Effects of Acid Whey Marination on Tenderness, Sensory and

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Effects of Acid Whey Marination on Tenderness,Sensory and Other Quality Parameters of Beef Eyeof RoundJason KimBrigham Young University

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Effects of Acid Whey Marination on Tenderness, Sensory

and Other Quality Parameters of Beef Eye of Round

Jason Kim

A thesis submitted to the faculty of Brigham Young University

in partial fulfillment of the requirements for the degree of

Master of Science

Michael L. Dunn, Chair Frost M. Steele

Laura K. Jefferies

Department of Nutrition, Dietetics, and Food Science

Brigham Young University

Copyright © 2018 Jason Kim

All Rights Reserved

ABSTRACT

Effects of Acid Whey Marination on Tenderness, Sensory and Other Quality Parameters of Beef Eye of Round

Jason Kim Department of Nutrition, Dietetics, and Food Science, BYU

Master of Science

The growth of the Greek-style yogurt market is causing many problems for dairy companies who are trying to handle the voluminous whey by-product. Acid whey, unlike sweet whey, has a low amount of protein and high amounts of lactic acid, calcium, and other minerals. Therefore, it has limited commercial value to the food industry and often requires additional processing for disposal. Lactic acid and calcium solutions have shown efficacy in increasing the tenderness of beef and other types of meat. The purpose of this project is to investigate the use of acid whey, with its high amounts of lactic acid and calcium, to tenderize beef (eye of round, IMP 171C) during marination.

This study evaluated the effects of marination of utilizing acid whey in improving quality

parameters of beef. 13 roasts (Top round steaks from USDA Select steers) were randomly assigned to one of six marination treatments: (1) calcium chloride, (2) lactic acid, (3) phosphate (4) acid whey (lot 1), (5) acid whey (lot 2), and (6) control. Steaks were marinated in vacuum pouches, aged for 48 hours, cooked to 70º C and evaluated by a consumer sensory panel and other quantitative tests (texture analyzer, colorimeter, collagen, cook loss, and pH). Marination with acid whey increased the tenderness and juiciness ratings without decreasing hedonic liking scores for the overall flavor or aftertaste of the beef samples. Keywords: acid whey, beef, eye of round, Greek yogurt whey, hedonic scale, colorimeter, liking, marination, tenderness, ta.xt2, sensory

ACKNOWLEDGMENTS

I would like to express my gratitude to the faculty and staff of the department of

Nutrition, Dietetics, and Food Science for their time in teaching and mentoring me throughout

my undergraduate and graduate degrees at BYU. I am grateful to Dr. Steele, Dr. Jefferies and Dr.

Dunn for their help on my committee, and Dr. Dunn for serving as my committee chair. I could

not have produced as fine of research without their expert guidance along the way. Thanks are

also in order for the students of the food research lab and my fellow graduate students who

helped me think through many problems.

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TABLE OF CONTENTS

TITLE PAGE ................................................................................................................................... i

ABSTRACT .................................................................................................................................... ii

ACKNOWLEDGMENTS ............................................................................................................. iii

TABLE OF CONTENTS ............................................................................................................... iv

LIST OF TABLES ......................................................................................................................... vi

LIST OF FIGURES ...................................................................................................................... vii

INTRODUCTION .......................................................................................................................... 1

Summary ..................................................................................................................................... 1

Acid Whey .................................................................................................................................. 2

Meat Tenderization ..................................................................................................................... 3

Objectives ................................................................................................................................... 5

Materials and Methods .................................................................................................................... 5

Product Selection ........................................................................................................................ 5

Treatments................................................................................................................................... 5

Packaging and Marination .......................................................................................................... 6

Cooking ....................................................................................................................................... 7

pH ................................................................................................................................................ 7

Cook Loss ................................................................................................................................... 7

Color ........................................................................................................................................... 7

Shear Force ................................................................................................................................. 8

Scanning Electron Microscopy ................................................................................................... 8

v

Collagen Content ........................................................................................................................ 9

Consumer Panel Analysis ........................................................................................................... 9

Statistical Analysis .................................................................................................................... 11

RESULTS AND DISCUSSION ................................................................................................... 11

Compositional Analysis ............................................................................................................ 11

Consumer Sensory Evaluation .................................................................................................. 11

Compositional and Textural Properties .................................................................................... 12

Color ......................................................................................................................................... 15

Electron Microscopy ................................................................................................................. 16

Conclusion ................................................................................................................................ 19

Complete References .................................................................................................................... 20

Appendix ....................................................................................................................................... 25

Texture Analyzer Test Settings ................................................................................................. 25

Collagen Method - Detailed ...................................................................................................... 25

Table 5 Complete Proximate Composition (%) and Mineral Content (ppm) of Acid Wheys .. 27

Table 6 Complete Mineral Analysis (ppm) .............................................................................. 27

Statistical Output ....................................................................................................................... 28

Sensory Comments ................................................................................................................... 84

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LIST OF TABLES

Table 1 Proximate composition (%) and mineral content (ppm) of acid whey ............................ 11

Table 2 Consumer acceptance and hedonic ratings for beef eye of round steaks marinated in

different solutions ......................................................................................................................... 12

Table 3 Means for soluble collagen, insoluble collagen, cook loss percentage, pH, and shear

force .............................................................................................................................................. 13

Table 4 Effect of acid whey and other marinade treatments on color of beef eye of round. ........ 15

Table 5 Proximate Composition (%) and Mineral Content (ppm) of Acid Whey ........................ 27

Table 6 Mineral Analysis (ppm) ................................................................................................... 27

vii

LIST OF FIGURES

Figure 1 Scanning electron micrograph (SEM) of raw beef muscles with different treatments

(control-no treatment, acid whey, calcium chloride, lactic acid, and phosphate) ......................... 18

1

INTRODUCTION

Summary

The expansion of Greek-style yogurt in the U.S. yogurt industry in the past ten years is

one of the most remarkable events in food production and sales in recent history (McCormack

2016). Greek-style yogurt is traditionally made by straining the fermented yogurt curd in a cloth

bag, removing the acid whey until it reaches a desired solids level. In the food industry this step

is achieved by mechanically separating the acid whey from the yogurt using a centrifugal

separator or a filtration membrane (Nsabimana and others 2005). The growth of this market has

caused a lot of problems for dairy companies who are attempting to handle large quantities of

this acid whey byproduct (Uderwerella and others 2017).

Greek-style yogurt acid whey, unlike sweet whey from traditional cheese production, is

low in protein and high in lactic acid, calcium, and other minerals. Ultrafiltration can be used to

recover the protein from this whey. However, the loss of membrane efficiency, or membrane

fouling, is a longstanding problem in facilities that utilize this technology (Berg and others

2014).

Membrane fouling during the processing of acid whey is attributed mainly to the

precipitation of calcium salts, especially calcium phosphates (Hanemaaijer and others 1989). The

high level of process inputs required to recover the low levels of protein in acid whey reduces its

commercial value to the food industry. Many companies in the dairy industry currently sell their

acid whey to farmers as animal feed. In efforts to maximize profits, and facilitate disposal, recent

research has explored new ways to apply or process acid whey for human consumption. Wojciak

and others. (2014), for example, found that use of acid whey in sausages could be used

2

effectively to improve the microbiological quality, and that it could produce a similar color to

sausage treated with a nitrite curing salt.

While the low protein content of acid whey makes it a low-value byproduct, the

abundance of lactic acid and calcium in the whey may lead to alternative uses in the food

industry. Lactic acid and calcium solutions have been found to be effective in increasing the

tenderness in beef and other cuts of meat (Berg and others 2001; Ostoja and Cierach 2003). The

purpose of this research was to investigate the use of acid whey in marinade solutions, as a

means to tenderize beef (eye of round, IMP 171C).

Acid Whey

Whey is the liquid byproduct of cheese and Greek-style yogurt manufacture. The whey

from hard cheeses is referred to as sweet whey, and is relatively high in protein, as well as

containing minerals and some acid. Sweet whey is the principal source of the high-value whey

protein powders that are in high demand worldwide. The whey derived from Greek yogurt

manufacture (or quark or cream cheese production) is referred to as acid whey. It is low in

protein, and contains appreciable amounts of lactose, minerals, and lactic acid. The disposal of

acid whey is a major problem worldwide. Ultrafiltration of both acid whey and sweet whey are

practiced (Ganju and Gogate 2017); however, there are many issues that arise due to membrane

fouling during processing (Berg and others 2014). Pretreatment of whey to remove calcium salts

has been shown to increase membrane productivity (Patel and Merson 1978, Patocka and Jelen

1991), however, these additional process steps reduce production capacities and increase cost.

The low protein yields with acid whey currently make this commercially non-viable.

