. You may contact Janice Harte with any questions (355-8474x105). Your response is
confidential and we will protect your confidentiality to the full extent of the law. You are free to
not answer any question you choose, but please try to answer every question. We are not able to

use incomplete responses nor are we able to provide the incentive for incomplete responses.

If you have any questions during your reading this consent form, or during or after your
participation, please do not hesitate to contact the on-site sensory evaluation leader and/or the
principle investigator. Feel flee to contact Dr. Janice Harte, the principle investigator, via phone
at 517-355-8474, ext. 105. She also can be reached via email at harte'a msu.edu for any inquiry
you might have due to your participation in the study.

In case you have questions or concerns about your role and rights as a research participant, please
feel free to contact Dr. Peter Vasilenko, Michigan State University's Chair of University
Committee on Research Involving Human Subject (UCRIHS) by phone: (517) 355-2180, fax:
(517)432-4503, email: ucrihs@msu.edu or regular mail: 202 Olds Hall, East Lansing, MI 48824.

PLEASE NOTE UPON YOUR SIGNING THIS CONSENT FORM, YOU
VOLUNTARILY AGREE TO PARTICIPATE IN OUR STUDY. YOUR SIGNATURE
INDICATES YOU HAVE READ THE INFORMATION PROVIDED ABOVE AND
THAT YOU HAVE HAD AN ADEQUATE OPPORTUNITY TO DISCUSS THIS
STUDY WITH THE PRINCIPLE INVESTIGATOR AND HAVE HAD ALL YOUR
QUESTIONS ANSWERED TO YOUR SATISFACTION. YOU WILL BE GIVEN A
COPY OF THIS CONSENT FORM WITH YOUR SIGNATURE FOR YOUR
RECORDS UPON YOUR REQUEST.

SIGNED DATE

 

60