3

As a result of the difficulty and costs of processing acid whey through filtration, there has

been interest in using whole, liquid acid whey as a functional ingredient in different applications.

Skrypolenk and Jasinska (2015) explored the idea of using acid whey as a base for creating

probiotic beverages. Vajda and others (2013) examined the effect of acid whey concentrate on

thermophysical properties of a milk based ice cream. Sady and others (2013) studied the

application of acid whey in orange drink production.

A limited amount of research has been conducted, looking at acid whey as a process

additive in meat products. Wojciak and others (2014) used acid whey with mustard seed to

replace nitrites in cooked sausages, with positive effects. Stadnik and Stasiak (2016) reported on

the physicochemical properties of pork loin marinated in acid whey prior to dry curing. The acid

whey marination, combined with sea salt, resulted in reduced browning, and was protective

against oxidation. Wojciak and others (2015) explored the use of acid whey as a marinade for

production of fermented beef eye of round. They found that acid whey decreased the pH,

increased the red color value as well as oxidative stability, but did not have appreciable sensory

effects in a limited eight-person trained panel. However, there has not been much research done

to look at the effects of acid whey on the tenderness of beef. The high amounts of acid and

calcium could potentially increase the tenderness.

Meat Tenderization

The sensory characteristics (texture, flavor, aroma, and color) of meat are important

attributes in determining its quality. Tenderness and associated juiciness are among the most

important quality attributes related to meat texture and eating quality. The texture of meat is

predominantly determined by the moisture and fat contents, as well as the types and amounts of

structural proteins (Aktas and Kaya 2001). Collagen is a major structural protein of

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intramuscular connective tissue that plays an important role in binding the myofibers to provide

structure. (Borg and Caulfield 1980; Chang and others 2010). It has been shown that the

toughness of a muscle is proportional to its intramuscular collagen content (Aktas 2003).

Many methods for altering meat tenderness have been evaluated, including the use of

marinades (Burke and Monahan 2003). Acidic marination involves the immersion of meat in a

solution containing vinegar (Kijowski 1993; Kijowski and Mast 1993), wine or fruit juice

(Arganosa and Marriott 1989; Burke and Monahan 2003), lactic acid (Aktas and Kaya 2001), or

other acidulants. Weak organic acids were tested for their ability to increase meat tenderization

either directly, through the physical weakening of muscle structures due to the swelling of

myofibers (Rao and Gault 1990) and direct weakening of the perimysial connective tissue (Lewis

and others 1991), or indirectly, through the activation of proteolysis by a release of cathepsins

from lysosomes (Berge and others 2001).

Presently, sodium phosphate is commonly used in meat processing, and has been

documented to increase protein solubility and the water-binding ability of meat (Hellendoorn

1962; Trout and Schmidt 1986). Smith and others (1984) concluded that injection of brine

containing sodium tri-polyphosphate into pork longissimus increased juiciness and reduced

Warner Bratzler shear values, and also increased juiciness when injected into beef

semimembranosus. While sodium phosphate is not a component of acid whey, calcium

phosphate (hydroxyapatite) is present at significant levels.

Several studies have evaluated calcium, in the form of calcium chloride, as a means for

increasing beef tenderness (Wheeler and others 1992; Whipple and Koohmaraie, 1993; Kerth

and others 1995). The tenderizing effects of marinating or injecting beef cuts with calcium

chloride, are postulated to relate in part to increased calcium- activated proteolysis (Cao and

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others 2012; Morgan and others 1991; Behrends and others 2005). However, a 10% injection of

0.3 M calcium chloride has been shown to have an adverse effect on palatability, imparting a

bitter, metallic and sour taste to the cooked product (Eilers and others 1994; Morris and others

1997). Calcium phosphate was shown to have less bitterness than calcium chloride, when applied

to cottage cheese (Puspitasari and others 1991), which suggests that the hydroxyapatite in acid

whey, may not be a significant cause of off-flavor development during marination.

Objectives

The exponential growth of the Greek yogurt industry led to the increase in supply of

Greek yogurt acid whey. This by-product has shown to be very difficult to process for food

consumption due to the low pH and high mineral content. Researchers have looked to explore

methods to improve processing and discovers new applications to use to acid whey. The

objective of this study is to evaluate the use of acid whey as a marinade for beef and its effects

on tenderness and other quality parameters.

Materials and Methods

Product Selection

Eye of round (semitendinosus) roasts (171C IMPS/NAMP) (n=13) were obtained from

grain fed cattle of about 15-18 months from a local beef packing facility. Vacuum packaged eye

of round roasts were transported to Brigham Young University and fabricated into steaks (2.54

cm thick). The steaks were then sub-divided into 3.00 cm x 3.00cm steak cubes prior to

application of treatments.

Treatments

The steak cubes from each roast were assigned randomly to either a control (no

marinade) or one of the five marination-treatments groups: calcium chloride, lactic acid, sodium

6

phosphate, acid whey (lot 1), and acid whey (lot 2). Calcium chloride marinade consisted of a pH

7.03 solution of (4.29 g/L) calcium chloride (Harris & Ford LLC, Indianapolis, IN) in distilled

water. The calcium concentration was designed to match the average calcium concentration of

the two lots of acid whey. Lactic acid marinade was prepared by adding a 50% lactic acid

solution (Purac INC, Blair, Nebraska) to distilled water to a target pH of 4.26. This pH matched

the pH of the acid whey lots collected from Greek yogurt production. The phosphate treatment

contained 2% sodium phosphate (Gusto M31 Sodium Phosphate, Formtech Solutions Inc,

Schenevus, NY) in distilled water, with a final pH of 7.32. This was prepared as instructed by

Formtech Solutions Inc. for use in meat marination. The two lots of acid whey were obtained

from a local Greek yogurt manufacturer, and were produced on different manufacturing days.

The steaks and marinades were combined in vacuum pouches to determine the effect of the

marination.

Packaging and Marination

386 steak cubes were cut from 13 different eye round roasts and those individual steaks

were vacuum packaged with nothing (control) or with the appropriated marination treatment

solution in 20.3 x 30.48cm vacuum bags (nylon/polyethylene) at 23.5º C (Vacmaster VP215).

The 386 steak cubes were evenly distributed among the 6 different treatments (minimum of 60

steak cubes per treatment). All marinades were added to the steak cubes to equal 25% of raw cut

weight (25% wt/wt). Steaks were marinated for 48 hours at 4º C. Following the 48-hour

marination, the samples were removed from the vacuum bag to have the pH and weight recorded

and the remaining liquid in the bag was discarded.

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Cooking

Steaks were cooked in an electric convection oven at 260º C (Model: JTP18; GE

Appliances) on a broiler pan. Temperature was monitored with a hypodermic temperature probe

coupled with a digital thermocouple thermometer (Fluke 52II Thermometer). As the geometric

center of each steak reached a final temperature of 71º ± 2 C they were removed from the oven

and placed on a cooling rack for 2 min. The steak samples were then vacuum packaged in a 20.3

x 30.48 cm vacuum bag (nylon/polyethylene) at 23.5º C (Vacmaster VP215) and stored at 4º C

until they were analyzed for weight, collagen, color, shear force, scanning electron microscope

and sensory.

pH

An Orion Star pH meter (A211 benchtop model 320) with an Orion 9120APAW

KNIPHE electrode, designed to measure surface pH, was used to test the pH of the meat before

and after each marinade treatment. The probe was calibrated using pH buffer standards before it

was used to determine the final pH. The probe was held on the surface of each cube of meat until

a final pH was recorded.

Cook Loss

Each cube (n=442) of meat was weighed before the marinade treatment, after the

marination, and after cooking. To calculate the percent loss the following formula was used:

{(weight after marination - weight after cooking)/weight after marination} *100.

Color

A model Colorflex EZ Hunter lab colorimeter, equipped with a 25 mm-diameter

measuring area, was used to determine the color change of the uncooked meat due to each

treatment (n = 423). The colorimeter was calibrated using color standard tiles before each day of

8

use. The instrument was set to illuminant A and Commission International de l’Eclariage (CIE)

L* (lightness), a*(redness), and b*(yellowness) values were recorded. To capture a complete

representation of the color, the samples were rotated after each measurement for a total of 5

measurements per sample.

Shear Force

Following cooking and refrigerated storage overnight at 4º C, a cylindrical core sample

was taken from the center of each cube (1.5 cm diameter) by cutting with a cork borer parallel to

the muscle fiber orientation (n= 385). Using a texture analyzer (TAXT2, Texture Technologies,

Hamilton, MA) with a Warner-Bratzler attachment, the core samples were sheared perpendicular

to the muscle fibers using settings suggested by the instrument manufacturer. Compression-mode

setting, with a contact force of 1 g and trigger force of 20 g was used with a test speed of 3.3

mm/sec over a 30-mm distance. After the test was completed the remaining portions of steaks

were refrigerated at 4º C and used later to determine soluble and insoluble collagen.

Scanning Electron Microscopy

Samples of uncooked marinated beef were produced by cutting a small section (<0.5 mm

thick) against the natural grain of the meat samples. The samples were placed in a buffer solution

(2% glutaraldehyde in 0.06 M sodium cacodylate) for 24 h, then rinsed with 0.03M sodium

cacodylate buffer 6 times at 10 min intervals. Rinsed samples were then fixed with 1% osmium

tetroxide (OsO4) for 1 h. Next, the samples were rinsed with distilled water to remove any

remaining OsO4. Subsequent samples were then dehydrated by gradient ethanol series (10, 30,

50, 70, and 95 vol.%) for 15 min in each solution and in absolute ethanol for 45 min. The

samples were then dried in a critical point dryer (Tousimis 931, Rockville, MD), and sputter

coated with gold/palladium (10nm). The specimens were examined and photographed using

9

environmental scanning electron microscopy (FEI XL30 ESEM FEG). The ESEM was

performed in “high vacuum” mode to avoid imaging noise in the picture. An accelerating voltage

of 10kV (spot size 3.0) and working distance of 10.2. The mounted samples were placed in a

chamber with a pressure of 1.0 Torr.

Collagen Content

Collagen content was determined in beef samples according to the AOAC (2000)

procedures (Method No. 990.26), with modifications stated in the procedure of Eilert and

Mandigo (1993). Steaks were chopped in a food processor and a 4g sample was placed in 22 ml

of Ringer’s solution and homogenized for 1 min using a tissue homogenizer at 20,000 rpm.

Homogenates were heated in a water bath for 15 mins at 50º C and subsequently centrifuged

(HN-SII Benchtop Centrifuge) at 2500 G at room temperature for 5 mins. The supernatant was

filtered through No. 5 filter paper (Thermo Fisher Scientific) into an Erlenmeyer flask labeled as

soluble collagen. The sediments were mixed with 10 mL of ¼-strength Ringer’s solution and

centrifuged again. The supernatant was filtered into the soluble collagen flask and the pellet and

filter paper were placed in a flask labeled as insoluble collagen. Sulfuric acid (3.5M/30 mL) was

added to both the soluble and insoluble portions. The flasks were covered and heated in an oven

at 105º C for at least 20 h. The collagen content of the sediment was determined as per the

method in the AOAC. Hydroxyproline standards were used to generate a calibration curve, and

collagenous connective tissue content was multiplied by 7.52 and 7.25 to determine soluble and

insoluble collagen, respectively (Cross and others 1973).

Consumer Panel Analysis

The sensory properties of the steaks prepared using different marinades, were evaluated

by a 110-member consumer taste panel conducted at the Brigham Young University Sensory

10

Laboratory. The panel was conducted in a single session in the afternoon. Panelists were

recruited from a database of university employees and students and were selected based on their

willingness to evaluate steak. Both genders were equally represented, with approximately equal

representation among age categories from age 20 to 60 y. The study was approved by the

university Institutional Review Board and panelists provided their informed consent. The steaks

were prepared by cutting each steak into 2.54 cm x 3 cm x 3 cm cubes. Each steak cube was

marinated and cooked as described previously. Cooked samples were placed in an insulated

steam table set at 71º C to keep them warm until serving. Fresh samples were prepared every 30

mins, by cutting each sample with the grain of the meat into four equal pieces, and then each

panelist was given two of the four pieces. The panelists were served five different samples, each

representing a different treatment. Samples were served in 2 oz. plastic cups, labeled with a

random 3-digit blinding code; and panelists were directed to consume the samples in order from

left to right. Sample presentation order was randomized to ensure that each sample saw an

approximately equal number of presentations in each position. Questions were presented one-at-

a-time on a computer screen and data were collected using Compusense® 5 (version 4.6)

software (Compusense Inc., Guelph, Ontario, Canada). Panelists evaluated appearance, aroma,

flavor, texture, and overall liking using a discrete 9-point hedonic scale, where 9 = like

extremely, 5 = neither like nor dislike, and 1=dislike extremely. They also evaluated the cooked

steaks from each treatment for tenderness and juiciness using a 5-point just about right (JAR)

ideality scale (1=definitely not juicy enough, 3=just about right, 5=definitely too juicy). The

question about overall liking was placed at the end of the questionnaire to obtain a response that

allowed time for panelists to consider all aspects of sensory quality (McEwan and others 2005;

Nielson and others 2006). Panelists were instructed to use a bite of unsalted cracker and a sip of

11

bottled water to refresh their sense of taste between samples. Panelists were compensated

monetarily for their time.

Statistical Analysis

Data were analyzed for significance using Statistical Analysis System software version

9.1 (SAS Inst., Inc., Cary, N.C., U.S.A.). Analysis of variance (PROC GLM) was used to

analyze color, collagen, percent loss, shear force, and pH. Sensory data were analyzed using a

mixed model repeated measures analysis of variance (PROCMIXED). Both models used the

Tukey–Kramer procedure to determine significant difference among means. Significant

differences were defined as P < 0.05.

RESULTS AND DISCUSSION

Compositional Analysis

The proximate composition and mineral content of the two different lots of acid whey are

presented in Table 1.

Table 1 Proximate composition (%) and mineral content (ppm) of acid wheys Acid

Whey

Moisture

Fat

Protein

Carb

Ca

K

Mg P Fe Zn

Lot 1 94.42 0.01 0.31 4.56 1154 1452 103 602 1.31 3.86

Lot 2 94.67 0.01 0.33 4.67 1226 1479 107 617 1.19 5.3

Both lots of whey were within expected values, and there was little chemical variation between

the two lots. The pH was 4.26 for lot 1 and 4.25 for lot 2.

Consumer Sensory Evaluation

The acid whey treated meat scored significantly better than the other marinades and not

significantly different than the control in overall acceptance, appearance, flavor and aftertaste in

12

the consumer sensory panel. The meat treated with acid whey was also rated as significantly

more tender and more juicy than the control, though not as tender as the calcium chloride or

phosphate treated samples. For tenderness, all samples scored in the “slightly not tender enough”

range (see Table 2), except for the phosphate treatment which was rated “just about right”. All

marinated samples, including acid whey, scored “just about right” in juiciness, whereas the

control scored “slightly not juicy enough.”

It is of particular interest that the acid whey marinade resulted in significantly better

flavor and a more acceptable aftertaste than all other marinade treatments, indicating that the

minerals and other components of the whey had a positive, rather than negative impact on flavor.

Table 2 Consumer acceptance and hedonic ratingsa for beef eye of round steaks marinated in different solutions Sample (n=110) Overall Acceptability Appearance Flavor Tenderness Juiciness Aftertaste

Control 7.03a 7.27a 7.01a 2.39b ± 0.61 2.32b± 0.68 6.65a

Acid Whey 6.87a 6.82a 7.11a 2.67ab ± 0.33 2.71a ± 0.29 6.67a

Lactic Acid 6.02b 6.19b 5.89b 2.61ab ± 0.39 2.91a ± 0.09 5.68b

Na Phosphate 6.37b 6.10b 6.35b 2.77a ± 0.23 2.96a ± 0.04 5.89b

Calcium Chloride 6.36b 6.25b 6.12b 2.71a ± 0.29 2.82a ± 0.18 6.03b

aMeans for sensory panel rating for overall acceptability, appearance, flavor, tenderness, juiciness and aftertaste (n=110). Acceptability, appearance, flavor, and aftertaste were calculated based on a 9-point hedonic scale,1=dislike extremely, 9=like extremely. Tenderness was calculated based on a 5-point JAR scale, 5=definitely too tender, 3=just-about-right, 1=definitely not tender enough. Juiciness was calculated based on a 5-point JAR scale, 5=definitely too juicy, 3=just-about-right, 1=definitely not juicy enough. Like super-scripts within a column represent no significant difference (p>0.05). Compositional and Textural Properties

The pH for the acid whey (5.18) treated beef was significantly lower than the control

sample and all other treatments. This result was unexpected because the lactic acid and whey

marinades were initially at the same pH. This difference in pH could possibly be explained by

the inherent microbial load of the acid whey, that may still have continued to produce lactic acid

13

while the meat was marinated; or alternatively the pH difference may have resulted from

presence of natural buffers in the whey samples.

The statistical differences for tenderness, exhibited in the consumer panel, were not

picked up using the Warner-Bratzler attachment on the texture analyzer. There was no significant

difference in peak force detected between any of the treatments (see Table 3). This is clearly a

case where the human sensory organ is more accurate than the instrument. We specifically used

the semitendinosus muscle in this study because it is a tougher cut of beef, which could show

potential enhancements from marination pretreatment. However, DeYonge-Freeman and others

(2000) reported no improvement in semitendinosus tenderness after calcium chloride injection.

Aktas and Kaya (2001), reported that longissimus dorsi decreased in peak force when treated

with lactic acid. However, this tenderization effect was attributed to the change in pH to below

4.0. Similarly, Ertbjerg and others (1995) showed that lactic acid injected at low concentration

(0.3 M), leading to a pH of 5.2 -- which is close to the isoelectric point of the major myofibrillar

protein, did not improve beef texture, while injection at 1.0 M resulted

Table 3 Meansa for soluble collagen, insoluble collagen, cook loss percentage, pH, and shear force. Treatment

Soluble Collagen (mg/g) n=346

Insoluble Collagen (mg/g) n=346

Cook loss (%)n=422

pH n=422

Shear Force (g) n=385

Control 0.058a ± 0.006 5.773a ± 0.268 29.08b ± 0.94 5.88b ± 0.07 4714.47a ± 254

Acid Whey 0.053a ± 0.006 6.008a ± 0.262 34.78a ± 0.92 5.18a ± 0.07 4928.18a ± 278

Lactic Acid 0.042a ± 0.006 5.913a ± 0.266 39.92c ± 0.92 5.91b ± 0.07 5180.85a ± 243

Na Phosphate 0.103b ± 0.006 6.235a ± 0.267 19.72d ± 0.93 6.99c ± 0.07 4847.75a ± 251

Ca Chloride 0.040a ± 0.006 6.020a ± 0.267 39.68c ± 0.94 5.89b ± 0.07 4937.44a ± 259

aPercent cook loss was calculated using weights taken immediately prior to and following cooking. pH was determined immediately after the 48 hour marination period using a surface pH probe. Shear force was calculated by recording the peak force applied to the sample core. Like super-scripts within a column represent no significant difference (p>0.05).

14

in a meat pH of 4.6 and decreased meat toughness. Since the meat pH for all the treatments

evaluated in our study did not fall below pH 5.0 (acid whey treatment being the lowest, at pH

5.18), it is possible that lower pH marinade treatments may have resulted in greater differences

in tenderness as manifested by texture analyzer peak force. Lower pH treatments were not

evaluated, due to our interest in evaluating the efficacy of untreated acid whey, at its native pH.

The only significant increase in soluble collagen content was observed in the phosphate

treatment, which showed a nearly 1.8-fold increase (see Table 3) in collagen solubility compared

to the control. There was no significant difference in the amount of insoluble collagen for any of

the treatments (see Table 3). Collagen has swelling properties under acidic or alkali conditions,

and swollen collagen can be more readily converted into gelatin at high temperatures such as

those encountered during cooking. In a number of previous studies, increased collagen solubility

under acidic or alkali conditions resulted in improvements of meat tenderness. For example,

Naveena and others (2011) reported that collagen solubility of buffalo meat increased with

increasing ammonium hydroxide concentrations. Oreskovich and others (1992) noted an increase

in soluble collagen values in beef marinated with 0.7M acetic acid (pH 2.50), compared to those

in control (non-buffer) and 0.1M NaCl (pH 6.50) marinated beef. Chang and others (2010)

suggested that marination with weak organic acids causes the denaturation of intramuscular heat-

soluble collagen. Even though the lactic acid and acid whey contained weak organic acids, the

concentration at which the treatments were applied to each sample possibly did not lower the pH

enough to result in a change in the soluble collagen content. While the acid whey and phosphate

pH levels were significantly different from control (the phosphate sample was near neutral, the

acid whey sample remained above pH 5), only the phosphate had an effect on the soluble

collagen.

15

As expected, the phosphate treatment had the lowest cook loss percentage (19.72%) and

was significantly lower than all the other treatments (p>0.05) (see Table 3). Phosphates are well

known for increasing water holding capacity and reducing cook loss in meat products (Roldan

and others 2014). However, it is interesting to note that, of all marinade treatments, the acid

whey treated sample had the next lowest percentage cook loss at 34.78% which was only about

5% higher than the control and significantly lower than the calcium chloride and lactic acid

treatments. Gault (1984, 1985), and Rao and Gault (1990), and Offer and others (1989) found

that meat treated with acidic marinades with a pH below 5.0 suffered less cooking loss. This may

help explain the lower cook loss for the acid whey treated sample, compared to the other non-

phosphate marinades.

Color

Before the samples were cooked, the acid whey treatment was significantly darker than

the control sample, but not significantly different from control on the red-green (a*) or blue-

yellow

Table 4 Effect of acid whey and other marinade treatments on colora of beef eye of round. Before Cooking After Cooking

Sample CIE L* CIE a* CIE b* CIE L* CIE a* CIE b*

Control 54.92a ± 0.70 7.60c ± 0.50 14.16b ± 0.34 42.59ab ± 0.94 8.74b ± 0.37 17.16ab ± 0.36

Acid Whey 47.93b ± 0.70 7.42c ± 0.50 13.95b ± 0.34 40.82bc ± 1.01 7.09c ± 0.41 15.29c ± 0.42

Lactic Acid 53.36a ± 0.71 8.79bc ± 0.51 14.26b ± 0.35 43.35a ± 0.96 9.11a ± 0.38 17.56a ± 0.37

Phosphate 42.94c ± 0.70 9.97b ± 0.50 13.45b ± 0.34 44.92a ± 0.99 9.03a ± 0.39 18.46a ± 0.38

Calcium

Chloride

38.33d ± 0.70 17.99a ± 0.52 18.20a ± 0.35 38.35c ± 0.96 10.24a ± 0.38 16.85bc ± 0.37

aThe three CIE color coordinates are defined as: L*, where 0 = black and 100 = white; a*, where negative values indicate green, while positive values indicate red; and b*, where negative values indicate blue and positive values indicate yellow. Like super-scripts within a column represent no significant difference (p>0.05).

16

(b*) scales (see Table 4). After cooking, the acid whey marinated meat was no longer

significantly darker than the control, but was significantly less red and less yellow. Compared to

the other treatments, the uncooked acid whey samples were also significantly darker than lactic

acid, but lighter than phosphate and calcium treatments, both of which were similar to the

control. The calcium chloride treatment was significantly darker than all other treatments and

control, though not significantly darker than the acid whey.

The color effects reported in Table 4, for the uncooked samples, differ somewhat from

color changes resulting from the natural decline in pH of beef as it ages. Generally, the natural

pH drop in beef during aging, leads to a lighter color (Wojciak 2014), whereas in our study, the

lowest pH product (acid whey marination) resulted in a darker colored product, compared to

control. The minerals and sugars present in the whey may have played a role in the darkening

effect, possibly by altering the oxidation state of the myoglobin.

Considering all the differences in cooked beef color across the sample spectrum, acid

whey treated beef was the only marinated sample that was at statistical parity with the control

sample for consumer acceptance of appearance, with the two most preferred samples being the

acid whey and control sample.

Electron Microscopy

Figure 1 shows an electron micrograph of uncooked muscle and connective tissue from

the bovine semitendinosus muscles 48 h after each treatment (control-no treatment, acid whey,

calcium chloride, lactic acid, and phosphate). Each image examines the intramuscular collagen

matrix; and the effects of each treatment on the collage structure can be seen by comparison to

the untreated control.

17

The unmarinated, raw control sample shows bundles of regular muscle fibers with an associated,

crisscrossed network of fairly tight collagenous fibers. The acid whey sample, by comparison,

shows a much looser, more open collagen structure, especially compared to the calcium chloride

sample. The lactic acid treated sample is quite similar to the control, while the phosphate treated

sample shows very loose, widely separated and disordered collagen fibers, very much unlike any

of the other samples.

The electron micrographs support the findings of the collagen extraction data in that there

are no dramatic differences in the appearance of the collagen in any of the samples, with the

exception of the phosphate treated sample. The control-no treatment, lactic acid and calcium do

not have any physical sign of collagen breakdown; however, the acid whey sample seems to

show signs that some of the collagen fibers are starting to breakdown.

18

Control Acid Whey

Calcium Chloride Lactic Acid

Phosphate

Figure 1 Scanning electron micrograph (SEM) of raw beef muscles with different treatments (control-no treatment, acid whey, calcium chloride, lactic acid, and phosphate)

19

Conclusion

Untreated acid whey appears to be a suitable base for beef marinade. Consumer

acceptance testing showed that marination of beef in acid whey resulted in the highest overall

flavor acceptance scores, and significantly improved the tenderness and juiciness of samples

relative to the control, without any indication of negative off-flavors. However, the increase in

tenderness and juiciness observed in consumer testing was not large enough for analytical

instrumentation to detect with statistical significance, and was not manifested in the

soluble/insoluble collagen results. Acid whey resulted in more significant cook loss than

control, though significantly less than the non-phosphate marinades evaluated. The color of acid

whey treated beef was different than control, but did not significantly affect consumer

acceptance. The effects of lactic acid and calcium were individually evaluated in other

treatments, and no apparent synergistic effects on meat tenderization with the combination of

calcium and lactic acid present in acid whey were observed. The main driver in tenderization

seems to be the change in pH from the marination.

20

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Obuz E, Dikeman M, Grobbel J, Stephens J, Loughin T. 2004. Beef longissimus lumborum, biceps femoris, and deep pectoralis Warner-Bratzler shear force is affected differently by endpoint temperature, cooking method, and USDA quality grade. Meat Sci. 68(2):243-8. Offer G, Knight P, Jeacocke R, Almond R, Cousins T, Elsey J, Parsons N, Sharp A, Starr R, Purslow P. 1989. The Structural Basis of the Water-Holding, Appearance and Toughness of Meat and Meat-Products. Food Microstruct. 8(1):151-70. Oreskovich DC, Bechtel PJ, McKeith FK, Novakofski J, Basgall EJ. 1992. Marinade pH affects textural properties of beef. J.Food Sci. 57(2):305-11. Ostoja H, Cierach M. 2003. Effect of calcium ions on the solubility of muscular collagen and tenderness of beef meat. Nahr.-Food 47(6):388-90. Patel P, Merson R. 1978. Ultrafiltration of Cottage Cheese Whey - Influence of Whey Constituents on Membrane Performance. J.Food Sci.Technol.-Mysore 15(2):56-60. Patocka G, Jeleen P. 1991. Calcium Association with Isolated Whey Proteins. Canadian Institute of Food Science and Technology Journal-Journal De L Institut Canadien De Science Et Technologie Alimentaires 24(5):218-23. Pietrasik Z, Dhanda J, Pegg R, Shand P. 2005. The effects of marination and cooking regimes on the waterbinding properties and tenderness of beef and bison top round roasts. J.Food Sci. 70(2):S102-6. Pringle TD, Harrelson JM, West RL, Williams SE, Johnson DD. 1999. Calcium-activated tenderization of strip loin, top sirloin, and top round steaks in diverse genotypes of cattle. J.Anim.Sci. 77(12):3230-7. Puspitasari N, Lee K, Greger J. 1991. Calcium Fortification of Cottage Cheese with Hydrocolloid Control of Bitter Flavor Defects. J.Dairy Sci. 74(1):1-7. Rao MV, Gault NFS. 1990. Acetic acid marinading - the rheological characteristics of some raw and cooked beef muscles which contribute to changes in meat tenderness. J.Texture Stud. 21(4):455-77. Roldan M, Antequera T, Perez-Palacios T, Ruiz J. 2014. Effect of added phosphate and type of cooking method on physico-chemical and sensory features of cooked lamb loins. Meat Sci. 97(1):69-75. Sady M, Jaworska G, Grega T, Bernas E, Domagala J. 2013. Application of Acid Whey in Orange Drink Production. Food Technol.Biotechnol. 51(2):266-77. Scanga J, Delmore R, Ames R, Belk K, Tatum J, Smith G. 2000. Palatability of beef steaks marinated with solutions of calcium chloride, phosphate, and (or) beef-flavoring. Meat Sci. 55(4):397-401.

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25

Appendix

Texture Analyzer Test Settings

Each day before it was used the texture analyzer was calibrated for height and force of 50 mm

with a return speed of 10 mm/sec and a contact force of 1 g. The following below are the settings

for the TAXT2 with the Warner Bratzler attachment.

Test Mode – Compression

Pretest Speed – 3.3 mm/sec

Test Speed – 3.3 mm/sec

Post Test Speed – 5 mm/sec

Target Mode - Distance

Distance - 30 mm

Trigger Type - Auto (Force)

Tigger Force - 20 g

Advanced Options – Off

Collagen Method - Detailed

The cooked steaks after the texture analyzer test were ground into a homogenous mixture

using a food processor for 1 min. Ground samples (4g) were placed into 50mL polycarbonate

centrifuge tubes in duplicate. Ringer’s solution (22mL; NaCl, CaCl, and KCl) was added to each

tube and the samples were homogenized in a Tissue Master Homogenizer 125 at 20,000 rpm for

1 min. The samples were then heated in a water bath at 50C for 15 min and subsequently

centrifuged (HN-SII Benchtop Centrifuge) at 2500 G at room temperature for 5 min. The

supernatant was filtered through #5 filter paper (Thermo Fisher Scientific) into an Erlenmeyer

26

flask labeled as soluble collagen. The pellet was rinsed with ¼ strength Ringer’s Solution

(10mL) and centrifuged as previously described. The supernatant was filtered into the soluble

collagen flask and the pellet and filter paper were placed in a flask labeled as insoluble collagen.

Sulfuric acid (3.5M/30 mL) was added to both the soluble and insoluble portions. The flasks

were covered and placed in an oven (Lab Line Imperial II Radiant Heat Oven) at 105C for at

least 20 h. The hot hydrolysate labeled insoluble was diluted to 100mL with water, mixed and

filtered through Fisherbrand #5 filter paper. The hot hydrolysate labeled soluble was taken out

and boiled until the volume was less than 25 mL and then it was diluted to 25mL with water,

mixed and filtered through Fisherbrand #5 filter paper. The samples were pipetted (insoluble;

0.36 mL diluted hydrolysate and 5.64 mL of water; soluble; 6 mL of diluted hydrolysate) into

borosilicate glass 13 x 100mm test tubes. One milliliter oxidant (citric acid monohydrate, sodium

hydroxide, sodium acetate trihydrate, 1-propanol, and chloramine-t, pH=6) was added and the

samples were mixed and allowed to stand for 20 min. One mL color reagent (perchloric acid, 4-

dimehtylaminobenzaldehyde, and 2-propanol) was added and mixed. The resulting mixture was

heated to 60 C in a water bath for 15 min and then allowed to cool in cold tap water bath for 3

min. Absorbance of samples was read at 558nm using a VWR UV-1600PC Spectrophotometer.

Hydroxyproline standards were used to generate a calibration curve and collagenous connective

tissue content was multiplied by 7.52 and 7.25 to determine soluble and insoluble collagen,

respectively (Cross, Carpenter, and Smith, 1973).

27

Table 5 Complete Proximate Composition (%) and Mineral Content (ppm) of Acid Wheys Acid

Whey

Moisture

Fat

Protein

Carb

Ca

K

Mg P Fe Zn

Lot 1 94.42 0.01 0.31 4.56 1154 1452 103 602 1.31 3.86

Lot 2 94.67 0.01 0.33 4.67 1226 1479 107 617 1.19 5.3

Table 6 Complete Mineral Analysis (ppm) Boron Calcium Copper Iron Potassium Magnesium Manganese Sodium Phosphorus Zinc

Acid Whey (Lot 1)

7.89 1154 .26 1.31 1452 103 .11 649 602 3.86

Acid Whey (Lot 2)

7.1n4 1226 .10 1.19 1479 107 .09 666 617 5.30

28

Statistical Output The SAS System 13:11 Friday, July 28, 2017 9

Analysis of shear force grams

The Mixed Procedure

Model Information

Data Set WORK.GOOD

Dependent Variable force_g

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Model-Based

Degrees of Freedom Method Containment

Class Level Information

Class Levels Values

roast_num 13 01 02 03 04 05 06 07 08 09 10

11 12 13

treatment 6 AW1 AW2 CAL LAC NTR PHO

Dimensions

Covariance Parameters 3

Columns in X 7

Columns in Z 87

Subjects 1

Max Obs per Subject 385

Number of Observations

29

Number of Observations Read 385

Number of Observations Used 385

Number of Observations Not Used 0

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 6829.29385406

1 3 6826.35854055 0.00001172

2 1 6826.31946666 0.00000026

3 1 6826.31866680 0.00000000

Convergence criteria met.

The SAS System 13:11 Friday, July 28, 2017 10


The Mixed Procedure

Covariance Parameter Estimates

Cov Parm Estimate

roast_num 66529

roast_num*treatment 70047

Residual 3533748

Fit Statistics

-2 Res Log Likelihood 6826.3

AIC (Smaller is Better) 6832.3

AICC (Smaller is Better) 6832.4

30

BIC (Smaller is Better) 6834.0

Type 3 Tests of Fixed Effects

Num Den

Effect DF DF F Value Pr > F

treatment 5 56 0.41 0.8374

Least Squares Means

Standard

Effect treatment Estimate Error DF t Value Pr > |t|

treatment AW1 4928.18 252.16 56 19.54 <.0001

treatment AW2 4919.98 304.01 56 16.18 <.0001

treatment CAL 4937.44 259.50 56 19.03 <.0001

treatment LAC 5180.85 243.11 56 21.31 <.0001

treatment NTR 4714.47 254.76 56 18.51 <.0001

treatment PHO 4847.75 251.88 56 19.25 <.0001

Differences of Least Squares Means

Standard

Effect treatment _treatment Estimate Error DF t Value Pr > |t| Adjustment

treatment AW1 AW2 8.2010 381.19 56 0.02 0.9829 Tukey-Kramer

treatment AW1 CAL -9.2582 346.90 56 -0.03 0.9788 Tukey-Kramer

treatment AW1 LAC -252.67 334.43 56 -0.76 0.4531 Tukey-Kramer

treatment AW1 NTR 213.71 342.97 56 0.62 0.5357 Tukey-Kramer

treatment AW1 PHO 80.4274 340.98 56 0.24 0.8144 Tukey-Kramer


31




The Mixed Procedure


Standard




treatment CAL LAC -243.41 340.65 56 -0.71 0.4779 Tukey-Kramer

treatment CAL NTR 222.97 348.84 56 0.64 0.5253 Tukey-Kramer

treatment CAL PHO 89.6856 346.58 56 0.26 0.7968 Tukey-Kramer

treatment LAC NTR 466.38 336.64 56 1.39 0.1714 Tukey-Kramer

treatment LAC PHO 333.10 334.43 56 1.00 0.3235 Tukey-Kramer

treatment NTR PHO -133.29 343.00 56 -0.39 0.6991 Tukey-Kramer


Effect treatment _treatment Adj P

treatment AW1 AW2 1.0000

treatment AW1 CAL 1.0000

treatment AW1 LAC 0.9737

treatment AW1 NTR 0.9888

treatment AW1 PHO 0.9999


32




treatment CAL LAC 0.9794

treatment CAL NTR 0.9875

treatment CAL PHO 0.9998

treatment LAC NTR 0.7354

treatment LAC PHO 0.9173

treatment NTR PHO 0.9988


Analysis of shear force newtons

The Mixed Procedure

Model Information

Data Set WORK.GOOD

Dependent Variable force_n







Class Levels Values

roast_num 13 01 02 03 04 05 06 07 08 09 10

33

11 12 13


Dimensions


Columns in X 7

Columns in Z 87

Subjects 1






Iteration History


0 1 3323.77500780

1 3 3320.83968576 0.00002738

2 1 3320.80061287 0.00000060

3 1 3320.79981305 0.00000000




The Mixed Procedure


34

Cov Parm Estimate

roast_num 6.3981

roast_num*treatment 6.7366

Residual 339.84

Fit Statistics






Num Den


treatment 5 56 0.41 0.8374

Least Squares Means

Standard


treatment AW1 48.3289 2.4728 56 19.54 <.0001

treatment AW2 48.2486 2.9814 56 16.18 <.0001

treatment CAL 48.4198 2.5448 56 19.03 <.0001

treatment LAC 50.8068 2.3841 56 21.31 <.0001

treatment NTR 46.2331 2.4984 56 18.51 <.0001

35

treatment PHO 47.5402 2.4701 56 19.25 <.0001


Standard











The Mixed Procedure


Standard








36





















Analysis of soluble collagen

The Mixed Procedure

Model Information

37

Data Set WORK.GOOD

Dependent Variable soluble







Class Levels Values

roast_num 14 1 01 02 03 04 05 06 07 08 09

10 11 12 13


Dimensions


Columns in X 7

Columns in Z 92

Subjects 1






Iteration History

38


0 1 -1318.54499304

1 3 -1339.05688676 0.00001527

2 1 -1339.07229360 0.00000003

3 1 -1339.07232096 0.00000000




The Mixed Procedure


Cov Parm Estimate

roast_num 0.000068


Residual 0.000928

Fit Statistics

-2 Res Log Likelihood -1339.1

AIC (Smaller is Better) -1333.1

AICC (Smaller is Better) -1333.0

BIC (Smaller is Better) -1331.2


Num Den


39

treatment 5 59 18.63 <.0001

Least Squares Means

Standard


treatment AW1 0.05416 0.005939 59 9.12 <.0001

treatment AW2 0.05225 0.005636 59 9.27 <.0001

treatment CAL 0.04004 0.005920 59 6.76 <.0001

treatment LAC 0.04250 0.005803 59 7.32 <.0001

treatment NTR 0.05787 0.005900 59 9.81 <.0001

treatment PHO 0.1036 0.005829 59 17.78 <.0001


Standard



treatment AW1 CAL 0.01412 0.007755 59 1.82 0.0737 Tukey-Kramer

treatment AW1 LAC 0.01166 0.007629 59 1.53 0.1319 Tukey-Kramer

treatment AW1 NTR -0.00371 0.007693 59 -0.48 0.6314 Tukey-Kramer

treatment AW1 PHO -0.04946 0.007669 59 -6.45 <.0001 Tukey-Kramer


treatment AW2 LAC 0.009750 0.007407 59 1.32 0.1932 Tukey-Kramer



The Mixed Procedure

40


Standard





treatment CAL NTR -0.01783 0.007718 59 -2.31 0.0244 Tukey-Kramer

treatment CAL PHO -0.06357 0.007675 59 -8.28 <.0001 Tukey-Kramer

treatment LAC NTR -0.01537 0.007597 59 -2.02 0.0477 Tukey-Kramer

treatment LAC PHO -0.06111 0.007562 59 -8.08 <.0001 Tukey-Kramer

treatment NTR PHO -0.04574 0.007638 59 -5.99 <.0001 Tukey-Kramer







treatment AW1 PHO <.0001






41


treatment CAL PHO <.0001


treatment LAC PHO <.0001

treatment NTR PHO <.0001


Analysis of insoluble collagen

The Mixed Procedure

Model Information

Data Set WORK.GOOD

Dependent Variable insoluble







Class Levels Values

roast_num 14 1 01 02 03 04 05 06 07 08 09

10 11 12 13


Dimensions


42

Columns in X 7

Columns in Z 92

Subjects 1






Iteration History


0 1 1173.36951524

1 2 1081.58740007 0.00047149

2 1 1081.47171732 0.00000720

3 1 1081.47005545 0.00000000



Analysis of insoluble collagen

The Mixed Procedure


Cov Parm Estimate

roast_num 0.5831

43


Residual 1.1369

Fit Statistics






Num Den


treatment 5 59 1.41 0.2355

Least Squares Means

Standard


treatment AW1 6.2048 0.2695 59 23.03 <.0001

treatment AW2 5.8102 0.2622 59 22.16 <.0001

treatment CAL 6.0204 0.2674 59 22.52 <.0001

treatment LAC 5.9125 0.2662 59 22.21 <.0001

treatment NTR 5.7734 0.2685 59 21.51 <.0001

treatment PHO 6.2346 0.2673 59 23.32 <.0001


Analysis of L_star

The Mixed Procedure

44

Model Information

Data Set WORK.GOOD

Dependent Variable L







Class Levels Values

roast_num 14 0 01 02 03 04 05 06 07 08 09

10 11 12 13


Dimensions


Columns in X 7

Columns in Z 93

Subjects 1





45


Iteration History


0 1 2397.67854979

1 2 2365.06382313 0.00001703

2 1 2365.04943227 0.00000014

3 1 2365.04931689 0.00000000



Analysis of L_star

The Mixed Procedure


Cov Parm Estimate

roast_num 1.2683


Residual 13.8671

Fit Statistics






Num Den

46


treatment 5 60 96.76 <.0001

Least Squares Means

Standard


treatment AW1 46.9736 0.7077 60 66.38 <.0001

treatment AW2 48.8839 0.7001 60 69.83 <.0001

treatment CAL 38.3260 0.7183 60 53.36 <.0001

treatment LAC 53.3579 0.7132 60 74.81 <.0001

treatment NTR 54.9156 0.6997 60 78.48 <.0001

treatment PHO 42.9406 0.7002 60 61.33 <.0001


Standard


treatment AW1 AW2 -1.9104 0.8944 60 -2.14 0.0368 Tukey-Kramer

treatment AW1 CAL 8.6476 0.9053 60 9.55 <.0001 Tukey-Kramer

treatment AW1 LAC -6.3844 0.9029 60 -7.07 <.0001 Tukey-Kramer

treatment AW1 NTR -7.9420 0.8910 60 -8.91 <.0001 Tukey-Kramer

treatment AW1 PHO 4.0330 0.8922 60 4.52 <.0001 Tukey-Kramer

treatment AW2 CAL 10.5579 0.9027 60 11.70 <.0001 Tukey-Kramer



Analysis of L_star

47

The Mixed Procedure


Standard




treatment CAL LAC -15.0319 0.9111 60 -16.50 <.0001 Tukey-Kramer

treatment CAL NTR -16.5896 0.8994 60 -18.45 <.0001 Tukey-Kramer


treatment LAC NTR -1.5576 0.8969 60 -1.74 0.0876 Tukey-Kramer

treatment LAC PHO 10.4174 0.8967 60 11.62 <.0001 Tukey-Kramer

treatment NTR PHO 11.9750 0.8859 60 13.52 <.0001 Tukey-Kramer




treatment AW1 CAL <.0001

treatment AW1 LAC <.0001

treatment AW1 NTR <.0001






48

treatment CAL LAC <.0001

treatment CAL NTR <.0001






Analysis of A_star

The Mixed Procedure

Model Information

Data Set WORK.GOOD

Dependent Variable A







Class Levels Values

roast_num 14 0 01 02 03 04 05 06 07 08 09

10 11 12 13


Dimensions

49


Columns in X 7

Columns in Z 93

Subjects 1






Iteration History


0 1 2131.86777556

1 2 2107.77186182 0.00000037

2 1 2107.77161266 0.00000000



Analysis of A_star

The Mixed Procedure


Cov Parm Estimate

roast_num 0


Residual 7.4463

50

Fit Statistics






Num Den


treatment 5 60 63.69 <.0001

Least Squares Means

Standard


treatment AW1 7.4765 0.5093 60 14.68 <.0001

treatment AW2 7.3554 0.5035 60 14.61 <.0001

treatment CAL 17.9946 0.5174 60 34.78 <.0001

treatment LAC 8.7856 0.5133 60 17.11 <.0001

treatment NTR 7.5975 0.5034 60 15.09 <.0001

treatment PHO 9.9728 0.5034 60 19.81 <.0001


Standard



treatment AW1 CAL -10.5181 0.7260 60 -14.49 <.0001 Tukey-Kramer

51



treatment AW1 PHO -2.4963 0.7161 60 -3.49 0.0009 Tukey-Kramer




Analysis of A_star

The Mixed Procedure


Standard




treatment CAL LAC 9.2089 0.7288 60 12.64 <.0001 Tukey-Kramer

treatment CAL NTR 10.3971 0.7218 60 14.40 <.0001 Tukey-Kramer

treatment CAL PHO 8.0217 0.7219 60 11.11 <.0001 Tukey-Kramer


treatment LAC PHO -1.1872 0.7190 60 -1.65 0.1039 Tukey-Kramer






52















Analysis of B_star

The Mixed Procedure

Model Information

Data Set WORK.GOOD

Dependent Variable B





53



Class Levels Values

roast_num 14 0 01 02 03 04 05 06 07 08 09

10 11 12 13


Dimensions


Columns in X 7

Columns in Z 93

Subjects 1






Iteration History


0 1 1696.76347810

1 2 1616.40808211 0.00003243

2 1 1616.39358612 0.00000023

3 1 1616.39348630 0.00000000


54


Analysis of B_star

The Mixed Procedure


Cov Parm Estimate

roast_num 0.6419


Residual 2.2175

Fit Statistics






Num Den


treatment 5 60 42.63 <.0001

Least Squares Means

Standard


treatment AW1 13.7292 0.3461 60 39.67 <.0001

treatment AW2 14.1771 0.3414 60 41.53 <.0001

treatment CAL 18.1973 0.3496 60 52.04 <.0001

55

treatment LAC 14.2598 0.3477 60 41.01 <.0001

treatment NTR 14.1562 0.3435 60 41.21 <.0001

treatment PHO 13.4534 0.3434 60 39.18 <.0001


Standard










Analysis of B_star

The Mixed Procedure


Standard




treatment CAL LAC 3.9376 0.3818 60 10.31 <.0001 Tukey-Kramer


56




treatment NTR PHO 0.7028 0.3719 60 1.89 0.0636 Tukey-Kramer



















Analysis of pH

57

The Mixed Procedure

Model Information

Data Set WORK.GOOD

Dependent Variable pH







Class Levels Values

roast_num 13 01 02 03 04 05 06 07 08 09 10

11 12 13


Dimensions


Columns in X 7

Columns in Z 90

Subjects 1





58


Iteration History


0 1 385.33780056

1 3 289.41176422 0.00134224

2 1 289.02933451 0.00005215

3 1 289.01560890 0.00000009

4 1 289.01558461 0.00000000



Analysis of pH

The Mixed Procedure


Cov Parm Estimate

roast_num 0.02252


Residual 0.08970

Fit Statistics






59

Num Den


treatment 5 59 152.53 <.0001

Least Squares Means

Standard


treatment AW1 5.2763 0.06991 59 75.47 <.0001

treatment AW2 5.0849 0.06722 59 75.65 <.0001

treatment CAL 5.8905 0.06883 59 85.58 <.0001

treatment LAC 5.9143 0.06776 59 87.28 <.0001

treatment NTR 5.8829 0.06905 59 85.20 <.0001

treatment PHO 6.9886 0.06807 59 102.66 <.0001

Differences of Least Squares Mean

Standard










60

Analysis of pH

The Mixed Procedure


Standard








treatment LAC PHO -1.0743 0.07586 59 -14.16 <.0001 Tukey-Kramer

treatment NTR PHO -1.1057 0.07700 59 -14.36 <.0001 Tukey-Kramer











61









Analysis of Difference

The Mixed Procedure

Model Information

Data Set WORK.GOOD

Dependent Variable Difference






Class Level Informatio

Class Levels Values

roast_num 16 01 02 03 04 05 06 07 08 09 10

11 12 13 5 7 8


62

Dimensions


Columns in X 7

Columns in Z 97

Subjects 1






Iteration History


0 1 2059.17520984

1 2 2020.78743165 0.22067827

2 3 2015.17839263 .

3 1 1972.79182564 0.03234612

4 1 1946.99105320 0.01974043

5 1 1931.64129158 0.01132416

6 1 1923.02193774 0.00581922

7 1 1918.69029247 0.00245977

8 1 1916.91582319 0.00071313



63

The Mixed Procedure

Iteration History


9 1 1916.42813935 0.00009423

10 1 1916.36886078 0.00000229

11 1 1916.36751662 0.00000000



Cov Parm Estimate

roast_num 58.5390


Residual 4.3283

Fit Statistics






Num Den


treatment 5 60 72.28 <.0001

Least Squares Means

Standard

64


treatment AW1 15.2339 1.9417 60 7.85 <.0001

treatment AW2 14.6047 1.9498 60 7.49 <.0001

treatment CAL 16.8317 1.9513 60 8.63 <.0001

treatment LAC 16.7133 1.9486 60 8.58 <.0001

treatment NTR 12.9773 1.9504 60 6.65 <.0001

treatment PHO 10.1558 1.9496 60 5.21 <.0001



The Mixed Procedure


Standard





treatment AW1 NTR 2.2566 0.4227 60 5.34 <.0001 Tukey-Kramer






treatment CAL LAC 0.1184 0.4243 60 0.28 0.7812 Tukey-Kramer

65



treatment LAC NTR 3.7360 0.4182 60 8.93 <.0001 Tukey-Kramer

















treatment LAC NTR <.0001




66

Analysis of Percent Loss

The Mixed Procedure

Model Information

Data Set WORK.GOOD

Dependent Variable percent_loss







Class Levels Values

roast_num 16 01 02 03 04 05 06 07 08 09 10

11 12 13 5 7 8


Dimensions


Columns in X 7

Columns in Z 97

Subjects 1




67



Iteration History


0 1 2930.72653354

1 2 2882.32125392 5.75891726

2 1 2850.53347033 2.18999730

3 3 2835.24856259 .

4 1 2790.86772424 0.02029831

5 1 2763.69420387 0.01242968

6 1 2747.33607263 0.00718642

7 1 2738.02921952 0.00375142

8 1 2733.26029633 0.00163612



The Mixed Procedure

Iteration History


9 1 2731.23759578 0.00050584

10 1 2730.64215266 0.00007672

11 1 2730.55880894 0.00000251

12 1 2730.55628130 0.00000000


68


Cov Parm Estimate

roast_num 619.98


Residual 29.5023

Fit Statistics






Num Den


treatment 5 60 83.48 <.0001

Least Squares Means

Standard


treatment AW1 47.6103 6.2927 60 7.57 <.0001

treatment AW2 45.9779 6.3130 60 7.28 <.0001

treatment CAL 51.1462 6.3162 60 8.10 <.0001

treatment LAC 51.3134 6.3107 60 8.13 <.0001

treatment NTR 40.4756 6.3144 60 6.41 <.0001

treatment PHO 31.1844 6.3127 60 4.94 <.0001

69



The Mixed Procedure


Standard














treatment LAC NTR 10.8379 1.1860 60 9.14 <.0001 Tukey-Kramer





70













treatment LAC NTR <.0001



The SAS System 14:10 Thursday, August 3, 2017 1


Analysis of L_star

The Mixed Procedure

Model Information

Data Set WORK.GOOD

Dependent Variable L


71






Class Levels Values

roast_num 13 01 02 03 04 05 06 07 08 09 10

11 12 13


Dimensions


Columns in X 7

Columns in Z 85

Subjects 1






Iteration History


0 1 3114.80564219

72

1 3 3069.01482958 0.00000021

2 1 3069.01459909 0.00000000



Analysis of L_star

The Mixed Procedure


Cov Parm Estimate

roast_num 5.0368


Residual 32.6313

Fit Statistics






Num Den


treatment 5 54 9.96 <.0001

Least Squares Means

Standard


73

treatment AW1 40.1934 0.9650 54 41.65 <.0001

treatment AW2 41.4492 1.2083 54 34.30 <.0001

treatment CAL 38.3509 0.9647 54 39.75 <.0001

treatment LAC 43.3548 0.9601 54 45.16 <.0001

treatment NTR 42.5889 0.9453 54 45.06 <.0001

treatment PHO 44.9239 0.9853 54 45.60 <.000


Standard










Analysis of L_star

The Mixed Procedure


Standard



74


treatment CAL LAC -5.0039 1.0318 54 -4.85 <.0001 Tukey-Kramer






















75



Analysis of A_star

The Mixed Procedure

Model Information

Data Set WORK.GOOD

Dependent Variable A







Class Levels Values

roast_num 13 01 02 03 04 05 06 07 08 09 10

11 12 13


Dimensions


Columns in X 7

Columns in Z 85

Subjects 1


76





Iteration History


0 1 2299.12804987

1 3 2274.94753058 0.00003877

2 1 2274.91922503 0.00000017

3 1 2274.91910733 0.00000000



Analysis of A_star

The Mixed Procedure


Cov Parm Estimate

roast_num 0.4946


Residual 6.1801

Fit Statistics




77



Num Den


treatment 5 54 11.79 <.0001

Least Squares Means

Standard


treatment AW1 7.3382 0.3805 54 19.29 <.0001

treatment AW2 6.8444 0.4960 54 13.80 <.0001

treatment CAL 10.2431 0.3803 54 26.93 <.0001

treatment LAC 9.1069 0.3781 54 24.08 <.0001

treatment NTR 8.7361 0.3709 54 23.55 <.0001

treatment PHO 9.0315 0.3903 54 23.14 <.0001


Standard








78



Analysis of A_star

The Mixed Procedure


Standard




treatment CAL LAC 1.1362 0.4578 54 2.48 0.0162 Tukey-Kramer














79











Analysis of B_star

The Mixed Procedure

Model Information

Data Set WORK.GOOD

Dependent Variable B







Class Levels Values

roast_num 13 01 02 03 04 05 06 07 08 09 10

80

11 12 13


Dimensions


Columns in X 7

Columns in Z 85

Subjects 1






Iteration History


0 1 2271.78562366

1 3 2248.88249469 0.00000214

2 1 2248.88099186 0.00000000



Analysis of B_star

The Mixed Procedure


Cov Parm Estimate

81

roast_num 0.3684


Residual 5.8076

Fit Statistics






Num Den


treatment 5 54 13.12 <.0001

Least Squares Means

Standard


treatment AW1 15.0550 0.3724 54 40.42 <.0001

treatment AW2 15.5306 0.4891 54 31.75 <.0001

treatment CAL 16.8479 0.3722 54 45.27 <.0001

treatment LAC 17.5609 0.3699 54 47.47 <.0001

treatment NTR 17.1602 0.3629 54 47.28 <.0001

treatment PHO 18.4622 0.3825 54 48.27 <.0001


Standard

82










Analysis of B_star

The Mixed Procedure


Standard






treatment CAL PHO -1.6143 0.4766 54 -3.39 0.0013 Tukey-Kramer





83

















84

Sensory Comments P r o d u c t s 1 - 935 935 No Treatment 2 - 146 146 Acid Whey 3 - 213 213 Calcium 4 - 527 527 Lactic Acid 5 - 798 798 Phosphate R e s u l t s 1 17 1-935 The taste was absent while the texture seemed unsettling. This was my least favorite

of the selections today.

1 19 1-935 tasted very good

1 40 1-935 It was really hard to answer all of these questions with the amount of sample that we

had. I didn't know how many more questions I was going to have to answer and two bites was

not enough to remember everything. If I could answer all of the questions at the same time, then

I could have gone through and rated everything about one sample, and then move on to the next

sample. ...I didn't know I was going to have to rank them, I think that is close to my opinon, but

there were so many samples and so many questions.

1 45 1-935 This sample is slightly over cooked. But still the best.

1 48 1-935 GReat. Could maybe use a touch of tenerizer or pounding.

1 51 1-935 this sample is the only one that really smelled like a steak to me.

1 55 1-935 This was the best of the five...

1 66 1-935 This sample was the best tasting as far as flavor, tenderness, and juiciness.

1 76 1-935 935 had the most/best flavor of the samples that I enjoyed.

1 90 1-935 I liked the doneness and flavor of 935 the best, but in all cases, l was surprised by

how watery all the steak tasted.

85

1 101 1-935 While 935 appeared the darkest, I thought it had the best flavor and about the right

level of juicyness.

1 107 1-935 This might've tasted the best by just a tiny bit, but the overall presentation brought it

down to 2nd for me.

1 109 1-935 I like it! good presentation and flavor, needed to be juicy to reach perfection.

1 14 2-146 Sample 146 was near perfect! It was juicier and tasted better - but wasn't as raw as

sample 798


1 33 2-146 very tough and difficult to chew

1 48 2-146 REALLY chewey

1 57 2-146 I like the outter sear.

1 71 2-146 Very well prepared and had an excellent taste.

1 80 2-146 This one felt like the clear winner to me. The others were either too dry or too bland.

1 102 2-146 Best overall sample, but all samples could have used a little salt.

1 109 2-146 Is correct the point of cooking.

1 18 3-213 A little on the rare side.

1 19 3-213 too much grizzel

1 38 3-213 too pink for me to eat :(

1 39 3-213 Looked too raw to me.

1 48 3-213 NExt best. Not as chewey as the other 3.

1 49 3-213 A little to red for me - didn't seem well done.

1 57 3-213 Very pale.

1 65 3-213 #213 wasn't cooked very well, but it had the best flavor.

86

1 109 3-213 Needs to bleed to be juicy. If it is 'rare'


1 37 4-527 Seems too rare.

1 38 4-527 Too pink to eat, and you can see a small vessel :(

1 48 4-527 chewy

1 49 4-527 Way too red, color turned me off a bit.

1 57 4-527 appearance is just right.

1 71 4-527 While I wouldn't have considered it as ''well done,'' this sample was exceptionally

good. The taste and moistness were very good.

1 89 4-527 Best one for juciness and flavor

1 107 4-527 This had hardly any taste and was way too rare for me.

1 109 4-527 It is assumed that needs to be juicy. It was not tender.

1 15 5-798 Looks scary. I think I always expect more color.

1 16 5-798 Sample 798 one looks under cooked, I had to really talk myself into eating it. There's

a strange sheen to the meat. The seared edges add flavor, this one didn't offer that seared flavor.

1 18 5-798 Does not appear cooked enough. Appears rare and too much juicy liquid.

1 19 5-798 Little bloodier but very good

1 48 5-798 chewy, but not quite as band as the last 2

1 49 5-798 Closer to well done, but still a little too pink.

1 53 5-798 This is the only sample I deliberately did not finish.

1 57 5-798 Very pale.

1 58 5-798 This one was my favorite

1 83 5-798 I felt this one was over cooked.

87

1 89 5-798 Dry

1 100 5-798 it was an off flavor not exactly meat off flavor

1 102 5-798 Toughest and driest of the lot.

1 107 5-798 This was my second favorite in taste by just a tiny bit, but the overall presentation

(appearance, juiciness, aroma, flavor, etc) made it the best for me.

Documents

Effects of Acid Whey Marination on Tenderness, Sensory and